The Green Mother Machine Reloaded

This project follows on from a previous project "The Green Mother Machine: a microfluidics device for cyanobacteria" to build a microfluidics device for observation of cyanobacteria.

The Idea

Cyanobacteria have the ability to exploit sunlight to convert atmospheric CO2 into carbohydrates, and possess a very simple but robust circadian clock. The relatively fast doubling times of cyanobacteria compared to plants and the simplicity of their circadian clock make cyanobacteria ideal organisms to study the interaction of the circadian clock with other biochemical networks (both endogenous and synthetic). These networks can interact in non-intuitive ways and often the mechanisms of such interactions are only apparent at the single cell level. Considerable efforts to build tools to observe bacteria at the single cell level have focused on other types of bacteria, such as E. coli or B. subtilis. These organisms have been preferred because of their ubiquity as laboratory model organisms and extremely fast growth rates.

In this project we want to build a microfluidic device which allows the observation of Synechococcus elongatus PCC 7942, a well-studied cyanobacterium, at the single cell level. A clever microfluidic design was developed in the Jun Lab at the University of California in San Diego for the study of E. coli [1]. In this device the bacteria grow in dead-end channels, which are slightly wider than the diameter of an E. coli cell. Thus the colony grows in a line, rather than randomly across a surface, which makes the segmentation and tracking of cells easier. Moreover, the cell at the bottom of the channel can be followed for very long times. Unfortunately, it is not possible to grow cyanobacteria in this traditional Mother Machine as S. elongatus cells are slightly larger than E.coli and their growth is inhibited when in direct contact with PDMS, the standard polymer used in microfluidic applications.

We started this project in 2015 when we received funding from SynBio Fund to build a Mother Machine type of microfluidic device optimized for cyanobacteria. We call this device the Green Mother Machine. With the funds from SynBio Fund we have been able to build a prototype of the Green Mother Machine. To get the cells to grow, we chemically removed the uncured PDMS and passivated the channels. However, the loading of the cells into the channels is still very poor, with, on average, fewer than one cell lineage per field of view.

In addition to building a better device with improved loading, we want to extend this project to build a Green Mother Machine that will allow for ultrafast switching of media on the device without generating any backflow. We intend to do this by exploiting the dial-a-wave valve pioneered by the Hasty lab at University of California in San Diego [2]. Backflow when two channels merge is a common problem in microfluidics, and this backflow causes fluctuations in the switching times. A traditional solution to this problem is to use a solenoid valve outside of the chip. These solenoid valves are expensive (£100- £1000), and the switching involves a lag time as the switch is located

The Team

Dr Christian Schwall,
Postdoctoral researcher, The Sainsbury Laboratory, Cambridge

Dr Philipp Braeuninger-Weimer,
Postdoctoral researcher, Department of Engineering, University of Cambridge

Dr Bruno Martins,
Postdoctoral researcher, The Sainsbury Laboratory, Cambridge

Dr Arijit Das,
Postdoctoral researcher, The Sainsbury Laboratory, Cambridge

Mr Chao Ye,
Graduate student, The Sainsbury Laboratory, Cambridge

Prof Anthony Hall,
Head of Plant Genomics, Earlham Institute, Norwich

Toby Livesey, Background: Plant Biology


Project Outputs

Project Report

Summary of the project's achievements and future plans

Project Proposal

Original proposal and application

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Project Resources


Progress report on the project: The Green Mother Machine Reloaded

 

Summary

In this project we want to build a microfluidic device which allows the observation of Synechococcus elongatus PCC 7942, a well-studied cyanobacterium, at the single cell level. We based our design on a well-established device called the mother machine and tailored it to the specific needs of Synechococcus elongatus. One of the biggest challenges in adapting the mother machine to Synechococcus elongatus is to keep the cells alive and to load the cells into the growth channels. Here we optimize the loading and survival of Synechococcus elongatus in the green mother machine by improving the loading protocol and the experimental setup. In addition, we tested various prototypes for the robust media switching between different media.

Report and outcomes

Initially we had less than one cell per channel during the loading process. After modifying the loading protocol by using a combination diffusion and spin loading (using a spin coater) almost all channels are fully loaded. To improve the survival of Synechococcus elongatus in the device we reduced the stress levels due to phototoxicity by using a very light sensitive sCMOS camera (Photometrics Prime). We also tried different designs of the Dial-a-Wave junction, a clever way of handling the fluids for robust switching. To achieve the switching, we had to upgrade our syringe pumps to two computer controllable pulse free pumps (fig 1).

   
  
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  Figure 1. Illustrating Pump setup.  
  
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Figure 1. Illustrating Pump setup.

After rigorous tests of different pumps, we ended up going for the KDS legato 200 as it has a good balance between performance and price. The switching with the Dial-a Wave junction worked but the growth channels in our initial prototype ended up being too high. Thus the cells could not be trapped in the growth channels. With the additional money we want to get the growth channel height right and investigate different channel designs.