SynBioSRI

The Green Mother Machine: a microfluidics device for cyanobacteria

Optimising a microfluidic chemostat for experiments using cyanobacteria, including growth and feeding channels.

The Idea

Cyanobacteria are ubiquitous autotrophic bacteria that have the natural ability to utilize sunlight to convert atmospheric CO2 into carbohydrates. Cyanobacteria’s photosynthetic abilities are very appealing to synthetic biology and biotechnology. Firstly, these organisms can be harnessed to produce compounds, such as biofuels, that they would not normally produce in the wild, but which have important economic value. Secondly, cyanobacteria possess a number of comparative advantages over land plants for this goal: their genomes are easier to manipulate, they have faster growth rates, and they can be grown in areas that are not suitable to agriculture (1). While viable biotechnological applications of cyanobacteria will ultimately require production to take place in large reactors and tanks, the optimisation of the underlying cellular process will hinge on an appropriate quantitative understanding of how biochemical networks (both endogenous and synthetic) operate intracellularly. Metabolic and regulatory networks can interact with each other in non-intuitive ways, and often the mechanisms of such interactions are only apparent at the single cell level. However, while systems and synthetic biologists have spent considerable effort in observing and building tools, such as microfluidic chemostats, to study microbes at the single cell level, these efforts have largely focused on other types of bacteria. Organisms such as E. coli and B. subtilis have been preferred because of their ubiquity as laboratory model organisms and extremely fast growth rates.

Microfluidic chemostats provide an ideal environment in which the environmental conditions can be controlled tightly. The media can be exchanged easily during the experiment thus allowing for switching between for example carbon rich and carbon poor media or media with and without specific stress inducers.

Clever microfluidics design, known as the ‘mother machine’, grows bacteria in dead ended channels with a diameter of approximately one micron (growth channels) (2). The diameter of the channels is chosen to match the average diameter of an individual rod-shaped bacterium (Figure 1).

Due to this geometry, the cells grow in a line and the cell at the bottom of the dead ended growth channel (mother cell) can, in principal, be studied for an unlimited period of time (Figure 1 A). Fresh media is supplied through a comparably wide (100 µm x 100 µm, width x depth) feeding channel. Hundreds of short (25 µm in length) growth channels are attached to the feeding channel in a 90° angle. Any bacterium that is pushed out of the growth channel as the cells divide is flushed away into the feeding channel. Fresh nutrients reach the end of channel by diffusion. The mother machine design (2, 3) has the following advantages compared to other chemostats:

  1. The mother cell can be followed for an unlimited period of time.
  2. As the cells grow in a line, rather than randomly orientated like in the case of a normal bacteria colony, image analysis (segmenting and single-cell tracking) is easier.
  3. The media can still be easily exchanged.

This device, and variations of it, are made out of polydimethylsiloxan (PDMS) and were fabricated from a patterned silicon wafer (master) using soft lithography (2–4). The masters themselves are fabricated by photolithography (2–4). The drawback of conventional photolithography is that its resolution limit is about 1 µm thus making the fabrication of micrometre sized growth channels very challenging. An alternative to photolithography in the fabrication of such small channels is electron beam lithography (EBL). Few devices have exploited EBL to fabricate mother machines due to the high cost of electron beam time and the EBL facilities compared to conventional photolithography. However the excellent control over the pattering of feature sizes down to 10 nm and the high success rate are of great advantage A mother machine that has been produced using EBL was presented by Moolman and co-workers. They patterned a positive photoresist with EBL and etched the channels into the silicon wafer (5) (Figure 2 A). As a result the pattern on the silicon wafer was not the negative of the final PDMS device and an intermediate mold made out of PDMS had to be fabricated (Figure 2 B) before the patterns could be transferred into PDMS (Figure 2 C).

In our project we will design a microfluidic device based on the mother machine, which will be optimized for the cyanobacterium S. elongatus PCC7942 and it will allow running experiments inmultiple mother machines at the same time on one device for 7 days. Rod-shaped cyanobacteria, such as S. elongatus, can be loaded onto and grown in existing microfluidics devices (6, 7), but have a small number of specific requirements that are not always met by devices optimised for, e.g., E. coli and B. subtilis. One important difference is S. elongatus cells differ in size, being typically longer and, more importantly, wider than other common types of rod-shaped bacteria (for example, and according to our own measurements, S. elongatus cells are 50% wider on average than B. subtilis cells). These cellular dimensions require matching channel dimensions for optimal experimental performance. Another important difference is cyanobacterial cells need a light source to proliferate. Under a typical widefield microscope, the light source is attached to the condenser lens and sheds light directly above the sample at very low illumination angles. Short distances between the light source and the sample, and low angles of incidence are problematic because it is then difficult to maintain even illumination across a modestly wide field of view. Cyanobacteria grow an order of magnitude slower than E. coli and B. subtilis, and so, in order to speed up the experimental process, it would be convenient if one could test multiple strains and conditions in the same device, while keeping light conditions comparable. This goal can only therefore be achieved by building a microfluidics device with separate, but closely arranged channels that maximise space on the chip (Figure 4). To account for these requirements specific to cyanobacteria we divide the project into two main focus areas:

  1. Using EBL to optimize the growth channel size to grow cyanobacteria
  2. Optimization of the feeding channel layout on a device to assure even illumination on the device and increase the throughput.

The Team

Christian Schwall
PhD Student, Sainsbury Laboratory

Philipp Braeuninger-Weimer
Research Student, Department of Engineering, Academic Division: Electrical Engineering, Research group: Solid State Electronics and Nanoscale Science

Bruno Martins
Post Doctoral Researcher, The Sainsbury Laboratory


Project Outputs

Project Report
Summary of the project's achievements and future plans

Project Proposal
Original proposal and application
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Cyanobacteria are ubiquitous autotrophic bacteria that have the natural ability to utilize sunlight to convert atmospheric CO2 into carbohydrates. Cyanobacteria’s photosynthetic abilities are very appealing to synthetic biology and biotechnology. Microfluidic chemostats provide an ideal environment in which the environmental conditions can be controlled tightly. The media can be exchanged easily during the experiment thus allowing for switching between for example carbon rich and carbon poor media or media with and without specific stress inducers. The mother machine design has the following advantages compared to other chemostats: 1. The mother cell can be followed for an unlimited period of time. 2. As the cells grow in a line, rather than randomly orientated like in the case of a normal bacteria colony, image analysis (segmenting and single-cell tracking) is easier. 3. The media can still be easily exchanged.

Cyanobacteria are ubiquitous autotrophic bacteria that have the natural ability to utilize sunlight to convert atmospheric CO2 into carbohydrates. Cyanobacteria’s photosynthetic abilities are very appealing to synthetic biology and biotechnology. Firstly, these organisms can be harnessed to produce compounds, such as biofuels, that they would not normally produce in the wild, but which have important economic value. Secondly, cyanobacteria possess a number of comparative advantages over land plants for this goal: their genomes are easier to manipulate, they have faster growth rates, and they can be grown in areas that are not suitable to agriculture (1).

While viable biotechnological applications of cyanobacteria will ultimately require production to take place in large reactors and tanks, the optimisation of the underlying cellular process will hinge on an appropriate quantitative understanding of how biochemical networks (both endogenous and synthetic) operate intracellularly. Metabolic and regulatory networks can interact with each other in non-intuitive ways, and often the mechanisms of such interactions are only apparent at the single cell level. However, while systems and synthetic biologists have spent considerable effort in observing and building tools, such as microfluidic chemostats, to study microbes at the single cell level, these efforts have largely focused on other types of bacteria. Organisms such as E. coli and B. subtilis have been preferred because of their ubiquity as laboratory model organisms and extremely fast growth rates.

Microfluidic chemostats provide an ideal environment in which the environmental conditions can be controlled tightly. The media can be exchanged easily during the experiment thus allowing for switching between for example carbon rich and carbon poor media or media with and without specific stress inducers.

A clever microfluidics design, known as the ‘mother machine’, grows bacteria in dead ended channels with a diameter of approximately one micron (growth channels) (2). The diameter of the channels is chosen to match the average diameter of an individual rod-shaped bacterium (Figure 1).

  Figure 1.  Overview of the mother machine. A) The mother cell is trapped at the bottom of a dead ended channel. As the cells divide they get pushed out of the growth channel into the feeding channel where they get flushed away. B) Sketch of the mother machine and a picture of the microfluidic device. C) Top view of mother machine. The large channel in the middle of the device is called feeding channel and the small channels branching out of the feeding channel are called growth channels. All figures were adapted from (1)

Figure 1. Overview of the mother machine. A) The mother cell is trapped at the bottom of a dead ended channel. As the cells divide they get pushed out of the growth channel into the feeding channel where they get flushed away. B) Sketch of the mother machine and a picture of the microfluidic device. C) Top view of mother machine. The large channel in the middle of the device is called feeding channel and the small channels branching out of the feeding channel are called growth channels. All figures were adapted from (1)

Due to this geometry, the cells grow in a line and the cell at the bottom of the dead ended growth channel (mother cell) can, in principal, be studied for an unlimited period of time (Figure 1 A). Fresh media is supplied through a comparably wide (100 µm x 100 µm, width x depth) feeding channel. Hundreds of short (25 µm in length) growth channels are attached to the feeding channel in a 90° angle. Any bacterium that is pushed out of the growth channel as the cells divide is flushed away into the feeding channel. Fresh nutrients reach the end of channel by diffusion. The mother machine design (2, 3) has the following advantages compared to other chemostats:

1. The mother cell can be followed for an unlimited period of time.

2. As the cells grow in a line, rather than randomly orientated like in the case of a normal bacteria colony, image analysis (segmenting and single-cell tracking) is easier.

3. The media can still be easily exchanged.

This device, and variations of it, are made out of polydimethylsiloxan (PDMS) and were fabricated from a patterned silicon wafer (master) using soft lithography (2–4). The masters themselves are fabricated by photolithography (2–4). The drawback of conventional photolithography is that its resolution limit is about 1 µm thus making the fabrication of micrometre sized growth channels very challenging. An alternative to photolithography in the fabrication of such small channels is electron beam lithography (EBL). Few devices have exploited EBL to fabricate mother machines due to the high cost of electron beam time and the EBL facilities compared to conventional photolithography. However the excellent control over the pattering of feature sizes down to 10 nm and the high success rate are of great advantage.

A mother machine that has been produced using EBL was presented by Moolman and co-workers. They patterned a positive photoresist with EBL and etched the channels into the silicon wafer (5) (Figure 2 A). As a result the pattern on the silicon wafer was not the negative of the final PDMS device and an intermediate mold made out of PDMS had to be fabricated (Figure 2 B) before the patterns could be transferred into PDMS (Figure 2 C).

  Figure 2.  Workflow of Moolman device. A) The patterns are etched into the silicon wafer after the negative resist has been patterned with the electron beam. B) A negative replica of the structures on the silicon wafer is transferred into a PDMS mold. C) The PDMS mold made in B) is used to pattern the final device onto the PDMS. The final pattern on the PDMS is identical with the pattern on the silicon wafer. The figure was taken from (5)

Figure 2. Workflow of Moolman device. A) The patterns are etched into the silicon wafer after the negative resist has been patterned with the electron beam. B) A negative replica of the structures on the silicon wafer is transferred into a PDMS mold. C) The PDMS mold made in B) is used to pattern the final device onto the PDMS. The final pattern on the PDMS is identical with the pattern on the silicon wafer. The figure was taken from (5)

In our project we will design a microfluidic device based on the mother machine, which will be optimized for the cyanobacterium S. elongatus PCC7942 and it will allow running experiments in multiple mother machines at the same time on one device for 7 days. Rod-shaped cyanobacteria, such as S. elongatus, can be loaded onto and grown in existing microfluidics devices (6, 7), but have a small number of specific requirements that are not always met by devices optimised for, e.g., E. coli and B. subtilis.

One important difference is S. elongatus cells differ in size, being typically longer and, more importantly, wider than other common types of rod-shaped bacteria (for example, and according to our own measurements, S. elongatus cells are 50% wider on average than B. subtilis cells). These cellular dimensions require matching channel dimensions for optimal experimental performance. Another important difference is cyanobacterial cells need a light source to proliferate. Under a typical widefield microscope, the light source is attached to the condenser lens and sheds light directly above the sample at very low illumination angles. Short distances between the light source and the sample, and low angles of incidence are problematic because it is then difficult to maintain even illumination across a modestly wide field of view.

Cyanobacteria grow an order of magnitude slower than E. coli and B. subtilis, and so, in order to speed up the experimental process, it would be convenient if one could test multiple strains and conditions in the same device, while keeping light conditions comparable. This goal can only therefore be achieved by building a microfluidics device with separate, but closely arranged channels that maximise space on the chip (Figure 4). To account for these requirements specific to cyanobacteria we divide the project into two main focus areas:

1. Using EBL to optimize the growth channel size to grow cyanobacteria

2. Optimization of the feeding channel layout on a device to assure even illumination on the device and increase the throughput.

Implementation:

1. Using EBL to optimize the growth channel size to grow cyanobacteria

Our plan is to use EBL to fabricate growth channels specifically tuned to grow cyanobacteria similarly to Moolman and co-workers (5). However instead of using a positive resist we will be using a negative photoresist. Positive photoresists become soluble to the developer in the areas that have been exposed to the light (Figure 3 A). Negative resists are insoluble to the developer in the areas that have been exposed to the light (Figure 3 B).

  Figure 3.  Overview of positive and negative resits. A) Positive photoresists become soluble to the developer in the areas that have been exposed to the light. B) Negative resists are insoluble to the developer in the areas that have been exposed to the light. The image was taken from Wikipedia (8).

Figure 3. Overview of positive and negative resits. A) Positive photoresists become soluble to the developer in the areas that have been exposed to the light. B) Negative resists are insoluble to the developer in the areas that have been exposed to the light. The image was taken from Wikipedia (8).

The advantage of negative resists is that the patterns can be easily transferred into PDMS as the pattern on the silicon wafer is the negative of the final device in PDMS. Thus by using a negative resists with the EBL the intermediate PDMS mold Moolman and co-worker needed for the fabrication of their device (Step 2 in Figure 2) would no longer be necessary. This would greatly facilitate the replica molding of the patterns from the silicon wafer to the PDMS. Unfortunately, the choice of negative EBL resists is very limited and a new process flow has to be developed. We have narrowed down our choice of negative resists to AZ 5214E and SU8 (one of the most commonly used negative photoresists). Once we have found a good photoresist we will test a variety of growth channel sizes to optimize the growth conditions of cyanobacteria in our mother machine.

2. Optimization of the feeding channel layout

On a typical mother machine there is only one feeding channel, which allows for one experiment at a time (Figure 4 A). This greatly limits the application of the mother machine to slow growing cyanobacteria. We therefore want to design a device with multiple mother machines packed closely to each other such that they can fit onto a chip of the size of a 2 pound coin (Figure 4).

  Figure 4.  Overview of mother machine designs we want to test. A) The classic mother machine device published by Wang and co-workers. B) This device has two mother machines on one chip. C) In this device two growth channels with different conditions and strains can be image in one field of view (radius = 600 µm). D) A combination of B) and C).

Figure 4. Overview of mother machine designs we want to test. A) The classic mother machine device published by Wang and co-workers. B) This device has two mother machines on one chip. C) In this device two growth channels with different conditions and strains can be image in one field of view (radius = 600 µm). D) A combination of B) and C).

To test the feasibility of our project we fabricated a mother machine entirely by using EBL to pattern channels of a constant depth of 1 µm and a width of 1 µm to 10 µm into AZ 5214E. The structures were replicable multiple times into PDMS without losing any of their resolution and we were able to load cells into the growth channels (Figure 5). Unfortunately we could not establish a good perfusion of media through the feeding channels as they were too shallow. Thus, we now want to fabricate feeding channels with a width of 100 µm and a depth of 100 µm as Wang and co-workers did (2). Pattering such large structures using EBL is tedious which is why we will exploit conventional photolithography in a second step before the growth channels have been deposited. For the photolithography step we will have to order various chrome masks to pattern the photoresists. These chrome masks are expensive and unfortunately there is no alternative as transparencies or emulsion glass masks do not have a high enough resolution.

 Figure 5. Micrograph of cells in the prototype mother machine.

Figure 5. Micrograph of cells in the prototype mother machine.

The work in the project will be divided as follows: - All the necessary clean room work e.g. the lithography work will be done by Philipp Braeuninger-Weimer who is an expert on EBL and photolithography. - The design of the microfluidic chips and the PDMS work will be done by Christian Schwall. - The devices will be tested with cyanobacteria by Christian Schwall and Bruno Martins.

Benefits and outcomes:

To check the feasibility of our project we made a promising prototype fabricated with electron beam lithography (EBL) in collaboration between the Department of Engineering (Philipp Braeuninger-Weimer) and the Sainsbury Laboratory (Christian Schwall and Bruno Martins). The funding for electron-beam time and access to the clean room facilities is already in place. In order to further pursue this project, what we require at this point is funding to design a few chrome masks so we can pattern the larger feeding channels using conventional photolithography. The new device would be specifically designed for cyanobacterium S.elongatus PCC 7942, which to our knowledge, is a device that does not yet exist. Further, having multiple mother machines very close to each other on the same chip would drastically increase the throughput of one experiment. This is of great importance when running experiments for 7 or more days.

The direct benefits of this project to the framework of synthetic biology are two-fold:

1. S. elongatus is one of the most widely studied cyanobacteria, because it is the model organism of choice for research on the prokaryotic circadian clock, and because it is a promising host of synthetic circuits for metabolic engineering (1).Despite their importance, specific single-cell analysis tools have, so far, prioritized other types of bacteria. We believe that, in order to optimally characterise and manipulate the interaction between a cyanobacterium ‘chassis’ (i.e., a cell) and an exogenous circuit, we must first study systems in a rigorously controlled environment at the single-cell level. Our device will provide such a level of control. On the other hand, while we will design and test our device with cyanobacteria, there is no impediment to it being tweaked and used to grow other bacteria.

2. Microfluidics has emerged as a pivotal technique to study organisms at single-cell or microcolony levels. However, fabricating the devices can be challenging and discourage usage by the wider community. It is therefore essential that cross-talks between advanced engineering and fundamental biology are maintained. Our proposal addresses this cross-talk by: i) partnering skills in materials engineering, physics and cell biology from two different Cambridge departments; ii) exploring the potential of EBL, a technology that has not yet been used extensively to fabricate devices for biology; iii) accomplishing all steps that link an engineering blue print to a real everyday experiment in a biology lab.

Other researchers will benefit from our project too: for any working design, we will share the CAD files and our gained expertise openly. We will produce a step-by-step manual, or equivalent documentation, which will be shared online. Anyone in the community will be able to use or modify our device for their own needs.

References:

  1. I. M. P. Machado, S. Atsumi, Cyanobacterial biofuel production. J. Biotechnol. 162, 50–56 (2012).
  2. P. Wang et al., Robust growth of Escherichia coli. Curr. Biol. 20, 1099–1103 (2010).
  3. T. M. Norman, N. D. Lord, J. Paulsson, R. Losick, Memory and modularity in cell-fate decision making. Nature. 503, 481–6 (2013).
  4. Z. Long et al., Microfluidic chemostat for measuring single cell dynamics in bacteria. Lab Chip. 13, 947–54 (2013).
  5. M. C. Moolman, Z. Huang, S. T. Krishnan, J. W. J. Kerssemakers, Electron beam fabrication of a microfluidic device for studying submicron-scale bacteria. J. Nanobiotechnology. 11, 12 (2013).
  6. J. R. Moffitt, J. B. Lee, P. Cluzel, The single-cell chemostat: an agarose-based, microfluidic device for high-throughput, single-cell studies of bacteria and bacterial communities. Lab Chip. 12, 1487 (2012).
  7. S.-W. Teng, S. Mukherji, J. R. Moffitt, S. de Buyl, E. K. O’Shea, Robust Circadian Oscillations in Growing Cyanobacteria Require Transcriptional Feedback. Science (80-. ). 340, 737–740 (2013).
  8. Cepheiden, Comparison positive negative tone resist. Wikipedia, (available at http://en.wikipedia.org/wiki/Photoresist).

System with bacteria metabolic and biochemical network information

The idea is to develop a computational system and corresponding web site to couple information on metabolic and biochemical networks within bacteria (focussing initially on cyanobacteria) with networks of protein evolutionary history and homology. The system is designed to be easily used by biologists with minimal computing experience. The main principle is that the variability in the enzymatic components of pathways across a large sample of different genomes provides a valuable resource for the understanding and manipulation of biosynthesis.

The Idea

The idea is to develop a computational system and corresponding web site to couple information on metabolic and biochemical networks within bacteria (focussing initially on cyanobacteria) with networks of protein evolutionary history and homology. The system is designed to be easily used by biologists with minimal computing experience. The main principle is that the variability in the enzymatic components of pathways across a large sample of different genomes provides a valuable resource for the understanding and manipulation of biosynthesis.

We aim to create a web­based tool to easily analyse the gene and protein families involved in the complete multi­organism network of biosynthetic pathways across hundreds of genomes. This will allow points of interest such as conserved, specialist and missing biosynthetic steps to be quickly and easily identified from within a clade of organisms. Thus the synthetic pathways within individual species can be better understood, and underpinned with concrete data relating to genes and homologues etc.

Importantly, the genome specific differences will enable identification of useful pathway components that can be recombined in novel ways to introduce foreign biosynthetic pathways into an organism of interest.

This project involves the interconnection of two networks of information 1) the traditional biochemical network of enzyme­driven metabolic pathways and 2) the evolutionary connection between homologous genes and proteins across a wide range of organisms. These networks can readily be described by a computational system that models each enzyme as a "node" with connections to other nodes. In the first case the connections will correspond to metabolite (product­substrate) links between genes, and in the second case the connections represent significant sequence similarity, from which homology can be inferred. These networks will be computed and/or curated and stored in a database that will then underpin the web­based tool. Gene families not involved in metabolic pathways will also be collated and stored in the same system, and though not presented in exactly the same way, they will be equally accessible.

The web site will display the information in a manner that aims to illustrate how different biosynthetic pathways differ between genomes. The presentation of data will be primarily visual, anchoring it to a metabolic pathway map; either a complete multi­genome "superset" map or just a subset, focussing on a particular pathway. The display of gene presence/absence and conservation across the genomes will also be graphical, including for genes not involved in a metabolic pathway. The user will then be able to investigate the full depth of sequence based information, connect to external bioinformatics databases (GenBank, UniProt etc.) and extract any family trees and alignments for any and all points of interest. All data will be downloadable as spreadsheets and in a variety of popular bioinformatics formats. The metabolic map may also be superimposed with other, orthogonal data (e.g. transcriptomics, ChIP­seq, metabolomics) that can be anchored to the genes, so the information can be displayed and analysed in a pathway­wide context, rather than as the more common linear genome or array­based representations. Free, open­source software allowing mapping of genomics data to a complete metabolic gene map (and also to genes not involved in metabolism) is currently not available and would make many biological analyses simpler.

The Team

David Lea­Smith: Postdoctoral associate in the Department of Biochemistry (Chris Howe lab). David specialises in microbial genetics and biochemistry with a focus on photosynthetic organisms (cyanobacteria and purple bacteria). He uses synthetic biology, genomics and genetic tools to understand bacterial physiology and metabolism and develops strains with increase biofuel or electrical output using energy derived from either photosynthesis or degradation of waste products. He will be primarily responsible for the creation of a holistic biosynthetic pathway map from a wide­ranging review of databases and published literature relating to metabolism and biosynthetic pathways. This will be completed for cyanobacteria in the initial instance. Eschirichia coli, the best annotated bacterium and the model cyanobacterium, Synechocystis sp. PCC 6803 and Nostoc sp. PCC 7120, will be used to create the anchor points for a consistent annotation of homologous gene clusters. David will also be involved in the preliminary testing of the web site and provide feedback to ensure that the eventual outcome suits the needs of the biological community. The clustering of genes into families naturally aims to make it clear what the homology relationships are between different proteins, e.g. to identify orthologues. Where the traditional naming of genes and proteins are either inconsistent or missing, combining knowledge about orthology and where a protein is likely to act in a particular pathway make the identity of any component unambiguous. Thus as a necessary side­effect, the system will be to generate a single consistent nomenclature for all of the enzymes in the network of pathways, across all the organisms of interest. In the future this could provide a basis for automatically annotating new genome sequences within the same scheme. 

Tim Stevens: Senior Investigator Scientist in the Munro Lab at the MRC Laboratory of Molecular Biology in Cambridge. Tim provides computational biology oversight, development and training within the LMB's Cell Biology Division. He researches several bioinformatics areas with a primary focus on protein homology detection methods, 3D genome structure and interactions (PMID:24067610), transmembrane proteins (PMID:20603021) and is author of the book "Python Programming for Biology" (ISBN­13: 978­0521895835). Tim will be responsible for the bioinformatics analyses pathway maps as a website. This work involves several sequential steps: comparison of all protein sequences from all genomes under study to generate a matrix of detectable similarities; the hierarchical clustering sequences into family groups; the detection of remote homology and identification of missing genes; the connection of clusters, and thus also individual genes, to anchor points on the biosynthetic pathway map. All of this information will be stored in an SQL database (as is standard) and be presented as an interactive, searchable, graphically­oriented web page. A hierarchical approach will be used for the clustering of protein sequences into family groups because the amount of conservation within a family can vary substantially from case to case. Also, by moving up and down a familial hierarchy a user of the website will be able to see how specialisation of function arises as species and sequences diverge. This will allow sequence variation (or absence) to be related to metabolic capabilities. Initial work will focus on cyanobacteria because it is a mainstay of the Howe lab and because a large amount of analysis on cyanobacterial synthetic pathways has already been performed, with a practical application towards generating biofuels. This group of bacteria will also serve as a test bed for the system, fixing any problems and refining the web site before the project is opened­up to bacteria at large. This project must be of limited scope to be achievable within a limited time period, and so will focus on a subset of Bacteria, but the technology would naturally be expandable to further clades, including those from Archaea and Eukarya.


Project Outputs

Summary of the project's achievements and future plans

Project Proposal
Original proposal and application

software.png

Project Resources


Implementation

The funding will be used primarily for the purchase of a rack­mount computer server (e.g. Dell PowerEdge R730) that will run the website and database, with a capability for on­the­fly informatics searches/analysis and provision for the database to be expanded as needed to encompass further information for more species. It will be housed within the Department of Biochemistry and top­level administration will be provided by the Bioinformatics and Computational Biology Service (http://computing.bio.cam.ac.uk/index.html), but all of the set­up and day­to­day running of the server and web site will be handled by the primary collaborators. Mid­way though the term of the project, a test version of the web site running on the server will be made available locally to the Synthetic Biology SRI and full public deployment will occur by the end of the term. The funding will also cover the costs of publishing this work in an appropriate peer reviewed journal.

Benefits and outcomes:

This project idea is underpinned by the collaboration of a University laboratory that specialises in understanding molecular evolution, metabolism and biochemistry of photosynthetic micro­organisms, with a specialist in computational biology from the LMB who can provide all of the necessary expertise in bioinformatics and the computer programming required to create on­line services and databases.

The eventual outcome of this project will be the creation of a tool to enable any researcher within the field of synthetic biology to investigate synthetic outcomes and plan genetic modifications in the context of a complete biochemical network. Syntheses of interest will be presented in an accessible manner based on a metabolic pathway map, while the full depth of information will be made available at every enzymatic point, for example to show sequence alignments and trees to illustrate how homologues have arisen, diverged and disappeared.

This tool will be built using open, web­based technologies and thus the final product will be readily shareable with the entire biological community. Coupled with this, all of the computational code that underpins the system will be made available as open source software, with the potential for future collaborative development with other groups.

The project would provide the synthetic biology field, and wider disciplines, with a validated set of protein families and a consistent nomenclature (which in isolation is not a trivial task). This could then naturally pervade newly sequence genomes, providing a robust means by which many gene sequences can be automatically and consistently annotated. Much of the preliminary proof­of­concept work for this project has already been achieved and so it is entirely realistic to complete the project, and present it as a functional web resource, within six months.

An initial database covering cyanobacterial genomes and proteome sequences has already been assembled. Also, all of the computational code required to perform exhaustive all­versus­all sequence based homology searches, and the clustering and hierarchical sub­clustering thereof, has been written.

Given our preliminary work on cyanobacterial genomes, this clade will be the first to be presented in a prototype web site. This prototype will be made available locally to the Synthetic Biology SRI, for testing and appraisal by anyone who wishes to try the system, thus promoting interaction within the entire SRI. In due course the situation will be expanded to cover further bacterial clades and then released publicly.

When completed, this work will be published in an appropriate peer reviewed journal.

Synthesis of novel optimised lux reporters for eukaryotic systems

This project would build on our previous work identifying and optimizing bacterial luciferases from a variety of marine bacteria. We propose to generate versions of these reporters for eukaryotic systems, including the production of Nanolantern-like systems for lux luciferases. 

The Idea

Bioluminescent reporters are a common tool in molecular biology. Luciferases from various sources (firefly, click beetle, dinoflagellate, sea pansy, copepod and bacterial) have been cloned and exploited as reporters, due to their fast time dynamics and their detectable and quantifiable outputs. Each luciferase has particular enzymatic properties and substrate requirements, leading to limitations on how assays can be performed. While all eukaryotic luciferases require the addition of an exogenous substrate (eg. luciferin, calcium ions) to produce light, bacterial bioluminescence generated by the lux operon has the benefit of autonomous luminescence [Engebrecht et. al, Cell 1983]. Yet, compared to fluorescent protein reporters, little engineering has been applied to improve and optimize the lux operon on a protein level.

Recently, development of the Nanolanterns [Takai et al. 2015] from Renilla luciferase variants has expanded the colour palette for luciferase reporters, as well as enhancing their brightness. This development enables real-time multi-channel luminescence measurements which can have significant applications for cell biology and gene expression analysis. These use reporters are also somewhat limited through their requirement of the addition of coelenterazine substrate.

The bacterial luminescence pathway has have been inserted into eukaryotic systems previously, with some success in yeast and mammalian systems [Close et al. 2010]. Often the luminescence yield is limited compared to the bacterial context due to the difficulty of expressing polycistronic operons in eukaryotic systems. In plant systems, previous studies have only inserted the luxAB luciferase, which would requires the addition of decanal as a substrate [Cui et al. 2014].

This project would build on our previous work identifying and optimizing bacterial luciferases from a variety of marine bacteria. Our work as part of the April 2015 SRI’s funding call has led us to generate a variety of autonomous lux operon reporters and luciferase reporters through directed evolution. In order to develop these reporters further for eukaryotic systems, we propose to generate versions of these reporters for eukaryotic systems, including the production of Nanolantern-like systems for lux luciferases. This will allow the use of highly efficient autonomous luminescence production for eukaryotic systems with multiple colors, which will provide new systems for gene expression and cell biology studies, as well as any other applications where light energy needs to be produced from chemical energy.

The Team

Bernardo Pollak
Graduate Student, Department of Plant Sciences

Anton Kan
Graduate Student, Department of Plant Sciences


Project Outputs

 

Project Report
Summary of the project's achievements and future plans

Project Proposal
Original proposal and application

software.png

Project Output
Project poster


Implementation

We will use the brightest evolved proteins obtained from our previous SRI project to design and synthesize codon-optimised versions of these lux reporters for a plant chassis. In our previous work, we reorganised the lux operon (luxCDABEG) into the luciferase component, luxAB, and the substrate pathway component, luxCDEG. To reengineer the reporters for the plant context, we will redesign the polycistronic substrate pathway operon into simple monocistronic transcriptional units. Recent work on Marchantia polymorpha has also discovered and tested a range of native promoters that can be used to carefully tune the expression of each of the components to their optimal levels. We will also design and generate monomeric versions of the luxAB reporter, and through translational fusions to fluorescent protein expanding of the colour palette for multi-channel imaging and detection. Due to the complexity of the genetic reorganisation, DNA synthesis provides a very attractive route to the generation of such constructs. We will proceed to test these systems in Marchantia polymorpha to demonstrate the use of bright and autonomous lux reporters in eukaryotic systems.

Benefits and outcomes

Our project aims to develop a range of novel bright, autonomous, multi-channel bioluminescent reporters, as well as vectors for their expression in eukaryotic plant systems. These will be made publicly available.

SynBio Student Society

To establish the Cambridge University Synthetic Biology Society (CUSBS) to promote the field of synthetic biology amongst the student community and within schools in Cambridge. To continue development of OpenScope and other open technology projects.

The Idea

The Cambridge University Synthetic Biology Society (CUSBS) aims to promote the field of synthetic biology (SynBio) amongst the student community and within schools in Cambridge. Particular focus will be given to its real-world applications and interdisciplinary nature. Expert speakers from different SynBio disciplines will be invited to give talks, and links to external SynBio events and networks will be developed. We aim to work closely with both the SynBio SRI and the Open Plant initiative, and strengthen their direct links with STEM students.

To give members hands-on experience in designing and documenting open-source hardware and software, CUSBS will run student-led projects. The possibility of wet-lab based work for future CUSBS generations will be explored, and suitable workshop and lab infrastructures put in place. Educational events for local schools relating to the themes of synthetic biology, genetic engineering, open-hardware and electronics will be held and teaching resources made openly-available.

The student-led projects set CUSBS apart from many other student societies, and the vast majority of the SynBio Fund grant would be used to cover these costs. As the longevity and success of CUSBS depends on getting students engaged in projects as soon as possible, we envisage beginning enrolment by January 2016. We also intend to apply for sponsorship from science-oriented companies, banks and firms that have supported other societies in the past. We would be keen to coordinate this with the SynBio SRI such that resources could be pooled and funds received would be directed into a central pot.

The Team

  • Atti English - Undergraduate student, Pembroke College, Natural Sciences, SynBio Student Society
  • Lorenzo Venturini - Undergraduate student, Queens' College, Engineering, SynBio Student Society
  • Souradip Mookerjee - Undergraduate student, St Catharine's College, Medicine, SynBio Student Society
  • Simon Swan - Undergraduate student, Clare College, Dept of Chemical Engineering and Biotechnology, SynBio Student Society
  • Ginny Rutten - Undergraduate student, Trinity Hall, SynBio Student Society
  • Ocean Haghighi-Daly - Undergraduate student, Clare College, Natural Sciencesm SynBio Student Society
  • Olivia Lala - Undergraduate student, New Hall, Natural Sciences, SynBio Student Society
  • Anastasia Ershova - Undergraduate student, St John's College, Natural Sciences, SynBio Student Society
  • Will Earley

Project Outputs

 

Project Report
Summary of the project's achievements and future plans

Project Proposal
Original proposal and application
Download PDF >> 

software.png

Project application slides
Design files and documentation


Outcomes, Outputs, Progress

The Cambridge University Synthetic Biology Society (CUSBS) aims to promote the field of synthetic biology (SynBio) amongst the student community, particularly outside of the biological sciences. We aim to do this by creating online resources for learning about synthetic biology, promotion of events and networks, e.g. iGEM, hosting of speakers from a range of fields within SynBio and organising of student-led projects.

In our first year, we have successfully managed to start the above objectives. We have created a website (www.CUSBS.co.uk) for the current and future hosting of resources. Café Synthetique has been promoted on our Facebook page and iGEM at our events. Dr Guy-Bart Stan from Imperial College London gave a presentation on synthetic biology design and control, in Easter term. Three other speakers have expressed an interest in presenting.

The major outcome of the first year has been an ongoing student-led project to design and build a scanning, automated microscope for multiple samples and time-lapse studies, to demonstrate the use of lower cost DIY alternatives for instruments in research labs. This has attracted students from across the scientific disciplines, with 30 people on the project associated mailing list.

Speaker Events and Networks

  • 1.1. The society hosted one speaker this year - Dr Guy-Bart Stan from Imperial College London. This talk was hosted in the engineering department and was well attended. Refreshments were provided, and all costs were covered by the society. Without specific attendance records, but from anecdotal evidence, there appeared to be a substantial amount of post-graduate attendance, with less undergraduates in attendance. The talk was mostly accessible, however may have been pitched above the general knowledge of an undergraduate from a non-biological discipline. However, given the popularity among post-graduates this does not appear to be a problem. Given the response the event was a success.
  • 1.2. Other speakers have been contacted and three have responded with interest. The SynBio groups at UCL, Nottingham and Imperial have been contacted. These include Mathematicians, Molecular Biologists, Engineers and Computer Scientists. In future the society plans to hold approximately three talks in each of the first two terms of the academic year. The possibility of talks during exam term will depend upon future assessment of interest.
  • 1.3. The society plans on expanding the range of speakers to include people working in industry / start-ups / intellectual property law / bioethics. In this way we would broaden the awareness of the implications of the field in terms of law, etc. to those from a scientific background, and attract new members of non-scientific disciplines, e.g. law.
  • 1.4. While the responsibility of organising these events to date fell to the president it is planned to give the publicity officer greater autonomy in order to achieve our objectives.
  • 1.5. Non-CUSBS SynBio related events have been advertised via our mailing list (cusbs-interest@srcf.net ; 59 members), on our Facebook page and at events. As the SynBio community grows in Cambridge, it is expected that promotion of these events will take up a greater amount of the publicity secretaries time.
  • 1.6. All members of the outgoing committee signed up for accounts with EUSynBioS, however it doesn’t seem to be active upon last inspection.

Educational Workshops and Outreach Events

  • 2.1. Whilst the outgoing publicity secretary attempted several to contact local schools, however none response was recieved. Should there be significant interest within the scoieties members, a new role will be created to organise these events. Given the experience of the previous committee the methodology may be changed to include the following:
  • 2.1.1. Use of college and department outreach resources, such as contacts and relationships with schools, as these are more legitimate from a school point of view.
  • 2.1.2. Use of combination of phone calls and email, rather than emails alone.

Student-Led SynBio Projects

  • 3.1. The projects have been the major activity within the society this year. Initially two projects, Project 1 was not persued due to a different group (WaterScope) working on the same project to a higher standard than was possible for a student-led project. Project 2, has been successful in attracting participants from a diverse range of sciences, and is ongoing. Several undergraduate physicists, all of whom are on the incoming committee, contributed significantly to the project.
  • 3.2. While project 2 is almost complete on the hardware side, more work is required to complete the software side. The time available to work on the project limited due to the parts not arriving until Easter term. Progress continued during Easter term before exams, with weekly 2-3 hour sessions, with an average attendance of about 7/8 people. Sessions became daily after the exams and May week when students had more time available. Additionally, some work has been done during the long vacation, primarily by Ptolemy Jenkins, incoming committee junior treasurer.
  • 3.3. Project work has been carried out in the Dyson centre of the engineering department, where the CUSBS has secured space. This space is available for any future hardware projects.
  • 3.4. Collaboration with WaterScope was frequent initially; however, it became apparent that any collaboration would be difficult to continue in the format of a student-led project. The Docubricks software has been used to partially document the project, with completion of the documentation expected to occur as the project continues.
  • 3.5. The documentation has, as yet, not been released as open-source due to time constraints. Publication of documentation is expected during next academic year.
  • 3.6. To date no further prototypes have been built. We plan to repeat the project next year. It would be expected to be more rapid, as documentation will have been generated. The goal would be to give some hardware experience to those members of the society from non-engineering backgrounds.
  • 3.7. In collaboration with BioMakeSpace, the society plans on running at least one wetlab project in order to broaden and deepen members experience SynBio

SynBio Hub

The SynBio Hub platform will allow the scientific community to monitor, review and discover the latest developments in synthetic biology Intellectual Property (IP). The open source platform will initially track all IP being published via the US patent office (USPTO) for relevance to synthetic biology.

The Idea

The SynBio Hub platform will allow the scientific community to monitor, review and discover the latest developments in synthetic biology Intellectual Property (IP). The open source platform will initially track all IP being published via the Us patent office (USPTO), currently the USPTO makes available all patent applications and granted patents as a weekly dump of around 14000 documents. Although the data is freely accessible it is hard to manage and view unless you have access to premium services and tools that can present this information more easily. Being able to monitor the stream of patent data will give a valuable resource to the synthetic biology community, allowing access to both detailed scientific information relating to inventions and detailed information on key players in the space, both inventors and companies, that can be useful in identifying collaboration opportunities. The platform will also allow for users to tune and edit their areas of interest allowing more relevant data ‘streams’ to be created and shared eg new genome editing techniques, applications featuring particular DNA ‘parts’. 

It is hoped that should the project be successful we could then develop the project further to include other sources of patent eg EPO (European Patent Office), WIPO ( World Intellectual Property Organisation) and non patent literature sources eg CrossRef, PubMed etc using ContentMine tools. Historic data could also be loaded into the system, giving a much more holistic view of the state of IP within domain of synthetic biology, however this would significantly increase the technical challenges for the system as the volume of data increases. We are proactively seeking funding and having a prototype would be beneficial.

The Team

Ben Pellegrini, ex CTO of CambridgeIP, responsible for online patent search platform with over 100 million scientific documents.

Dr John Liddicoat, Philomathia Post Doctoral Fellow in Intellectual Property Law and Genomics, Centre for Law, Medicine and Life Sciences, Faculty of Law

Dr Ben Tregenna, PhD in Quantum Computing at Imperial, early member of Autonomy with specialist skills in Information Retrieval (IR), data search and indexing systems.

Dr Peter Murray-Rust, Emeritus Reader at the Department of Chemistry and Shuttleworth Foundation Fellow, Founder of ContentMine a project providing open source software and training for text and data mining.


Project Outputs

software.png

Implementation

The USPTO publishes around 14000 patent applications and grants per week, meaning during the 6 month project we will likely have to search and look at 336,000 documents, it is therefore vital the system is designed to scale with the all the implications of big data analysis. A cloud-based infrastructure will be utilised, on AWS (Amazon Web Services), to allow for quick deployment of servers and allow for potentially rapid growth. Data ingestion and migration routines will be written to transform the structured data dumps (XML) to a NOSQL store to allow for better management of the big data and more flexibility in managing the data going forward. Once the data is accessible through the NOSQL data store we will create indexes, to make the documents easily accessible and searchable. At this point we will be able to start filtering the data for synthetic biology related documents using the search algorithm we will develop, it is assumed this algorithm will be initially broad enough to allow the capture of all documents. The algorithm will primarily consist of using broad patent classification filters and keyword filters, use of standard ontology’s, identifiers and custom search criteria, possibly in the form of regular expressions generated in collaboration with synthetic biologists. Tools will then be added to front end to allow users to fine tune the data they see. Once we have reduced the document data down to relevant documents we will need to present this data to end users through an easy to use interface. The ability to specifically filter the data for patents in a narrower field of interest such as microbial, plants or biomedical patents would also be possible using the same technologies and user interface.

The front end will be delivered in Ruby on Rails, making use of freely available gems and open source plugins to deliver maximum functionality in time available (eg account management, analytics, search, filter, export etc).

Red Katipo will develop the system and meetings will be arranged at regular intervals with John Liddicoat, Peter Murray-Rust, members of the Synthetic Biology SRI and potentially wider stakeholders to ensure the system design is useful to potential users.

Benefits and Outcomes

This monitoring service, and eventual corpus of synthetic biology related literature which would be freely available and accessible in a centralised place would allow people to comment and share content and information would act as a step towards developing routes for determining freedom to operate in synthetic biology. It would complement efforts promoting IP-free sharing, development of standardised Open MTAs (Material Transfer Agreements) and exploration of innovative licensing arrangements.

We see an opportunity to embed such a platform in the technology and knowledge transfer workflow of research institutions, providing a storefront for IP.

We propose to use this grant to seed fund the early development of SynBio Hub. We will seek additional funding and have had positive responses to the idea from SynbiCITE. Business models to ensure its long-term sustainability are being explored and this would most likely be through a freemium model offering value added services but the basic platform developed during this phase will be open source and provide open data for all.

Reliable IP-free system for inter-chassis transfer of the high molecular weight DNA

This project aims to develop an IP-free system for transfer of the high molecular weight DNA from E. coli to M. polymorpha. This includes development of the plant-specific iBACs for the reliable transfer and integration of the high molecular weight DNA into the M. polymorpha genome. Alternative methods for interchassis DNA transfer, such as integrative and conjugative elements (ICEs) will be also explored.

The Idea

The ability to manipulate and transfer the high molecular weight DNA molecules between different chassis constitutes one of the main bottlenecks of the rational genome engineering. Bacterium Escherichia coli and plant Marchantia polymorpha are both well-characterized model organisms that are frequently used as chassis for biotechnology and synthetic biology applications. Although the vast majority of the good DNA assembly and editing tools are in E. coli, M. polymorpha is better host for certain applications. Novel tools combining the benefits of M. polymorpha chassis and the reliable DNA recombineering approaches developed for E. coli are critical for developing a robust synthetic biology toolkit. Bacterial artificial chromosomes (BACs) based on the E. coli fertility factor (F-factor) are frequently used for engineering high molecular weight DNA fragments in E. coli. We have recently engineered integrative bacterial artificial chromosomes (iBACs) that can accept virtually any high molecular weight DNA fragment for integration into B. subtilis chromosome and allow rapid selection of transformants by B. subtilis-specific antibiotic resistance and the yellow fluorescent protein (mVenus) expression. This project aims to develop an IP-free system for transfer of the high molecular weight DNA from E. coli to M. polymorpha. This includes development of the plant-specific iBACs for the reliable transfer and integration of the high molecular weight DNA into the M. polymorpha genome. Alternative methods for interchassis DNA transfer, such as integrative and conjugative elements (ICEs) will be also explored.

The Team

Dr. Mario Juhas
Research Associate, Department of Pathology

Dr. Dave Willey

Christian R. Boehm
PhD Candidate in Plant Synthetic Biology, Department of Plant Sciences


Project Outputs

 

Project Report
Summary of the project's achievements and future plans

Project Proposal
Original proposal and application
Download PDF >> 

software.png

Project application slides
Design files and documentation


This project led to a number of educational resources and useful DNA parts for the transfer the high molecular weight DNA between synthetic biology chassis - contributing to overcoming a major bottleneck of rational genome engineering.

Outcomes, Outputs and Progress

Bacterial artificial chromosomes (BACs) are frequently used for manipulating high molecular weight DNA fragments in E. coli. To facilitate integration of the high molecular weight DNA into M. polymorpha plastid, we engineered the well-characterized BAC pBeloBAC11 by inserting sequences homologous to the trnG-trnfM intergenic region of the M. polymorpha plastid DNA. Two integration target sequences introduced into pBeloBAC11, the leading (5') integration sequence (mppl1) and the trailing (3') integration sequence (mppl2) are 922 bp and 742 bp long, respectively. Any high molecular weight DNA inserted between the leading and trailing integration sequence will integrate into M. polymorpha by homologous recombination following microprojectile bombardment (biolistic method). Furthermore, the engineered iBAC (iBAC_Mp_mTurq) harbours cyan fluorescent protein-encoding gene (mTurq) and the spectinomycin resistance-encoding gene aadA, located between two flanking mppl integration sequences. The homologous sequences to the trnG-trnfM intergenic region of the M. polymorpha plastid DNA, cyan fluorescent protein-encoding gene (mTurq) and aadA conferring resistance to spectinomycin were obtained from plasmid pCSCL0*b (Boehm et al, Plant Cell Physiol, 2015).      

We used Gibson Isothermal Assembly (Gibson et al, Nat Methods, 2009, Merryman and Gibson, Metab Eng, 2012) to assemble iBAC_Mp_mTurq. Briefly, PCR amplified pBeloBAC11 backbone and the gene cassette comprising mTurq and aadA flanked by mppl integration target sites were joined to generate iBAC_Mp_mTurq. E. coli cells harbouring correctly assembled iBAC_Mp_mTurq grew on selective medium containing spectinomycin. The engineering of iBAC_Mp_mTurq was confirmed by diagnostic PCR with the flanking primers and sequencing. The engineered iBAC_Mp_mTurq can accept virtually any high molecular weight DNA for integration into the M. polymorpha plastid.

Follow Up Plans

The iBAC_Mp_mTurq-mediated system for the high molecular weight DNA integration into the M. polymorpha plastid allows rapid selection of transformants by spectinomycin resistance and the cyan fluorescent protein, mTurquoise, expression. In the follow up work we would like to adapt this system for expression of 17 different fluorescent proteins and chromoproteins. These fluorescent proteins and chromoproteins will be engineered for maximal expression in the M. polymorpha plastid.

Novel bioluminescent reporters

Finding, testing and generating optimised bioluminescent reporter vectors for use in various organisms.

The Idea

Bioluminescent reporters are a common tool in molecular biology. Luciferases from various sources (firefly, click beetle, dinoflagellate, sea pansy, copepod and bacterial) have been cloned and exploited as reporters, due to their fast time dynamics and their detectable and quantifiable outputs. Each luciferase has particular enzymatic properties and substrate requirements, leading to limitations on how assays can be performed. While all eukaryotic luciferases require the addition of an exogenous substrate (eg. luciferin, calcium ions) to produce light, bacterial bioluminescence is generated by a single lux operon which has the benefit of autonomous luminescence [Engebrecht et. al, Cell 1983].

Yet, compared to fluorescent protein reporters, little engineering has been applied to improve and optimize the lux operon on a protein level. Our idea is to sequence the genomes of various wild free-living marine bioluminescent bacterial species, in order to discover novel genes and gene variants responsible for bioluminescence and use these as novel luminescent reporters.

Seminal research has demonstrated important aspects about the structure of the lux operon in bioluminescent bacteria and has highlighted fundamental regulatory elements for light production

[Bassler et al., Mol Microb. 1993; Fuqua et al., J Bacteriol. 1994]. Recently, the 2010 Cambridge iGEM team provided an excellent demonstration of the use of synthetic biology practices to implement lux operon derived bacterial bioluminescence in E. coli. The team expressed a codon­optimized lux operon cassette driven by the arabinose inducible pBAD promoter, free of the endogenous quorum sensing regulation, and they were able to achieve high levels of tunable bioluminescence.

Although research has focused on symbiotic bioluminescent bacteria, many free­living marine bacteria exhibit bioluminescence to a highly variable degree. Whereas some strains can be seen only after several minutes in a dark room, bright strains are distinguishable by a blue glow even in low light levels. Bacterial bioluminescence has been studied since the late XIX century [Beijerinck, Arch Neerl des Sci Exact et Nat 1889], however, there have been few attempts to systematically quantify the luminosity of free­living environmental bioluminescent bacterial strains and search for the underlying genetic differences that might account for this variance of luminosity. Our plan is to systematically study a collection of such bacteria that have been gathered and isolated from marine environments around the world, in order to gain insight into their light producing mechanisms and generate reporters.

After sequencing the genomes and finding the sequences of bioluminescent genes, we plan to test them in a standardised E. coli context, in order to gain insight into the structure­function relationships. On the basis of this data, we will then generate optimised bioluminescent reporter vectors for use in various organisms.

The Team

Bernardo Pollak 
Graduate Student, Department of Plant Sciences

Anton Kan 
Graduate Student, Department of Plant Sciences

 


Project Outputs

Project Report
Summary of the project's achievements and future plans

Project Proposal
Original proposal and application
Download PDF >> 

Project Output
Follow-up project


Finding, testing and generating optimised bioluminescent reporters.

  Figure 1.  Environmental bioluminescent bacteria isolation. A, Plate after O/N incubation at RT plated with 1mL of shore seawater. B, Propagation plate with streaked colonies for isolation of bioluminescent bacteria. C, Gram strain micrograph of an isolated strain (63x). D, Multiple isolated bioluminescent bacteria streaked on a single plate for cross-comparative visualization of bioluminescence levels.

Figure 1. Environmental bioluminescent bacteria isolation. A, Plate after O/N incubation at RT plated with 1mL of shore seawater. B, Propagation plate with streaked colonies for isolation of bioluminescent bacteria. C, Gram strain micrograph of an isolated strain (63x). D, Multiple isolated bioluminescent bacteria streaked on a single plate for cross-comparative visualization of bioluminescence levels.

Outcomes, Outputs and Progress

Shore seawater was sampled from the Pacific, Atlantic, Caribbean oceans and the Mediterranean sea and 1mL of seawater was plated on LSW-70 agar plates. Colonies were then screened for luminescence and luminescent colonies were streaked out into new plates progressively until single luminescent colonies were distinguishable (Figure 1). Colonies were propagated into liquid media and stored at -80ºC in in 20% glycerol stocks. Species were identified by 16S sequencing (Figure 2).

  Figure 2.  16S phylogeny for isolated strains. A phylogenetic tree was produced using the 16S sequences of the strains by using the Geneious tree builder on a Global-alignment with free-end gaps alignment type with a 65% similarity matrix by the neighbour-joining method and the Tamura-Nei genetic distance model.

Figure 2. 16S phylogeny for isolated strains. A phylogenetic tree was produced using the 16S sequences of the strains by using the Geneious tree builder on a Global-alignment with free-end gaps alignment type with a 65% similarity matrix by the neighbour-joining method and the Tamura-Nei genetic distance model.

For each strain, a single colony was inoculated into 10mLs of LSW-70 media and cultures were  grown overnight at room temperature. Genomic DNA was extracted using the Life Technologies Genomic Purelink Genomic DNA extraction kit according to manufacturer’s instructions and DNA concentration was measured using the QuBit dsDNA HS Assay kit. Samples were diluted to 2.5ng/uL and sent for library preparation and Illumina sequencing. Library preparation was carried out using the Nextera XT DNA Library Preparation Kit and paired-end sequencing was performed on an Illumina Miseq platform using the 500 cycles v.2 program (2x250bp). Reads were screened and trimmed of adapter contamination using the bbduk command-line program from the bbtools open-source software package with the following parameters:

$bbduk.sh -Xmx1g in1=<reads_1.fq> in2=<reads_2.fq> out1=<filtered1.fq> out2=<filtered2.fq> ref=<adapters.fa> ktrim=r k=23 mink=11 hdist=1 tpe tbo

Filtered reads were then assembled using the SPAdes 3.5.0 genome assembler (Bankevich et al., 2012) with parameters for long-reads:

$python spades.py -o new_assembly -t 16  --pe1-1 <trimmed1.fq> --pe1-2 <trimmed2.fq> --careful

Assembled contigs were then blasted to a blastn database containing curated lux operons from a range of bioluminescent bacteria and hits were screened for ORFs on the Biomatters Geneious 8.1.2 software by the annotate from database function against the curated lux operon set. Sequences were then aligned by pairwise alignment using the Geneious alignment algorithm for global alignment with free-end gaps using a 65% similarity matrix.

Whole lux operons and luxAB genes were then cloned into a pSB3K3 vector by Gibson assembly, driven by a constitutive promoter J23101 (Biobricks registry). We also generated a restructured operon construct, in which we have split the luciferase genes (luxAB) from the substrate synthesis genes (luxCDEG or luxCDEFG) into separate operons on the pSB3K3 vector, driven by the R0011 and J23101 promoters respectively (Biobricks registry).

Screening of positive clones on agar plates and measuring of luminescence was performed on a refurbished gel documentation box mounted with the Photometrics Evolve 512 EMCCD camera using a Nikon 15-80mm camera lens controlled with the Micro-Manager open-source microscopy software. Imaging settings for luminescence measurements were 2000 ms of capture time with 500 EMCCD gain, and images were captured with the box door half open and completely closed to obtain both bright images of the plate and colonies positions and luminescence from lux operon/luciferase expressing colonies  (Figure 3).

  Figure 3. &nbsp;Bioluminescence in E. coli. A, bright image of a plate with E. coli transformants after cloning of a lux operon to the pSB3K3 vector. B, Luminescent positive clones expressing the cloned lux operon. C, bright image of a E. coli transformants after a round of evolution of the lux operon. D, luminescent colonies showing variable brightness due to different evolved lux operons being expressed. Scale bar (in D) &nbsp;measures 1 cm.

Figure 3. Bioluminescence in E. coli. A, bright image of a plate with E. coli transformants after cloning of a lux operon to the pSB3K3 vector. B, Luminescent positive clones expressing the cloned lux operon. C, bright image of a E. coli transformants after a round of evolution of the lux operon. D, luminescent colonies showing variable brightness due to different evolved lux operons being expressed. Scale bar (in D)  measures 1 cm.

Luminescence yield from the bacteria was quantified more accurately in a Clariostar plate reader (BMG Labtech), in which cells were grown overnight and their luminescence and absorbance measured every 10 minutes. The environmental bacteria were quantified in the plate reader in LSW-70 media at 25°C. This found a 2000-fold variation in maximal luminescence yield per cell density between the brightest and weakest strains. The operons and luciferases were also measured in E. coli on the pSB3K3 vector, grown at 30°C in M9 minimal media supplemented with 0.2% casamino acids and 0.4% glucose, with the luciferase-only constructs being supplemented with 0.1% decanal as a substrate.

The bioinformatics analysis also revealed three main types of operons from our sequenced genomes, with at least 58% sequence homology, as determined by the Clustal Omega algorithm (Sievers et al., 2011). This provides a rich pool of sequence variation whilst maintaining enough homology for a recombination based directed evolution approach to create a range of novel luminescent constructs. The evolution was performed by the DNA-shuffling method, in which DNA fragments from each lux operon were digested by DNAseI, and reassembled randomly by a primerless PCR, followed by a primer based PCR to obtain fragments to insert into the plasmid backbone. Bands showing the expected size were gel purified and cloned into the pSB3K3 vector through Gibson assembly and transformed into E. coli (Figure 3.C, 3.D). The colonies showed a high amount of variation in luminescence, and the brightest colonies were then selected. Sanger sequencing verified that the evolved constructs contain segments of several operons. We are currently performing more rounds of evolution to select for even brighter constructs. 

Conclusions

Our project has successfully isolated, sequenced and cloned the lux operons from 19 diverse marine environmental bacterial species. The bioinformatics results have shown that we have three main clusters of lux operons, with significant variation within each cluster. These variations also broadly correlate with a wide range of luminescence yield from the environmental strains. We cloned all the operons into an E. coli context, and performed quantification in a better defined DNA context, which found luminescence from all of our constructs. The diverse pool of sequences for functional lux operons from the environmental species also allows for a powerful directed evolution approach that can robustly explore large part of the sequence space to evolve optimized lux reporters. To this end, we have successfully implemented the DNA-shuffling method and in the process of evolving the decanal-specific luxAB genes, and autonomous lux reporters through the evolution of the restructured lux operons.

For the follow on outreach to the grant, we are collaborating with artists and fashion designers from the RCA to create an art installation to exhibit at the Cambridge e-Luminate festival. In this installation, we will display some of the brightest environmental bioluminescent bacteria in a setting that will allow the general public to engage with our work and create a discussion about the use of biology as technology.

We have submitted the sequencing and assembly data produced as part of of our project to the NCBI public repository and will be available following curation by the NCBI staff. Links are provided on the end of this document. Upon the completion of our evolution experiments, we will publish our quantification data and constructs publicly. 

Interactive web­-based software for genetic circuit design

This project aims to develop a rigorous design-modeling web-based tool for synthetic biology using a novel design methodology that has been recently developed in the Control Group (Engineering Department).

The Idea

Synthetic biology aspires to design and construct novel biological systems to reprogramme the cell for biomedical or biotechnological purposes. Despite many achievements in the last decade, the sheer complexity of interactions in biological systems has presented insurmountable challenges. The field currently lacks systematic principles for modelling, designing and constructing synthetic circuits that are robust, tunable and scalable. Current design approaches do not take advantage of key links between experimental and theoretical knowledge, and instead rely either on trial and error experimental redesigns of systems (with a limited number of available biological parts and components), or make experimentalists responsible for working with advanced mathematical modeling. 

This project aims to develop a rigorous design-modeling web-based tool for synthetic biology using a novel design methodology that has been recently developed in the Control Group (Engineering Department) as part of Peyman Gifani’s PhD project. This design method hides the mathematical complexity of design and modelling and makes it easily understandable for non-technical users. We intend for the method to be accessible and practical for both experimentalists and designers, to help researchers from a variety of backgrounds to tackle the nonlinear dynamics of biological systems. The SynBioFund will help our team to develop essential interactive web-based software that will guide users in designing target circuits, therefore ensuring that the method is freely accessible to the synthetic biology research community.

Using the method, biologists will be able to translate their experiential knowledge into meaningful mathematical representations that will enable them to both predict and control synthetic systems’ behaviours in silico. The software developed with SynBio funding guides the user to gradually translate a qualitative input-output relation to a quantitative representation (nonlinear feedback loops required for the target circuit) via a graphical user interface (GUI). This GUI hide the complexity of underlying dynamical systems theory and makes the required theoretical knowledge accessible to experimentalists. The design tool can be used in two ways: to design new circuits with increased complexity, robustness and tunability and to analyze and explore the capability of currently available circuits to redesign them for better functionality. 

The design framework engages nonlinear dynamical systems theory via a novel graphical method in which the user first develops a detailed qualitative description of a target circuit, specifying design criteria or goals for particular cellular input-output relations. The method then guides the user in transforming these design criteria into a specific representational form via a unique graphical palette. This graphical representation is converted into a qualitative mathematical representation by modeling the system’s trajectories in a phase space. The design framework then specifies building blocks with meaningful biological interpretations, which with the user can translate the qualitative mathematical representation into a mathematical formula representing the circuit structure and its parameters. This formula can then be interpreted as the system’s ordinary differential equation (ODE). This allows the construction of ordinary differential equation-based models, and provides a potential blueprint for implementation of a biological device based on standardised parts. The framework is intended to be used as an iterative process, whereby the qualitative and quantitative representations are gradually refined in order to best fulfil the design criteria. 

The proposed project will be a multidisciplinary collaboration between the Engineering Department (Control Group) and the Computer Lab (Rainbow Group), capitalising on the unique intersection between control theory, machine learning, artificial intelligence and synthetic biology. We will consult researchers from the Plant Science and Genetics departments to optimise the user interface design process. 

This project provides a valuable contribution to the current field. Designing a synthetic circuit from the interconnection of parts or devices can be significantly facilitated by using systematic in silico modeling and design relying on the separation of the design from the actual implementation. In this approach, various designs are first optimised and their properties are assessed using mathematical analysis and model-based computer simulations. 
This project also can be used as an educational tool for teaching nonlinear dynamical systems theory to student with biological science background. Biologists can understand better the role of nonlinear feedback loops in genetic regulatory systems by designing system using the proposed tool.

The Team

Peyman Gifani
Research Student, Information Engineering

Saeed Aghaee
Software Engineer, Telensa, Postdoctoral Research Fellow, Computer Lab

 


Project Outputs

Project Report
Summary of the project's achievements and future plans

Project Proposal
Original proposal and application
Download PDF >>


High Performance Mechanisms for Low Cost Science

Developing fluorescence and phase contrast functions in a low-cost, 3D-printed microscope and demonstrating its use in an incubator.

The idea

Optical microscopy is fundamental to biology, and relatively high performance microscopes can now be made very cheaply. Positioning the sample and focussing the objective, however, is difficult without expensive translation stages: a microscope is mostly mechanics. Many other tasks in biology require tiny, accurate motion ? achieved with expensive hardware such as mechanical micromanipulators and piezoelectric actuators. We have used inexpensive, 3D printed parts to make high performance mechanisms for low cost science, and we propose to apply this technology to problems in synthetic biology.

Our best example is a microscope small and cheap enough to be left in an incubator or fume hood for days or weeks. This will enable new science, for example by observing cells as they grow in an incubator ? experiments which are currently impossible to do on a large scale due to the time and resources required. We will improve this microscope?s biological imaging capabilities (adding fluorescence and phase contrast) and demonstrate its use in an incubator at the Light Microscopy Facility in the Cancer Research Institute. This will then allow us to study phototoxicity by monitoring cultures of cells over several days.

Plastic flexure technology could also reduce the cost of mechanical micromanipulators by three orders of magnitude, opening up a range of possibilities. When combined with open-source Arduino microcontrollers, it is even possible to automate these devices for around ?100. We will develop and test plastic micromanipulators for microinjection or electrophysiology, and assess their precision and stability. We will also investigate the use of ABS plastic as a potential replacement for PLA, as it has the potential to further improve the performance of printed mechanisms.

Finally, these low cost devices present obvious opportunities for science outreach, and this funding would enable us to create a class set of microscopes that can be taken (or lent) to schools as part of outreach activities, along with some fixed samples and lesson plans for easily-prepared specimens.

The Team


Project Outputs

Project Report

Project Proposal

Project Resources


Flexible, low cost, live­ cell imaging platform

Continued development of a flexible, low cost, live-imaging platform for long term monitoring of cell behaviour in vitro

The Idea

At present, long term in vitro imaging is costly, limited and closed. With this proposal, we seek funding to support the continued development of a flexible, low cost, live-imaging platform which was initially built to fully quantify the evolution of the cellular network structure and the establishment of the beating dynamics of human cardiomyocytes over extended periods of time, with the ultimate goal to better understand Hypertrophic Cardiomyopathy (HCM). 

Background: HCM is the most common form of Mendelian-inherited heart disease, affecting approximately 0.2% of the global population and it is the most common cause of sudden cardiac death in individuals younger than 35 years of age. The advent of somatic cell reprogramming to generate human induced pluripotent stem cells (hiPSC) has provided a new model system for studying cellular function and signalling in tissues which would have otherwise required highly invasive procedures from patients. R. Shakur and J. Kadiwala (Sanger Institute) have developed an efficient cardiomyocyte differentiation protocol for hiPSC and are now able to differentiate these cells into spontaneously beating ventricular myocytes that spatially self-organise into an intricate network. Despite this, the role of mechanical transduction and cell migration in the generation of mature electrically stable cardiomyocytes during the differentiation of such cells into beating networks remains unknown, due in most part to the lack of a flexible and affordable commercial imaging solution. 

Objectives: The current imaging device comprises an array of lenses/CCD sensors able to monitor up to six wells in a standard plate, is compact (250x200x150mm) enough to fit in an incubator, and requires no additional manual intervention across the monitoring period after setup. With respect to our primary objective, it is anticipated that the present imaging platform will allow us to record the evolution of morphological and dynamic characteristics and analyse them in the light of environmental conditions and cellular states in this model tissue. The work will help optimise the generation of such tissue culture work at high throughput, with direct clinical application for the development of future therapeutics. However, the platform itself should be of wide interest for any application that requires the long term monitoring of cell behaviour in vitro, a technique previously unavailable to the vast majority of synthetic biology labs. The project will in particular address current limitations of this setup and make it much more versatile by including different types of imaging and provide a flexible user interface.

The Team

Fergus Riche
Sensor CDT student, Department of Chemical Engineering and Biotechnology

Junead Kadiwala
Stem Cell Research Scientist, Department of Surgery

Rameen Shakur
Cardiologist, Laboratory for Regenerative Medicine, Department of Surgery, Clinical researcher, Wellcome Trust Sanger Institute

Alexandre Kabla
University Senior Lecturer, Department of Engineering, Emmanuel College


Project Outputs

Project Report
Summary of the project's achievements and future plans

Project Proposal
Original proposal and application
Download PDF >> 


Faster engineering for cyanobacteria

Developing a series of plasmids and a method to rapidly increase production of cyanobacterial mutants with multiple alterations.

The Idea

Cyanobacteria (oxygenic photosynthetic bacteria) are evolutionary ancient organisms and significant primary producers found in almost every environment on Earth. Several species, including the genetically tractable Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis), are used as model systems to study both photosynthesis and cyanobacterial metabolism and physiology. Synechocystis is also increasingly being considered for chemical and biomass production due to their highly efficient conversion of water and CO2 to biomass using solar energy and growth on non-arable land with minimal nutrients. 


We have already developed a series of tools that allow us to generate ‘unmarked’ mutants in Synechocystis, that is genetically modified strains containing no foreign DNA, unless when desired. To generate mutant strains, plasmids containing two fragments identical to regions in the cyanobacteria chromosome flanking the gene to be deleted (termed the upstream and downstream flanking regions are first constructed. Two genes are then inserted between these flanking regions. One of these encodes an antibiotic resistance protein, the second SacB. In the first stage of the process, marked mutants are generated. The plasmid construct is mixed with Synechocystis and the DNA is naturally taken up by the cell. Transformants are selected by growth on agar plates containing the appropriate antibiotic and the mutant genotype verified by PCR. To generate unmarked mutants, the marked mutant is then mixed with a second plasmid containing just the flanking regions or the flanking regions with an expression cassette between the inserts. Selection is via growth on agar plates containing sucrose. As sucrose is lethal to cells when the sacB gene product is expressed, only cells in which a second recombination event occurs, whereby the sucrose sensitivity gene, in addition to the antibiotic resistance gene are recombined out of the chromosome and onto the plasmid, will survive. In exchange the flanking regions and when applicable the DNA between them is inserted into the chromosome. 

There are a number of significant advantages to generating unmarked mutants. Because the incoming DNA has been removed in the unmarked mutant, the entire process can be repeated multiple times in the same strain. Therefore it is possible to make as many alterations to a strain as desired. In addition, the absence of foreign DNA, particularly genes encoding antibiotic resistance proteins, in the mutated strain is desirable as it avoids the possibility of ‘escape’ of organisms containing foreign genes into the environment. The one great disadvantage is the time in which it takes to generate a mutant strain, typically 5-6 weeks. The purpose of this project is to develop a series of plasmids and a method which will rapidly increase the speed at which cyanobacterial mutants with multiple alterations are constructed. 

The Team

Dr David Lea-Smith
Postdoctoral Researcher, Department of Biochemistry.


Project Outputs

Project Report
Summary of the project's achievements and future plans

Project Proposal
Original proposal and application
Download PDF >>

Project background slides

Design files and documentation.


Engineering of self­ cloning brewer’s yeast for novel terpene profiles in beer

Engineering of self­ cloning brewer’s yeast for novel terpene profiles in beer.

The Idea

Terpene flavor compounds are an important target for food improvement in a number of areas. One such area is the brewing of beer, particularly newer “craft” beers that feature large quantities of newer American varieties of hops bred for their aroma and flavor characteristics rather than their bittering properties. Beer fermentation by brewer’s yeast (Saccharomyces cerevisiae) results in changes to the profile of terpene compounds extracted from hops in a number of ways including degradation of flavor-active compounds, transformation between different classes, and hydrolysis of glycosidically bound precursors to make them flavor-active. All of these processes present targets for modification of yeast to change the profile of flavor compounds in finished beer. Genetically modifying yeastfor a food product is problematic due to EU regulations on such products but by using so-called “self-cloning” techniques to transfer sequences from one region of the yeast genome to another (i.e. cisgenesis), we can make useful modifications without creating an organism that is considered a “GMO” by current regulation. Our project will be composed of a laboratory component involving molecular biology for the creation of new yeast strains and gas chromatography combined with mass spectrometry (GC-MS) to analyze terpeneprofiles, a modeling and data analysis component in which we try to understand how terpene profiles influence flavor perception, and a legal component in which we attempt to create a legal pathway for bringing self-cloning yeast to the home brewing community.

The Team

Paul Grant
Contract Research Staff, Department of Plant Sciences

Sebastian Ahnert
Royal Society University Research Fellow, Department of Physics, King's College, Fellow and Director of Studies

Orr Yarkoni
Post Doctoral Research Fellow, Department of Pathology


Project Outputs

Project Report
Summary of the project's achievements and future plans

Project Proposal
Original proposal and application
Download PDF >>


Outcomes and progress:

Sequencing and cloning of exg1 and regulatory regions from homebrew strains

  1. We purchased homebrew yeast strains WLP001 (California Ale Yeast), WLP002 (English Ale Yeast), and WLP500 (Monastery Ale Yeast).  We performed DNA extractions from these strains and sequenced the WLP500 strain at the exg1 locus.  We found the sequence to be nearly identical to the published S. cerevisiae lab strain genome so primers designed against published sequence were likely to be valid in homebrew strains.  We PCR-amplified the regulatory regions (~1kb upstream of the start codon of exg1) from each of the homebrew strains and cloned them upstream of mCherry fluorescent protein to create reporter constructs.  We were hoping to find a diversity of regulatory sequence from which to choose variants for differing levels of Exg1 expression but sequences from WLP001 and WLP002 were identical (and identical to the published sequence) while WLP500 contained a total of 5 single nucleotide polymorphisms with respect to the other sequence within the 1kb cloned.  These regulatory regions were therefore not likely to be useful as reporter constructs as their expression levels were likely to be very similar.
  2. Development of a fluorescence assay for Exg1 activity.  Exg1 is an exo-beta-1,3-glucanase enzyme and as such should be secreted into growth media where its activity can be measured during growth of cultures.  We developed a platereader-based assay using 4-methylumbelliferyl-beta-d-glucoside (4-MUG) as a fluorescent substrate to monitor the activity of extracellular glucanase activity in growing cultures of the three homebrew yeast strains.  Consistent with the sequencing data, we did not observe measurable differences between the levels of fluorescence produced by the different strains.

Future work

We have performed the groundwork for analysing exg1 expression in homebrew strains of yeast but did not move forward into the engineering phase as planned as there was no evidence of differences in expression in the strains we analysed.  Obviously this was a very small-scale pilot and future work would require casting a wider net for source material from which to perform ‘self-cloning’ transfers of genetic material.  Another possibility is to expand into other genera such as Brettanomyces, which is reported to have higher glucanase activity.  Utilizing CRISPR-mediated homologous recombination would provide a way of inserting genetic material without having to use selection constructs. 

Outputs

Reporter constructs containing exg1 regulatory regions fused to mCherry and all sequencing data can be made available on request from Paul Grant (pg384@cam.ac.uk).

DIY Biolab

DIY Biolab plan to build and document open hardware for molecular biology. 

The Idea

Open source (OS) is reliant on a free and efficient exchange of concepts and products, and has been most successful in computing where the internet and common coding languages have enabled widespread dissemination of and participation in software projects. Open biology will require a similar set of systems in order to prosper. 

In lab hardware terms OS allows for the creation of tailored solutions using modifiable equipment rather than general purpose lab equipment. Many lab processes have common physical (eg heating/cooling for PCR, incubation, hot blocks) and software (eg peak detection, feedback control) components. 

In the most recent “Hack The Lab” workshop as part of the University of Cambridge Synthetic Biology initiative, there has been a great interest in building a do-it-yourself (DIY) biolab to make genetic research more accessible. Public biolabs already exists in a number of other cities. Our proposal is to investigate the currently available OS designs for basic laboratory equipment (PCR, pipette, centrifuge, microscope, electrophoresis system, fume hood) in order to build the first open labware DIY biolab. We plan to build and assess the function of the systems to generate a local resource bank of example equipment, subassemblies, suppliers, expertise, and documented software and hardware component libraries that will be useful to university laboratories as well as the open biology movement.

The Team

Tobias Wenzel
PhD candidate - Winton scholar for physics of sustainability, Department of Physics.

Isabella Gariboldi
Gates Scholar, Cambridge Centre for Medical Materials, Department of Materials Science and Metallurgy

Ian McFarlane, Biologist, CamGenomics Ltd

Aleksandar Tomic, Calibration specialist, ThermoFisher


Project Outputs

Project Report
Summary of the project's achievements and future plans

Project Proposal
Original proposal and application
Download PDF >>


Plant ProChip

Development of a microfluidic device for high-throughput analysis of genetic circuits in plant protoplasts.

This project aims to develop a high-throughput screen for the analysis of promoter sequences driving expression of a reporter gene in plant protoplasts. We envisage this device to be applicable to a range of plant species and cell types, and by coupling it to libraries of regulatory elements identified by DNase-Seq, it will rapidly increase the rate of identifying promoters for biotechnological applications.

The Idea

DNA regulatory elements are fundamental parts for genetic circuit design. The generation and characterization of promoter libraries can greatly facilitate fine tuning of gene expression within a circuit. We have used DNase-Seq (Meyer and Liu 2014) to identify candidate regulatory elements (>50,000) controlling cell-preferential gene expression within maize leaves (Burgess and Reyna-Llorens; unpublished data). Current validation techniques involve fusing sequences to a reporter and analysing expression in planta, which requires testing each element in an individual plant, either through transient biolistic transformation or the generation of stable transgenics (Brown et al. 2011). Applying these techniques to whole regulatory element libraries is not feasible at a laboratory scale.

This project aims to develop a high-throughput screen for the analysis of promoter sequences driving expression of a reporter gene in plant protoplasts. Protoplasts will be transformed by plasmid reporter constructs consisting of a regulatory region fused to a minimal promoter and a fluorescent protein. The expression level of fluorescent reporter within each protoplast will be questioned by laser in situ excitation in the sorting microfluidic device which will be also used to separate out individual protoplasts. Protoplasts will be sorted into two pools according to a user-specified threshold of fluorescence intensity (Abalde-Cela et al. 2015). In analysing a library of elements, this procedure will be performed iteratively at ever decreasing thresholds of fluorescence intensity to sort regulatory elements by their promoter activity. To determine which regulatory regions are present in each fraction, DNA will be extracted and sequenced using Illumina technology for each pool. We envisage this device to be applicable to a range of plant species and cell types, and by coupling it to libraries of regulatory elements identified by DNase-Seq, it will rapidly increase the rate of identifying promoters for biotechnological applications.

The Team

Dr. Steven Burgess,
Contract Research Staff, Department of Plant Sciences

Mr. Ivan Reyna-Llorens
Graduate Student, Department of Plant Sciences

Mr. Christian R. Boehm
PhD Candidate in Plant Synthetic Biology, Department of Plant Sciences

Dr. Sara Abalde-Cela
Postdoctoral Research Associate (PDRA) at Microdroplets Group, Department of Chemistry

Dr. Paul Bennett
Research Associate, Laboratory for Scientific Computing


Project Outputs

Project Report
Summary of the project's achievements and future plans

Project Proposal
Original proposal and application


Plant ProChip: Development of a microfluidic device for high-throughput analysis of genetic circuits in plant protoplasts.

Abstract

DNA regulatory elements are fundamental parts for genetic circuit design. The generation and characterization of promoter libraries can greatly facilitate fine tuning of gene expression within a circuit. Current validation techniques involve fusing sequences to a reporter and analysing expression in planta, which requires testing each element in an individual plant, either through transient biolistic transformation or the generation of stable transgenics (Brown et al. 2011). Applying these techniques to a considerable number of genetic circuits can be very laborious or not even feasible at a laboratory scale. The aim of this project was to use microfluidics to develop an inexpensive and high-throughput screening method for the analysis of genetic circuits in plant protoplasts. 

Progress

  • Generated a fluorescent reporter construct using Golden Gate Cloning.
  • Fabricated a PDMS microfluidic device for encapsulation of plant protoplasts.
  • Fabricated a PDMS microfluidic device for sorting of plant protoplasts.
  • Established of a procedure for protoplast isolation from maize, arabidopsis and Nicotiana benthamiana.
  • Established of a procedure for encapsulation of protoplasts.
  • Established a procedure for PEG transformation of protoplasts followed by encapsulation.
  • Established that protoplasts are stable in a microfluidic device.
  • Established of a laser setup for analysis of fluorescence from plant protoplasts.
  • Established a procedure for sorting protoplasts based on fluorescence intensity. 

Outputs

  • A blog which detailed project progress.
  • Presentation at OpenPlant Forum, JIC, 2016
  • Presentation at Cafe Synthetique, Cambridge, 2016
  • Online protocols for protoplast isolation and encapsulation on protocols.io

Plans for follow up

  • Develop an on-chip method of protoplast transformation to reduce DNA input requirements.
  • Write up and submission of methods article to an open access journal.

     

CELLUWIN: 3D printing for cellulose

For a feasibility study on creating 3D structures, using raw cellulose as a starting material, as a proof of principle for the use of cellulose as a modern building material.

The Idea

Cellulose is the world’s most abundant polymer; it forms the basis for paper, cotton and wood. The vast majority of cellulose is found in the biomass of plants and algae; making cellulose an environmentally friendly, renewable, biopolymer. The mechanical strength of cellulose is due to the self-assembly of individual polymers into tight fibres. Unfortunately bundling of the cellulose polymers renders the cellulose inert and insoluble – making it problematic for use.

Cellulose can be functionalised via dissolution of cellulose fibres into individual polymers via chemical treatment. This allows the cellulose polymers to be manipulated and re-formed into tailor made cellulose composites. This has the additional benefits of decreasing the use of oil derived plastics, but also would allow the use of cellulose from food waste (such as the juicing industry).

Initially this was proposed as a renewable source of material for 3D printing machines, as a replacement / substitute for plastic materials. Preliminary discussion about the use of cellulose in 3D printing quickly revealed an extended audience that would like to further develop the use of cellulose as a modern material.


The Team

Thomas Torode (Postdoctoral researcher, SLCU) thomas.torode@slcu.cam.ac.uk

Marco Aita (Postdoctoral researcher, SLCU) marco.aita@slcu.cam.ac.uk

Ward Hills (CEO, OpenIOLabs) Ward.Hills@OpenIOLabs.com


Project Outputs

Project Report
Summary of the project's achievements and future plans

Project Proposal
Original proposal and application

Project slides
Design files and documentation for Celluwin: 3d printing for cellulose


Project report

Abstract

This project has combined biochemical knowledge about cellulose (the world’s most abundant biopolymer) and advanced 3D printing techniques. The overarching aim is to advance the use of photosynthetically derived polymers in 3D printing as a replacement (where suitable) to widespread use of petroleum derived materials. Cellulose is an inert polymer which during this project has been: activatedinto a usable form, formulated into a working material suitable for storage and, in-activated (cured) into a stable material.

Outcomes, Outputs, Progress

Initial attempts at the activation and formulation of cellulose used citrus (lemons and orange) fruits as a starting material. This was aimed at using a mimic of a current waste product from these fruits, which have been sequentially juiced and extracted for pectins, leaving a cellulose-rich pulp as a waste product. This was a terrible idea as it made the initial activation and formulation a nightmare. As this project aims to facilitate the interface between cellulose and 3D printing, a lack of a working product limited the progression of the entire project. It was decided to use a pure cellulose source as a starting material, and re-visit the idea of waste-cellulose utilization at a later stage.

Using the bacteria Gluconacetobacter xylinus (which naturally forms cellulose biofilms) as a source of pure cellulose, we conducted a range of activation and formulation experiments to develop a protocol for the production of our initial product, affectionately termed “cellu-poop” (it looks fairly disgusting). With prior permission from Prof Jim Haseloff, we have omitted exact details due to the future outlook of the project. However, in brief: cellulose was activated in a range of ionic liquids, mixed in various ratios of activated/raw cellulose, at a range of concentrations, in various buffers.

The most promising outcome from these experiments was the complete solubilisation of cellulose in ionic liquids, which can then be precipitated via contact with water to deposit a cellulose structure. Sadly this approach is vastly un-suited for our laboratory and would require more extensive chemical engineering-style equipment and expertise – but would be ideal to explore in the future.

The current working cellulose 3D material “cellu-poop” is stable at 4oC and can be cured via removal of water to leave a pure-cellulose structure. Thus far we have achieved this via freeze-drying – but with the release of the remainder of the grant we are now working to modify a 3D printer to deposit and cure “cellu-poop” using peristaltic pumps and focused drying techniques (hot air blowers, IR lasers). This marks a large step forward for this project, as now we have the combined might of Marco doing his mechanical magic on the 3D printer and Tom creating cellulose cocktails.

  Figure 1) &nbsp;3D structures made of “cellu-poop”. The structure are very light, and have shown no collapse / faults so far (March 2017).

Figure 1) 3D structures made of “cellu-poop”. The structure are very light, and have shown no collapse / faults so far (March 2017).

The completed state of the project (due to lack of available time / equipment) is a great start, and has generated preliminary findings to support a larger research grant to continue this project. In short, a systematic approach to formulation, combined with access to a wider range of equipment would be ideal. An exciting side route is the possibility to cure the material via enzymes, allowing for a more biosynthetic approach.

Expenditure

The grant has been spent predominantly on chemicals for the activation and formulation of the cellulose, having tried for 5 months to get a working protocol to create “cellu-poop”. The extra £2k has been spent on 3D-printing equipment and additional chemicals. The grant also covered 6 months membership at MakeSpace, for Marco to build custom components, and light refreshments for networking.

CamOptimus

Self-contained user-friendly multi-parameter optimisation platform for non-specialist experimental biologists

The Idea

Biological problems are usually complex due to their multi-parametric nature and to the fact that these parameters are often interdependent. A commonly employed approach in attacking such problems relies on the use of background knowledge, or informed guesswork, to prioritise these parameters. For novel systems there may be insufficient background knowledge to enable successful prioritisation. Moreover, identifying and testing the effect of individual parameters is often an ineffective strategy because it ignores the interactive effects of mutually dependent parameters.

CamOptimus developed a hybrid approach to solve multi-parametric experimental design problems and to develop a simple-to-use and freely available graphical user interface (GUI) to empower a wider audience of experimental biologists to employ GA in solving their optimisation problems. 


The Team

Dr Duygu Dikicioglu 
Post Doctoral Research Associate, Department of Biochemistry

Dr Ayca Cankorur-Cetinkaya
Post-doctoral Research Associate, Department of Biochemistry

Dr João ML Dias
Bioinformatician, Research Associate, Department of Haematology and Sanger Institute

Dr John Kendall
Principal scientist, PhD, ZuvaSyntha Ltd


Project Outputs

Project Report
Summary of the project's achievements and future plans

Project Proposal
Original proposal and application

Project application slides
Design files and documentation 


CamOptimus: Self-contained user-friendly multi-parameter optimisation platform for non-specialist experimental biologists 

Abstract

Biological problems are usually complex due to their multi-parametric nature and to the fact that these parameters are often interdependent. However, the available methodologies for the optimisation of biological processes are usually impractical, since they generate an explosion in the number of experiments to be performed, and also difficult to perform, since they lack easy‐to‐use software. The latter restricts their practice to specialist users who are experienced in handling complex algorithms. To address both these problems, we have constructed a simple‐to‐use and freely available graphical user interface to empower a broad range of experimental biologists to employ evolutionary algorithms to optimise their experimental designs. The platform first adopts a Genetic Algorithm to scan a wide range of possibilities, thus ensuring that the search leads to the discovery of the subspace where the optimal combination of parameters resides. Symbolic Regression is then employed to construct a model to evaluate the sensitivity of the experiment to each parameter under investigation. We believe this tool to be an attractive alternative to commercially available software for both academic users and for experimental users in SMEs whose limited funds do not allow them to employ dedicated statisticians to carry out such tasks or to purchase commercial software.

Outcomes

The first task of the project involved demonstrating the applicability of the proposed approach and the second involved developing the software tool.

The applicability of our hybrid approach was demonstrated by optimising cultivation conditions for recombinant protein production by an inducible strain of the yeast Pichia pastoris (Komagataella phaffii). For the set-up under investigation, where the expression of a model protein (Human Lysozyme) was investigated under an inducible alcohol oxidase (AOX) promoter in a microbial host, the objectives were determined as: (1) the maximisation of cell growth until the induction of protein production, in order to facilitate the optimal use of resources to make as much of the cell population available for protein production as possible in the post-induction stage; (2) the maximisation of the overall protein activity at the end of the induction period; (3) the minimisation of cell growth during the period of induction in order to allow the resources to be preferably used by protein production rather than growth, and (4) the maximisation of productivity (protein activity/cell) in order to achieve the environmental configuration that allowed the cells to operate in the most efficient way. These objectives were assigned equal weights as there was evidence in the literature to suggest the dominance of one or more of these objectives over the others within this context.

The next stage in the process was the selection of the parameters that were considered to contribute substantially to these objectives. The preliminary experiments detailed above highlighted the importance of maintaining pH at a fixed value as an environmental effector (in the range of 3-7 using suitable citrate/phosphate buffer) and several medium components including ammonium and glycerol; the growth-related macronutrients, methanol and sorbitol; the induction-related macronutrients, as well as magnesium, calcium, potassium, and iron, whose concentration values were reported to vary considerably in the literature. We investigated this 9-parameter system with each parameter defined at 32 different levels. The cultivation conditions were selected such that the tests could be conducted in defined medium using loose-cap tubes to prevent anaerobiosis; the experiments were run in triplicate.

A genetic algorithm, which was based on the evolutionary ideas of natural selection and genetics, was employed as the adaptive heuristic search algorithm to conduct the optimization study. The algorithm used a population of possible solutions to a problem to evaluate the feasible solution space and, over generations, successive populations would be fitter and therefore more adapted to their environment, as dictated by their objective function. The initial step of the algorithm was concerned with the generation of an initial population of solutions, which provided the randomly generated values for the environmental parameters within a specified range. The experiments were conducted for each of these populations in triplicate and the fitness of each population as defined by the objective function was determined. The fitness values were evaluated and the best-performing individuals were selected. The population values for the best-performing individuals were ‘mated’, random ‘mutations’ were introduced and a new generation of populations was created. The fitness was evaluated and the procedure was repeated until a satisfactory convergence was observed in the objectives, which were represented by the convergence of the productivity and the protein activity values.  

A convergence in the performance metrics was observed after conducting three generations of experiments for 150 individuals and therefore the iterative procedure was halted due to this convergence. The optimised medium composition was further investigated for fine-tuning and the elimination of problems regarding precipitation via population profiling. The performance of this optimised set of environmental conditions was then verified by benchmarking its performance against other conditions reported in the literature. The new set of conditions was shown to yield a more than 80% improvement in Human Lysozyme activity and an over 55% improvement in productivity on average over the generations. The new recipe was shown to perform better to boost productivity and r-protein activity than any other medium reported for K. phaffii.

Having determined the optimal solution in that sub-space, we constructed a regression-based model to describe the interdependencies among the input parameters, which affect the output parameters and to investigate the sensitivity of the objectives to the input parameters. Multiple linear regression (MLR) models, commonly employed in Design of Experiments failed to describe our search space acceptably since a large number of factors and their interactions contributed to the construction of this search space under investigation. So, as the number of factors under investigation increased, more complex models were required to represent the possible interactions among those factors and to explain the variability in the output. Although non-linear models could provide a useful alternative in such instances, our lack of knowledge of the model structure opened up an infeasibly large number of possibilities to be tested. This led us to explore Symbolic Regression (SR) as an alternative approach to handle high-dimensional modelling problems with an unknown model structure. SR proved to be a powerful tool since it did not require any a priori knowledge of the model structure or the provision of such information to the algorithm ab initio. We then constructed models to describe our four different objectives: final cell density during the growth-promoting phase, further growth during the protein production phase, enzyme activity, and specific productivity. These models allowed us to explain the variation in each individual objective by the 9 factors under investigation. The residual sum of squares (R2) was selected as the metric to represent the proportion of variance in the models. The performance of these models in representing the variance in the dataset was compared to that of the MLR models. SR outperformed MLR in explaining the variance of the dependent variables, i.e. the individual objectives. The SR models could explains the variation in the cell growth until the induction of protein production, cell growth during the period of induction, overall protein activity and the productivity by 64, 75, 80 and 88 %, whereas for MLR models, these values were 12, 33, 60 and 61 %, respectively.

Having constructed the models, we conducted sensitivity analyses employing those batches of models to determine how sensitive each individual objective was to a small variation in each factor. We shifted the value of each factor from its determined optimum by 10% and investigated whether a similar response of 10% or higher was observed in individual objectives in their respective model pools, denoting such factors as major contributors. The sensitivity analysis revealed the distinction between what we call “operation-related” factors and “cell culture-related” factors in the experimental design, although the process of model construction was blind to the nature of the factors under investigation. We identified all dependent variables in the objective function to be highly sensitive to variations in the pH of the working culture. In this way, it was found to be imperative to have strict control over the pH of the cultivation during HuLy production by K. phaffii under the control of the alcohol oxidase promoter.

This task provided us with an optimised set of cultivation conditions and a novel choice of model-building strategy along with a descriptive model for the selected case study.  We used the results of this first task in developing the Graphical User Interface (GUI) for the tool, which constitutes the second proposed task. CamOptimus is a tool for applying Genetic Algorithm (GA) to solve multi-parametric optimisation problems and Symbolic Regression (SR) to obtain models using the data generated during optimisation procedure to investigate the effect of individual parameters on the system of interest. The source code for the compiled software and the Graphical User Interface (GUI) of the application are available under free licensing (GNU General Public License) and they are submitted to the Open Data Repository at the University of Cambridge (www…) along with the User Manual of the GUI. The permanent link to the software has also been shared on the Cambridge Systems Biology Website (www.) to increase the visibility of the tool. The MATLAB Runtime environment (version 9.0.1) is needed to run the executable versions (the compiled version and the GUI) on computers with Windows OS or Mac OSX. The MATLAB Runtime is available under free licensing at http://uk.mathworks.com/products/compiler/mcr/.

The user first selects the action to be taken in the main page of the GUI; using Genetic Algorithm to solve a multi-parametric optimisation problem, or conducting Symbolic Regression analysis to investigate a given solution sub-space. Each action is comprised of successive steps and the decisions made in each step feeds into the next one, guiding the user through the course of action to be taken. The Genetic Algorithm interactive interface allows actions to be taken in a given order so as to guide the user through the steps of the procedure. It is comprised of two blocks of information; one for setting up the experiment and another for evaluating the results. There is a set of information required to be determined and fixed constant throughout the application and therefore, this should be set up as the initial step. The information the experimenter is asked to provide are regarding the objective(s) of the experiment and the factors of interest, which are thought to have an impact on the objective(s). There are several GA-associated parameters, which the user might wish to alter, and the interface allows these changes under the “Advanced settings”.

The first decision to be made in an optimisation experiment is to define the measurable objective(s) that need to be optimised. If there is more than one objective, the first question that the user needs to address is whether these objectives are equally important, and hence whether or not they can be assigned equal weights. The user also has to provide a name for each objective and select whether that target objective should be maximised or minimised. Combinations of objectives where some are minimised whilst others are maximised are allowed. As soon as any objective function is defined, modifications are then allowed in the next section, which asks the user to identify all factors that need to be optimised to achieve the described combined objective function.

The experiments to be conducted will be generated as a report of this initial setup. The user may wish to include additional parameters, which will not be optimised in the procedure but will be kept at a fixed value, just to have them appear on the generated reports for convenience. Such an example would be that the user may wish to optimise only a given fraction of the medium components whereas, in the lab, all components need to be included in the actual experiments. Including these unchanged parameters in the factors list will allow a full medium recipe to be generated automatically to facilitate lab work. The interface will allow additional factors to be included. For each individual factor, the user has to select whether this new factor will be a factor to be optimised or will just be monitored. For factors that will be optimised, a range in which values will be allowed to vary should be defined. For those factors that will only be monitored, a single value and a corresponding unit should be introduced.

Genetic Algorithm is a search heuristic that can be successfully applied to many problems. Therefore the parameters intrinsic to its mode of operation may need to be adjusted, might need to be adjusted, perhaps based on similar type of problems to which it has previously been successfully applied. Therefore, the user is allowed to make a selection either to accept the default parameters or to provide values for the mutation rate, the crossover rate, the number of bits and the selection probability. At this point, the user is ready to generate the first set of experiments in the optimisation procedure. Once these steps outlined above are designed and the experimental procedure has been initiated, they cannot be changed or modified in any way. The interface also ensures this. The file that the generated experiments are saved is designed in such a way that the user can take the printout to the lab to carry out the experiments. The measurable outputs for the objectives under investigation (initially generated as zeroes, later to be modified by the user) will also be recorded in the same worksheet. In case the experiments are carried out in replicates, the mean or the median values (as seen fit by the user) should be recorded in the spreadsheet. Once the experiments are carried out and the objective outcomes are recorded in the worksheet, the user is then ready to use the tool for evaluating their results and generating the next set of experiments if they see necessary.

The initial setup and the experiments files are uploaded for accessing the software for the upcoming generations The individual non-normalised scores for each objective, which can be used as an indicator of the improvement of the system over time, as well as an absolute frequency plotdisplaying (i) the distribution of values employed by a given factor over the generations, and (ii) the distribution of values employed in the best performing fraction of the most recent generation are plotted by the software Using these plots, the user then makes a decision on whether if they would like to proceed with a next generation of experiments, based on how satisfactory the convergence of the factors and the scores were. Once the optimisation procedure is terminated as a result of convergence, the user is then ready to take their results to the next stage to investigate their solution space by selecting Symbolic Regression in the main page. As in Genetic Algorithm section, the interactive interface allows actions to be taken in a given order so as to guide the user through the steps of the procedure.

The regression analysis is designed to follow the optimisation protocol employed in Genetic Algorithm section, and therefore the structure is designed to make use of the data generated in the earlier stages. However, the tool will accept any other spreadsheet, which was prepared in a similar format to that generated by the Genetic Algorithm Section of the Tool. The first column of the spreadsheet is recognised as the identifier column for the experiments, and by selecting the number of columns dedicated to the factors under investigation, the user classifies the columns into the factors and the objectives, which then appear in their cognate boxes. The user then selects which parameter(s) would be employed in describing which objective by the constructed model.

Once the model parameters and the objective to be modelled are determined, the user then has to decide on the purpose of the model to be constructed. The tool is suitable for constructing both descriptive models and predictive models. The exploratory model option employs as much of the experimental data made available as possible to describe the solution space and through evolutionary approaches, attempts to reach an optimal model structure as well as regression coefficients in order to best fit a function to the available data. The goodness-of-fit of the model is described by how well the model fits the experimental data. The predictive model option retains a pre-defined percentage of the complete dataset as the validation dataset in a completely randomised manner and employs the remaining fraction (the training dataset) for constructing the model with the best fit available. This time, the goodness-of-fit of the model is described by how well the model constructed using the training dataset fits the validation dataset and this is an indicator of its predictive success, by definition.

Following the selection of the type of model to be constructed, the advanced settings for the symbolic regression can be adjusted by prompting the user to accept or change the default settings provided in the software. Although some suggested default values for the population size, number of generations, maximum number of genes and the maximum depth are provided in the tool itself, we urge the user to change these default settings in order to construct models with improved goodness-of-fit. Increasing population size and the number of generations improves the model fitness, whereas increasing the maximum number of genes and the maximum depth increases the complexity of the constructed model, and may also contribute to the fitness of the model.

It is worthwhile to note that the parameter values provided here should be considered as independent of those discussed in the Genetic Algorithm Section. The concepts and the parameters employed in describing these concepts are similar for genetic algorithms and other approaches including but not limited to symbolic regression, which employs genetic programming, and therefore should be evaluated within their own context. 

Once the settings are determined, the tool is then ready to construct the model. Depending on the parameters selected, the program may take a while to run and once the model is constructed, the user has the best-fitting model for running further sensitivity analysis as well as the model prediction and actual data plots for each data point and the scatter plots – prediction vs actual data for the training and the test sets along with the room mean squared (RMS) error and the regression coefficient (R2) values for each set. The significance of each factor (gene) is also provided.

Follow Up Plans

We would like to claim the additional £1000 for two main purposes; (a) full training and Q&A session for the use of the CamOptimus GUI aimed for all potential users – as proposed initially, and (b) completing the study based on the suggestions and the feedback we have received during the dissemination events. One major concern raised regarding the methodology was that only one test case was conducted to validate the approach. Due to the time limitations of the project, a comprehensive extension of the experimental validation could not be facilitated but an additional 6-months’ time would be sufficient to extend the work to a different experimental setup. Another concern was regarding the benchmarking. Since the area of application is industrial biotechnology, a scale-up study and performance comparison was suggested by the audience. Therefore, we would like to focus on these 3 major aspects during the follow-up. The training and Q&A sessions will be held for researchers working in SMEs and other industrial collaborators, who are interested in the tool and for academic researchers separately. Scale-up experiments for further benchmarking will be carried out within the first month of the follow-on period. The remaining 5 months will be allocated to conducting an optimisation study, again with biotechnological significance. The system under investigation will be a continuous cultivation system, which has huge challenges with respect to its optimisation, both in academia and in industry, which employ both mammalian cultivations and microbial fermentations. The first 3 months of the allocated period will be spent on optimisation of growth environment and the remaining 2 months will be spent on employing the optimised setup on real systems and adapting the scalable characteristics from batch cultures mimicking continuous systems (during exponential phase of growth) to real continuous setups running in fully controlled mode. This will also allow the investigation of the flexibility of the approach by extending its limits. 

Progress

The experimental task of the project was completed within the first 3 months and the optimised set of conditions was benchmarked against previously reported conditions during the 4th month as initially proposed. During the first three months of the project, we carried initial discussions with Dr John Kendall from ZuvaSyntha Ltd, to understand industrial perspective on the Design of Experiments and the typical use of commercially available DoE software (such as Jamp®) in solving multi-parametric optimisation problems. With these concerns and suggestions in mind, the first draft of the GUI for the tool, which consisted only of the Genetic Algorithm Section was made available. We had a chance to present the idea to the academic audience as a poster in a large conference (Annual Conference of the Microbiology Society 2016) and as an invited talk in a small focused meeting on Synthetic Biology (3rd Meeting of Applied Synthetic Biology in Europe). Both meetings allowed us a chance to receive very useful feedback from academia as initially proposed. The Annual Conference of the Microbiology Society 2016 allowed us to meet members of the Kay lab (MRC Laboratory of Molecular Biology, UK), who agreed to become beta testers for the tool, which helped substantially during the development process. The networking during the 3rdMeeting of Applied Synthetic Biology in Europe resulted in an invitation to present CamOptimus in the 17th European Congress of Biotechnology (ECB) 2016 through a short talk. Since this was a meeting with more than 1000 registrants, it allowed us to reach a wide audience with broad areas of interest. The remaining three months of the project was focused on exploring different modelling schemes for the exploration of the solution space and the implementation of the most suitable approach into the GUI. The tool was finalised and a manuscript has been drafted for the results. A pre-submission enquiry has been made for PLoS Biology and the Editorial Board invited us for the submission of a full manuscript for a Methodology Article.

The progress of the project deviated from the initial Proposal based on the feedback we have received during the course of the Project. We had to reshuffle the way we allocated the budget accordingly. In the first task, where we verified and demonstrated the applicability of the methodology, the convergence towards the optimised set of conditions was achieved much earlier than initially expected, resulting in allocation of less resources (time and expenditure) towards the experimental work. However, we were invited to present our final work in ECB2016, before our proposed dissemination plan. The feedback we have received during the dissemination helped us to shape the follow-on work in a different way, as will shortly be discussed.

Outputs

  1. Poster presentation: S16/P2 - CamOptimus: A self-contained, user friendly multi-parameter optimisation platform for non-specialist experimental biologists, Ayca Cankorur-Cetinkaya, Duygu Dikicioglu, Joao Dias, Jana Kludas, Juho Rousu, Stephen G. Oliver – Annual Conference of the Microbiology Society, 21–24 March 2016 (abstract book and poster attached – not available online) (https://www.synbio.cam.ac.uk/synbiofund/CamOptimus_project_folder/dissemination-in-the-annual-conference-of-the-microbiology-society-2016) – SynBio Fund acknowledged
  2. Short talk: CamOptimus: A self‐contained, user‐friendly multi‐parameter optimisation platform for non‐specialist experimental biologists, Duygu Dikicioglu – 3rd Meeting of Applied Synthetic Biology in Europe, 22-24 February 2016 (http://www.efb-central.org/Synthetic/Documents/prg.pdf) – SynBio Fund acknowledged
  3. Short talk: A roadmap to improve yield of the recombinant proteins production: a case study employing Komagataella phaffii as the host organism, Ayca Cankorur-Cetinkaya – 17th European Congress on Biotechnology, 3-6 July 2016 (http://ecb2016.com/wp-content/uploads/2016/07/FINAL-ECB-Programme-plus-Final-ECB-Programme-270616.pdf) – SynBio Fund acknowledged
  4. Manuscript draft: Full title - A Tool for Exploiting Complex Adaptive Evolution to Optimise Protocols for Biological Experiments (invited for full submission to PLoS Biology), Ayca Cankorur-Cetinkaya, Joao Dias, Jana Kludas, Juho Rousu, Stephen G. Oliver, Duygu Dikicioglu (yet confidential) – SynBio Fund acknowledged
  5. CamOptimus source codes, GUI for MS OS and MAC OSX, User Manual: http://dx.doi.org/10.17863/CAM.700
  6. Demo for the CamOptimus GUI on action: (https://www.synbio.cam.ac.uk/synbiofund/CamOptimus_project_folder/camoptimus-demo/view) (attached)

Expenditure

1- Replacement of laboratory consumables from the SGO lab for the first task – consumable plasticware (aerated loose cap tubes, micropipette tips, microcentrifuge tubes (Eppendorf), Stericup Filter Units (Millipore), PVC spectrophotometer cuvettes, 96-well plates (Nunc)):£229.02 (– from this Fund)

2- 1 x Portable computer
£1258.51

3- 2 x Attendance to 3rd Meeting of Applied Synthetic Biology in Europe
£1595.72

4- 1 x Attendance to the 17th European Congress on Biotechnology
£879.15

5- Travel and meeting expenses for industrial partner feedback on the development of the GUI of the software (meetings held at ZuvaSyntha Ltd., Welwyn Garden City)
£37.60

Bio-Hackathon

The Cambridge University Technology and Enterprise Club (CUTEC) has been running events to support young technologists for the last 13 years. This year our committee is passionate about supporting the transition from idea to prototype in biology and with this in mind we ran a Bio-Hackathon in collaboration with the London Biohackspace and with Bento Bio Works.

The Idea

50 competitors from across the world came together in Cambridge to try to solve some of the biggest challenges facing biology. Teams came with expertise as diverse as Computer Science, Genetics, Medicine, Art/Design and Performing Arts. This meant that the event was a wonderful learning experience and focused on dynamic solutions. Our overall winners focussed on building a web interface to enable to design and fabrication of novel DNA parts using a "cloud-laboratory" Transcriptic.


The Team

Freddi Scheib 
Freddi scheib is a PhD student in the Department of Archaeology and Anthropology and is an expert in the extraction, sequencing, and analysis of DNA from ancient human remains.

Peter Choy 
Peter is an undergraduate student of St Johns College, Cambridge.

Dr Thomas Meany
Dr Thomas Meany is Thomas is an Interdisciplinary Fellow, jointly hosted by CEB and the Plant Sciences department, developing microfluidic techniques for genetic engineering. His previous role was as a research scientist for the Toshiba Cambridge Research Labs as a Marie Curie Fellow developing quantum technologies.  

External Collaborators


Project Outputs

Project Report
Summary of the project's achievements and future plans

Project Proposal
Original proposal and application

Project Output
BioHackathon Winners

Biosynthage website
BioSynthage Website


The BioHackathon

50 competitors from across the world came together in Cambridge to try to solve some of the biggest challenges facing biology. Teams came with expertise as diverse as Computer Science, Genetics, Medicine, Art/Design and Performing Arts. This meant that the event was a wonderful learning experience and focused on dynamic solutions. Our overall winners focussed on building a web interface to enable to design and fabrication of novel DNA parts using a "cloud-laboratory" Transcriptic. (Tom Meany posted his original write-up here. )

 An agar 3D-printed microfluidic chip developed by Team Liquid during the Biohackathon (Credit: CUTEC)

An agar 3D-printed microfluidic chip developed by Team Liquid during the Biohackathon (Credit: CUTEC)

72 sleepless caffeinated hours saw 50 competitors from across the world try to solve some of the biggest challenges facing biology. They used software, hardware and, for the first time during any hackathon, used bioware to tackle problems. The goal was to make biology automated and reproducible with the long term application of creating disease resistant crops, new antibiotics, new products and novel medicines.

Teams came from all over the world with expertise as diverse as Computer Science, Genetics, Medicine, Art/Design and Performing Arts. This meant that the event was a wonderful learning experience and focused on dynamic solutions. Every member of a team thought differently and that breadth of experience led to some fantastic solutions (learn more about our teams at 1min 30s in video below). One team of artists thought about how to integrate design-thinking with biology, another built an ultra-low-cost prototype microfluidic system, one group thought about a way to compare low cost DNA synthesis. There was also a number of med-tech ideas, including a sensing toothbrush and  a household plant that can filter air. The team who came up with an idea of how to efficiently data mine information on drug-drug interactions won the award for the Best Scale Up Potential provided by Deep Science Ventures . Read more in this recent Labiotech article by Dani Bancroft

 Left: Bethan Wolfenden, co-founder of the Bento Bio, showing off the Bento Lab. Right: Stephen O’Connell, Assistant Director of IndieBio EU, with Trevor Nicks, the CSO of algae drink startup Spira. P.S. Nice tee, Stephen… (Credit: CUTEC)

Left: Bethan Wolfenden, co-founder of the Bento Bio, showing off the Bento Lab. Right: Stephen O’Connell, Assistant Director of IndieBio EU, with Trevor Nicks, the CSO of algae drink startup Spira. P.S. Nice tee, Stephen… (Credit: CUTEC)

Our overall winners focussed on building a web interface to enable to design and fabrication of novel DNA parts using a "cloud-laboratory" Transcriptic (listen to winner Evgeny Saveliev on BBC radio - 1hr 41 mins here!). This team (Evgeny Saveliev,Shannon DoyleHana JanebdarPablo LubrothClaus WeilandKelvin Zhang) will now enter the Cambridge Judge Business School Accelerate Cambridge Programme. They hope that their invention can be taken forward as a product to help the next generation of bio-entrepreneurs. 

Check out Cambridge TV's coverage of our event starting at 1min 30s. 

We had some intense mentorship from passionate, talented and engaged experts from a range of disciplines. Chris Grant from London based startup Synthace taught us how to code biology using Antha, a new programming language for automating biology. Helene Steiner from Microsoft Research spoke about how design thinking should be a part of every single step on the product or prototyping cycle. Cambridge Consultants taught teams how to prototype in the real world with hands on advice from their amazing team of world class engineers and product designers. Beth Wolfenden from startup Bento Bio taught teams how to use portable technology to study biology in resource limited locations. Eduardo Gianni of the London Biohackspace taught teams how to problem solve on a shoe string! John Cumbers inspired teams into action through his community driven startup SynbiobetaNassia Inglessis and Sydney Schaefer joined us to talk about interactive inventions and how physics meets biology and then meets design. Founders Elliot RothTrevor Nicks and Seki Anderson taught teams what it means to be a bio-entrepreneur from first hand experience at their startup Spira which is now based in the Indiebio accelerator. 

 OpenPCR and 3D printers like the ‘Ultimaker’ are enabling a generation of entrepreneurs to develop prototypes and come up with truly innovative ideas from outside the close-knit Biotech community (Credit: CUTEC)

OpenPCR and 3D printers like the ‘Ultimaker’ are enabling a generation of entrepreneurs to develop prototypes and come up with truly innovative ideas from outside the close-knit Biotech community (Credit: CUTEC)

OpenPCR and 3D printers like the ‘Ultimaker’ are enabling a generation of entrepreneurs to develop prototypes and come up with truly innovative ideas from outside the close-knit Biotech community (Credit: CUTEC)

This event could not have been possible without the amazing support of some passionate academics, local businesses and institutions. First of all a huge thanks to the Cambridge University Technology and Enterprise Club (CUTEC) who made this event possible. Team members Freddi, Chow, Nelly, Carolina Jeroen, Vaska, Kadi, Peter, Sanjana, Shiqing, James, Sarah, Ron and Akshara made this event a success. Thank you!

Thanks to the University of Cambridge Department of Plant Science teaching Lab (especially Barbara Landamore and Sue Aspinall), the University Synthetic Biology Strategic Research Initiative (especially Jenny Molloy and Jim Haseloff), the Cambridge Judge Business School (especially superstar Maximilian Ge!), the Biochemical Society and SynbiCITE. Local businesses and startups helped every step of the way, Cambridge Consultants (especially the amazing Synbio team Richard, Pari, James, Mary, Alim and Nick), IndieBio (especially Steven O'Connell), Spira (teamElliot RothTrevor Nicks and Seki Anderson), Cloud Laboratory Transcriptic (especially Ben Miles for your patience, guidance and support!) and Gilson (Susanna Lovell for the fantastic demo of PIPETMAX).

Thanks goes to the amazing participants who came with such passion, drive and excitement! Make sure to keep in touch and follow us on twitter @CUTEC to learn more about our future activities. 

An extended DNA recombinase toolkit for mammalian systems

Identifying, screening and optimising a novel recombinase toolkit for mammalian cells. 

The Idea

DNA recombinases perform excision, integration and inversion events on recognition of pairs of cognate DNA recombinase recognition sites. The outcome of the recombination event is directed by the orientation of the two recognition sites. These functions make valuable tools for a wide variety of applications including DNA assembly, control of gene expression, mutation, information processing (e.g. logic gates) and gene delivery/therapy. The field of synthetic biology has used these enzymes to build a variety of interesting devices, including memory modules, cellular counters and the full gamut of logic gates. However, the more sophisticated recombinase-based devices have so far been limited to use in prokaryotic organisms. This is in part due to the limited number of recombinases with high efficiency in other (e.g. mammalian) systems. 

We are particularly interested in the application of recombinases to investigating the neural circuits that subserve specific brain functions and associated disorders. A principal means of investigating this is to express reporter or effector genes in specific pathways at specific times either by delivery of synthetic genetic constructs via viruses or by generating transgenic animal models. This relies on a genetic reporter construct containing recombinase recognition sites designed such that reporter gene expression occurs only in the presence ofrecombinase activity (e.g. excision or inversion). The combination of injection of a retrograde virus carrying a recombinase expression cassette into brain structure A and injection of a second virus containing the reporter construct into brain structure B results in specific labelling of neurons in structure B that project to A. This technique has yielded interesting data on neuroanatomy. However the complexity of brain networks is such that structures typically receive inputs from several other loci. In these circumstances, multiple recombinases are required in order to simultaneously map these multiple circuits and their associated function. Moreover, the efficiency of theserecombinases will become increasingly important where outputs depend on the spatial intersection of their activity. 

We are currently limited in our designs by the small number of high-activity recombinases available. There are currently just two tyrosinerecombinase - Cre and Flp - with high activity across common cell types. Many recombinases have been identified, for example in microbial and phage genomes, however, their activity in mammallian cells is likely to be poor without sequence optimisation. We therefore propose to identify, screen and optimise (e.g. GC content, codon usage) a set recombinases whose activity in mammalian cells have not yet been reported. 

We proposed to: 
1) Identify candidate recombinases (principally from literature) 
2) Optimise the DNA sequences of the recombinsases in silico (GC content, codon usage etc). 
3) Have the optimised DNA sequences synthesised and clone into a repoter construct. 
4) Test the activity and orthogonality (i.e. test for specific for each recombinase for its cognate target sequence vs those of otherrecombinases) in fibroblasts using a transient transfection assay. 
5) Test the most promising recombinases in other mammalian cell lines. 

The recombinase activity data would be made public through publication online and the physical DNA sequences would be made available via Addgene. 

We think the availability of a larger set of high-efficiency recombinases would extend the utility of these versatile tools to a new range of varied applications including those application mentioned. The proposal also has the potential to benefit applications in eukaryotic systems beyond mammals, since the process of optimisation of recombinases from prokaryotic systems would likely involve similar hurdles (e.g. GC content). 


 The Team

Peter Davenport
Research Assistant, Department of Pathology

Maxime Fouyssac
PhD student, Department of Pharmacology

Haydn King 
Graduate student, Department of Pathology

 

Project Outputs

Project Report
Summary of the project's achievements and future plans

Project Proposal
Original proposal and application
Download PDF >> 


Outcomes, Outputs and Progress

Serine-type site-specific DNA recombinases are valuable tools for conditional expression of genetic constructs. We have a particular interest in mammalian neurobiology, where the currently small number of efficient serine recombinases limits the role these enzymes can play in elucidating (for example) neural connectivity. The core aim of this project is to identify and optimise serine recombinases for function in mammalian cells.

We have 1) constructed plasmid-based fluorescence reporters assays for recombinase activity, 2) demonstrated the function of these reporter assays in human fibroblast cells, 3) designed codon-optimised open-reading frames for a set of 6 recombinases whose functions have not previously been tested in mammalian cell lines. Having validated our reporter system, we plan to have these codon-optimised genes synthesised and to insert them into our validated vector backbones for characterisation.

Genetic constructs

Figure 1. Design of recombinase reporter and expression systems. Co-transfection of a recombinase expression plasmid (e.g. pCAG-Cre) with its cognate reporter plasmid (e.g. pCAG-loxP-STOP-loxP-ZsGreen) should result in recombination of the reporter and hence expression of ZsGreen fluorescent protein.

In order to assess recombinase efficiency we tested two designs (see figure 1):

1)      A terminator-excision system consisting of a transcriptional termination region flanked by recombinase recognition sites: removal of the transcriptional terminator facilitates RNA polymerase progression through the reporter ORF.

2)      An ORF-inversion system consisting of an inverted reporter ORF (ZsGreen) flanked by recombinase recognition sites.

We have assembled and tested a terminator-excision system for Cre (a tyrosine recombinase) and an ORF-inversion system for Bxb1 (a serine recombinase system). We chose Cre and Bxb1 for our test systems because these recombinases are among the most efficient DNA recombinases for excision and inversion reactions, respectively. We are using these systems to validate our strategy and will use them as references for the activities of other recombinases (see below). We in the process of constructing a terminator-excision system for Bxb1 for comparison.

Validation of reporter constructs

Figure 2. Validation of reporter and recombinase expression constructs. A) Co-transfection of Cre reporter (pCAG-loxP-STOP-loxP-ZsGreen) and recombinase constructs (pCAG-Cre). Green cells indicate successful recombination events. B) Relative recombination efficiencies of Cre and Dre reporter/expression systems.

The above plasmids were transfected into NIH 3T3 human fibroblasts to test for recombinase activity. Images of cells were captured using epifluorescence microscopy at 24 hours post-transfection. Transfection with reporter plasmids alone resulted in low background fluorescence that could easily be distinguished from constitutively fluorescent cells. As hoped, co-transfection of recombinase and reporter plasmids resulted in populations of brightly fluorescence cells, illustrated in figure 2A. Green cells in figure 2A indicate successful recombination of plasmid pCAG-loxP-STOP-loxP-ZsGreen by Cre recombinase expressed from plasmid pCAG-Cre. Similar results were obtained for the Bxb1 ORF-inversion system, though with lower efficiency (see figure 2B).

Ongoing work

  • Characterize the reporter constructs using flow cytometry.
  • Construct optimised recombinase expression cassettes.
  • Characterize the full set of expression cassettes by microscopy and flow cytometry.
  • Share reporter and recombinase plasmids via Addgene as well as associated online methods for open access to the scientific community.

A piezoelectric bio-platform to image and stimulate cellular interactions

To develop a novel piezoelectric platform to probe mechanobiological interactions. This pilot project serves to validate the basic process and has several key objectives. The first goal is to successfully grow a viable cell colony on the piezoelectric matrix. If that can be achieved, then we can determine whether or not the traction forces exerted by the cell culture can be detected and monitored as the culture grows.

The Idea

Mechanobiology is an emergent field of research concerned with the mechanical interactions between biological systems at a cellular level. It is becoming increasingly evident that the mechanical environment of a cell is crucial in determining its behaviour, and the subsequent long range morphology of multicellular systems. This proposal aims to probe these interactions using a novel piezoelectric platform.

If the mechanisms behind mechanical interactions can be determined, then the possibility exists to manipulate the function of cells by precisely controlling the biophysical forces they experience. The development of a platform that allowed manipulation of biophysical forces independently from the metabolic and signalling pathways engineered inside a cell would be a powerful aid in the development of synthetic biology. 


The Team


Project Outputs

Project Report
Summary of the project's achievements and future plans

Project Proposal
Original proposal and application
Download PDF >> 

Source code for test circuit
C code for matrix addressing scheme to control the brightness and flashing rate of 6 LEDs using 2 PWM signals and 5 pins. This will be transferred to control of piezoelectric material 'pixels'.


A touch screen for cells: Exploring the uses of piezoelectric materials for interfacing with biology

The aim of this project is to investigate if piezoelectric materials can be used to interface directly with biological systems by changing aspects of their mechanical environment. Piezoelectric materials can both detect and apply forces, therefore could be used to create some form of 'touch screen' for cells. Various aspects towards this aim have been investigated, including creating and characterising nanowires of a piezoelectric polymer: poly-L-lactic acid. Aerosol Jet Printing has been investigated as a possible fabrication route for the device, and a matrix addressing scheme has been developed for device control. A working prototype is currently being constructed. Results from the investigation into poly-L-lactic acid nanowires were presented at the Nanogenerators and Piezotronics conference (Rome, June 2016) and are currently being prepared for publication. The follow-on funding available for this project will allow for this manuscript to be published in an open access format.

Outcomes, Outputs and Progress

Biological systems are responsive not only to chemical changes, but also changes in their mechanical environment. This 'mechanobiology' is believed to regulate some important cell functions, such as how stem cells differentiate and the direction in which brain cells can grow. Understanding exactly how these mechanical cues can influence behaviour could lead to developments in regenerative medicine, for example.

The forces that occur between cells do so over very short length scales and are exceptionally small. In order to mechanically interface with these cellular systems, it is necessary to create a sensing element that is equally small and sensitive.

Nanowires of piezoelectric polymers are possible candidates for this. Piezoelectric materials can be used both to detect and apply forces, and by using a polymeric material and making the structures at the nanoscale, the sensitivity of the structures can be improved. The ultimate goal is to incorporate these nanowires into a grid of electrodes, allowing some spatial resolution to the measurements - therefore creating a touch screen for cells - as outlined in Figure 1. This was deemed a more appropriate name, rather than 'piezoelectric bioplatform' as was initially proposed.

 Figure 1. Schemactic of the proposed device

Figure 1. Schemactic of the proposed device

The majority of the time on this project has been spent developing the active piezoelectric component of the proposed device, with the remainder of the time used to develop the interface electronics as well as possible manufacturing techniques. These elements are being brought together to create a working prototype.

Template grown poly-L-lactic acid nanowires for piezoelectric sensing and actuation 

Poly-L-lactic acid (PLLA) is a piezoelectric polymer that is biologically derived, and as such inherently compatible with biological systems. PLLA was investigated for this project because of its biological nature, but also because the nature of its piezoelectricity is somewhat novel and is interesting to study in its own right.

Nanostructures have previously been made from PLLA using a technique known as electrospinning. The equipment required for this technique is complex, expensive and was not available for this project. Instead, a more straightforward ‘template wetting’ method was developed. This involves infiltrating a nanoporous membrane (or ‘template’) with polymer solution. The pores in these membranes, which are used commercially as particulate filters, are around 200 nm in diameter and the membranes themselves are nominally 60 μm thick. Once the solution has crystallised, the template material can be etched away to reveal polymer nanowires with the same dimensions as the pores. This method has been used extensively for creating nanowires of other piezoelectric polymers, but not before been used successfully with PLLA. Scanning Electron Microscopy (SEM) images of the nanowires at various points in the growth process are shown in Figure 2.

 Figure 2: Template grown nanowires of poly-l-lactic acid

Figure 2: Template grown nanowires of poly-l-lactic acid

It was found that changing some parameters of the growth process can influence aspects of the nanowire crystal structure. In order to optimise the sensing performance of the nanowires, it seemed reasonable to tune these growth conditions to target the crystal structure with strongest piezoelectric output. However, whilst PLLA is recognised as a piezoelectric polymer, the crystallographic origins of piezoelectricity in this material are not that well understood. The crystal structure of PLLA has been extensively studied, but never in the context of its piezoelectric properties. Therefore it was not possible to explicitly state which crystal phase, and thus which growth conditions, should be used.

In order to inform the choice of growth conditions, the piezoelectric properties of the nanowires had to be investigated and correlated with characterisation of the material’s structural. The intention was to answer the following questions:

  1. Of the 4 crystal phases of PLLA, which (if any) is responsible for its piezoelectric properties

  2. To what extent is alignment of crystallites and/or non-crystalline polymer chains important for revealing piezoelectricity?

  3. Does the template wetting method favour the formation of a particular crystal phase?

  4. Does the template wetting method lead to any preferential alignment of the polymer, either as crystallites or polymer chains?

Given the nature of piezoelectricity in PLLA, Piezo-Force Microscopy (PFM) was used to probe the piezoelectric response of individual nanowires, as shown in Figure 3. Achieving quantitative results from PFM images of nanowires is challenging, especially in the case of PLLA given its specific piezoelectric properties. Characterising the piezoelectric properties of PLLA nanowires using this technique has taken longer and proved more challenging than expected, which has delayed the progress of the project somewhat. However, the characterisation of PLLA nanowires in this manner has not before been achieved and has proved to be exceptionally informative.

X-ray diffraction (XRD) and Differential Scanning Calorimetry (DSC) have been used to characterise the structure of the nanowires. The results from the structural and piezoelectric characterisation can then be compared to reveal if any correlation exists. The results of this investigation were presented at a recent conference (NGPT - Rome, June 2016) and the slides from this investigation are freely available, attached to this report. A manuscript detailing the outcomes of this investigation is also under preparation. It is hoped that using the follow on funding available from the SynBio fund that the results can be published in an open-access journal.

 Figure 3: Piezo-Force microscopy a) amplitude and b) phase images of an individual PLLA nanowire

Figure 3: Piezo-Force microscopy a) amplitude and b) phase images of an individual PLLA nanowire

Biological aspects 

The motivation of this project is inherently biological, therefore some consideration towards this must be made. Initially, there were ideas to use E. coli bacteria as test cells for the prototype. However, as the length scales that might be easily achievable with respect to device fabrication became apparent, it seemed reasonable to choose a larger cell line.

We approached the Cambridge Centre for Medical Materials here in the Materials Science department and discussed the possibility of using some mammalian cells. Fibrosarcoma cells were suggested, which are cells taken from a fibrous tumour and are known to exert a reasonable degree of traction force whilst being relatively straightforward to culture \cite{Rasheed1974}.

Poly-L-lactic acid is widely used in biomedical applications and its biocompatibility is broadly accepted. However, the term ‘biocompatible’ is rather broad and extends beyond intrinsic material properties; the morphology of a material is also very important. Therefore, in order to test that the PLLA nanowires created here are indeed suitable, fibrosarcoma cells were grown on the samples produced. This was only a simple test, but initial results seemed promising with the cells able to survive and adhere to the nanowire structure.

The cells were imaged optically, as shown in Figure 4, however the topography of the nanowire samples meant that the images obtained from this investigation technique were of reasonably low quality. An alternative imaging method must therefore be used. The possibility of using fluorescence microscopy is currently being investigated. Another alternative is to dehydrate the cells and observe the samples using Scanning Electron Microscopy (SEM).

 Figure 4: Fibrosarcoma cells on a) an empty template b) a template filled with PLLA nanowires andd c) PLLA nanowires after the template has been removed

Figure 4: Fibrosarcoma cells on a) an empty template b) a template filled with PLLA nanowires andd c) PLLA nanowires after the template has been removed

Interface electronics

In order to simplify the project, it was decided to initially focus only on applying signals to the piezoelectric material. In order to achieve some spatial resolution to the applied signals, an array of ‘pixels’ is required. Each pixel should be individually addressable so that the physical stimulation can be targeted and varied across the device.

A matrix addressing scheme was developed to achieve this. This method allows for an array of m x n elements to be controlled using m + n electrodes and is very similar to the scheme used in smartphone touchscreens and LCD displays. A test circuit with LEDs at each pixel was constructed around a micro-processor development board (Sparkfun FreeSOC2 Development Board – Cypress PSoC5LP chip). A schematic of the circuit is shown in Figure 5. This allowed for the brightness and breathing rate of 6 LEDs to be controlled independently using just 5 electrodes. The source code used for this is available in the attached main.c file. The aim is to transfer this to a working prototype of the device, with piezoelectric material replacing the LED at each pixel.

 Figure 5: A schematic of the circuit used to develop the matrix addressing scheme

Figure 5: A schematic of the circuit used to develop the matrix addressing scheme

Fabrication methods

The uses of Aerosol Jet Printing (AJP) within this device have also been investigated. AJP is an additive manufacturing technique that can be used for wide-area deposition of fine-scaled features. It is typically used to pattern circuits in consumer electronics, and therefore could be used to print the electrodes required for this device. Since the system installed in the department – see Figure 6 - is relatively new, much work has had to be carried out to optimise and understand the deposition characteristics.

Investigation has also been carried out into producing polymer inks that could form a 'functional' piezoelectric ink. This could replace or perhaps augment the nanowires outlined above as the active piezoelectric component. Achieving a stable ink with appropriate viscosity is a significant challenge here, but work is progressing steadily.

 Figure 6: Photo and schematic of the aerosol jet printer

Figure 6: Photo and schematic of the aerosol jet printer

Expenditure

One of the most significant costs in this project was the PFM tips. These are similar to standard AFM tips, but must be doped to make them electrically conducting. Furthermore, the stiffness of these cantilevers must be optimised to probe the properties of polymers, which are generally reasonably compliant.

The travel funding allowed attendence to an Aerosol Jet Printing user group meeting hosted by Optomec, the manufacturers of the system in the Materials Science department. Given our relative inexperience with this technology, the meeting proved invaluable for learning new techniques and information regarding ink formulation.

Request and justification for follow on funding

We would like to request the additional funding available for this project. A manuscript detailing the findings of the poly-l-lactic acid nanowire investigation is currently being prepared and the follow on funding will allow for this to be published in an open access journal. It is hoped that these results will help to clarify the origins of the piezoelectric effect in this material. We do acknowledge that the aims of this investigation are mainly concerned with fundamental material properties, rather than synthetic biology, however the relevance of poly-l-lactic acid within biology – both in the context of this project but also more generally for biomedical implants – means that PLLA and its properties are worth investigating.

Conclusion

While a working protoype has yet to be achieved, key milestones towards this goal have been reached. This includes material fabrication and characterisation, biological and electronic interfacing, and optimising the device design. The outcomes of the material investigation have been presented at a recent conference (Nanogenerators and Piezotronics - NGPT - Rome, June 2016) and a manuscript detailing the results is currently being prepared. The follow-on funding available for this project will allow for these results to be presented in an open access journal.

The various aspects of the project are now at a stage where they can begin to be brought together. Whilst there will inevitably be future challenges to overcome, we are confident that a working device can be produced soon.

Open source 3D-printed microscope

Many tasks in biology require tiny, accurate motion – achieved with expensive hardware. We have used inexpensive, 3D printed parts to make high performance mechanisms for low cost science. Our best example is a microscope small and cheap enough to be left in an incubator or fume hood for days or weeks. This will enable new science, for example by observing cells as they grow in an incubator. We will improve this microscope’s imaging capabilities (adding fluorescence and phase contrast) and demonstrate its use in an incubator. We will also show that printed mechanisms can be used for other tasks, for example the mechanical manipulation of micropipettes for microinjection or patch clamping.

The Idea

Optical microscopy is fundamental to biology, and relatively high performance microscopes can now be made very cheaply. Positioning the sample and focusing the objective, however, is difficult without expensive translation stages: a microscope is mostly mechanics. Many other tasks in biology require tiny, accurate motion – achieved with expensive hardware such as mechanical micromanipulators and piezoelectric actuators. We have used inexpensive, 3D printed parts to make high performance mechanisms for low cost science, and we propose to apply this technology to problems in synthetic biology.

Our best example is a microscope small and cheap enough to be left in an incubator or fume hood for days or weeks. This will enable new science, for example by observing cells as they grow in an incubator – experiments which are currently impossible to do on a large scale due to the time and resources required. We will improve this microscope’s biological imaging capabilities (adding fluorescence and phase contrast) and demonstrate its use in an incubator at the Light Microscopy Facility in the Cancer Research Institute. This will then allow us to study phototoxicity by monitoring cultures of cells over several days.

Plastic flexure technology could also reduce the cost of mechanical micromanipulators by three orders of magnitude, opening up a range of possibilities. When combined with open-source Arduino microcontrollers, it is even possible to automate these devices for around £100. We will develop and test plastic micromanipulators for microinjection or electrophysiology, and assess their precision and stability. We will also investigate the use of ABS plastic as a potential replacement for PLA, as it has the potential to further improve the performance of printed mechanisms.

Finally, these low cost devices present obvious opportunities for science outreach, and this funding would enable us to create a class set of microscopes that can be taken (or lent) to schools as part of outreach activities, along with some fixed samples and lesson plans for easily-prepared specimens.


The Team

Dr Richard Bowman 
Richard is a Physics Research Fellow in the Nanophotonics Centre at the Cavendish Laboratory and has a background in optical tweezers and microscopy.

Dr Stefanie Reichelt 
Stefanie is Head of Light Microscopy at Cancer Research UK.

Dr Hugh Matthews
Dr Hugh Matthews is Reader in Sensory Physiology and his research interests include phototransduction and olfactory transduction. Calcium homeostasis in vertebrate photoreceptors and olfactory receptors, and its role in modulating their electrical responses. Light-induced calcium release within the photoreceptor outer segment.  

Prof Jeremy Baumberg
Jeremy Baumberg is a leader in nanoscience and nanotechnology, working for much of his career at the interface between academia and industry. He is Professor of Nanophotonics and Director of the Nanophotonics Centre.


Project Outputs

Project Report

Summary of the project's achievements and future plans

Project Proposal

Original proposal and application Download PDF >> 

OpenFlexure Scope DocuBricks

Full open source documentation for the hardware build on the DocuBricks site (also supported by SynBio Fund.

OpenFlexure Scope on github

Design files and documentation for the OpenFlexure Scope.

Open Access Paper

Sharkey, James P., et al. "A one-piece 3D printed flexure translation stage for open-source microscopy." Review of Scientific Instruments 87.2 (2016): 025104.

ArXiv | Cambridge | Publisher

Project Impact and Further Activities

  • Waterscope: Richard Bowman and co-founders started WaterScope, a social enterprise developing the microscope for water contamination testing in developing countries. They have since gone on to win several business and enterprise awards and obtain funding for a full-time employee and field work in Tanzania, where local partner STICLab have started printing the microscopes in-country. The team now also sell microscope kits.
  • OpenScope: The Cambridge-JIC iGEM 2015 team built on the Open Flexure scope with their OpenScope project, redesigning the stage for inverted microscopy and fluorescence. View their documentation on the iGEM wiki and follow the progress of the project via the Cambridge University Synthetic Biology Society, founded in 2015 to continue the work of the iGEM team and offer up opportunities for student led engagement with synthetic biology projects.

Project progress update

Abstract

Many tasks in biology require tiny, accurate motion – achieved with expensive hardware. We have used inexpensive, 3D printed parts to make high performance mechanisms such as a compact mechanical stage, enabling an inexpensive microscope. This project aimed to improve the microscope (higher resolution imaging, fluorescence, phase contrast) and also to demonstrate other devices including a micro manipulator, suitable for use in electrophysiology for patch clamping or microinjection.

Goals

The project had the following goals for outcomes:

  1. Improving microscope’s biological imaging capabilities (adding fluorescence and phase contrast) The basic configuration of our microscope uses transmission illumination: this gives bright-field images, suitable for observing many transparent samples. Adding a condenser lens in a printed holder allowed dark-field and basic phase contrast imaging, allowing a greater range of samples to be observed (See Figure 1).
    The inverted design of the microscope means that samples are generally imaged through slides or coverslips. Inverted microscopes work well with a wide range of samples, including cell cultures and microtomed specimens. Adding fluoresence imaging is still a work in progress
  2. Use in an incubator at the Light Microscopy Facility in the Cancer Research Institute. 
  3. Plastic micromanipulators for microinjection or electrophysiology
  4. Use of ABS plastic as a potential replacement for PLA, as it has the potential to further improve the performance of printed mechanisms.
  5. Science outreach kit

Outcomes

1. Open-source documentation

There are now full, illustrated assembly instructions available at docubricks as well as all source files available from GitHub. The GitHub repository contains the latest releases, in DocuBricks format, as well as the cutting-edge development options which include designs for use with RMS standard microscope objectives, and fluorescence filter mounts.

An extensive mechanical characterisation was performed by James Sharkey during his Part III project, which formed the basis of a publication describing the microscope’s mechanical design. This has been made open-access thanks to the SynBioFund grant.

2. Improved Imaging

The basic microscope design employs a webcam lens, taken from v1 of the Raspberry Pi camera module.  v2 of the camera module already represents significantly improved resolution (by about 50%) due to its superior optics.  However, for many scientific and medical applications it’s necessary to go beyond what is possible with a webcam lens.  There is now a larger version of the microscope, capable of accepting an RMS standard objective (4x-100x have been tested, and work).  This is used together with a 40mm planoconvex lens to correct for the shorter tube length and small sensor, to make the Raspberry Pi sensor cover the same field of view as a camera sensor 14.4x10.8mm in size (somewhere between a 1” and 4/3” video sensor) connected to a conventional lab microscope.

Fluorescence was demonstrated during Darryl Foo’s vacation project on the microscope, and has been re-integrated into the high-resolution objective-based design in a branch of the GitHub repository. This design will be used by the OpenPlant project “Establishing 3D Printed Microfluidics for Molecular Biology Workflows”.

Dark field and basic phase contrast imaging have been demonstrated in prototype designs, however we have not yet standardised and documented the build process for these. Both of these enhancements should be able to be made much more reproducible with our printed, adjustable condenser module which is present in the development branches on GitHub although not yet part of the documented releases.

 Figure 1. A microtomed section of Pollia condensata fruit imaged in (a) bright field and (b) dark field modes. (c) The imaging optics in the microscope, showing optional condenser lens and dark field stop. Removing the dark field stop converts the microscope to bright field mode, and removing the condenser lens decreases the brightness but does not prevent the microscope from working.

Figure 1. A microtomed section of Pollia condensata fruit imaged in (a) bright field and (b) dark field modes. (c) The imaging optics in the microscope, showing optional condenser lens and dark field stop. Removing the dark field stop converts the microscope to bright field mode, and removing the condenser lens decreases the brightness but does not prevent the microscope from working.

3. Micromanipulation

A number of variations of the OpenFlexure microscope have been produced to give full 3D motion of an object. Initial designs proved insufficiently stiff, and suffered greatly from coupling between translational and rotational motion. One of these, a delta robot design, was improved and used by the 2015 Sensors CDT cohort as part of their group project, creating an Optical Projection Tomography system(http://cdt.sensors.cam.ac.uk/news/team-project-optical-projection-tomography).

More recently, a new variation on the design incorporating two-stage mechanical reduction has allowed 100nm-scale motion with very low wobble and good orthogonality between axes.  It is currently designed in the form factor of a fibre-coupling stage, such as the flexure stages found in many optics laboratories that inspired the original flexure-based microscope.  Initial testing suggests it is a very promising design, with sufficient accuracy and stability to be used in aligning single mode optical fibres.  This design is on GitHub in development form, though it is yet to be packaged and documented for release.

Outreach

The primary impact of this project so far is the creation of WaterScope, a not-for-profit star-up aiming to use microscopy to bring about better diagnostics for public health and sanitation in the developing world.  We are working together with makers in Tanzania (STICLab) and others affiliated with the Tech for Trade network, such as AB3D in Nairobi, to investigate the financial and technical feasibility of producing microscopes locally, freeing them from reliance on imports and the often unreliable supply chain that entails.

We have also worked with Public Lab, a US based organisation aiming to use the microscope as part of an air quality monitoring project.  They have reproduced our high-resolution microscope, and are investigating creating a ruggedised case.

Finally, we have taken the microscope to various workshops and school visits, and have worked with a number of collaborators in Cambridge and beyond to test it out widely.  We are currently refining the design and working to collate teaching materials around the microscope, which we aim to make available soon.

Expenditure

The initial £4000 budget was divided into:

£1400: open access publication fee for Review of Scientific Instruments paper

£1600: purchase of Ultimaker 2 3D printer (this enabled faster prototyping and had better axis orthogonality than the RepRap machine.  It also made some successful ABS prints, and we intended to compare these to PLA ones particularly as regards stability at 37C.  Unfortunately the ABS microscopes did not print as well so their performance couldn’t be meaningfully compared.)

£400: purchase of various optical components for testing purposes to achieve higher resolution imaging and fluorescence.  Support in kind from WaterScope enabled sourcing of inexpensive objectives from Chinese manufacturers.

£600: consumables for constructing microscopes and testing them, including Raspberry Pis, Camera modules, plastic filament and nuts and bolts.

Plans for follow up

We intend to use the follow-on funding of £1000 to purchase the electronics and hardware to create a set of demo microscopes that might be lent to schools or other organisations in support of outreach or collaboration activities.  This will fund the purchase of enough hardware to produce 5-10 microscopes, complete with Raspberry Pis and screens as necessary, which will make it much easier to take demonstrations into schools, etc.