Project Proposal: Ambient temperature preservation of cell-free TX-TL reagents for use in synthetic biology
Commercially-available cell-free TX-TL reagents are relatively costly and although inexpensive preparation of crude cell-free lysates has been described (1), such materials still require expensive distribution via a cold chain and storage at –80 °C. This precludes their use in many contexts.
If a method of preserving cell-free TX-TL reagents at ambient temperature could be developed, this would dramatically reduce the cost of the technology and make it more widely accessible, especially in the field, to low-resource areas and for school and undergraduate education.
Two methods — freeze-drying and desiccation — are widely used to preserve biological materials, including whole microbial cells and isolated biological molecules. Pardee et al. (2, 3) have described the application of freeze-dried TX-TL systems to medical diagnostics and to on-demand biomolecular manufacturing. Desiccation could be, however, a much simpler procedure, requiring no special facilities or equipment. Both preservation methods result in some loss of biological activity and their optimisation is usually a process of trial-and-error.
We propose to investigate methods of stabilising commercially-produced cell-free TX-TL preparations at room temperature by desiccation, using trehalose to protect the labile biological components. Trehalose is a disaccharide that has proven utility in the preservation of both isolated biomolecules (DNA, enzymes and other proteins) and whole organisms (4, 5, 6).
Trehalose is synthesised by bacteria, fungi, plants and invertebrates. It is implicated in the ability of various organisms, including fungi, Selaginella lepidophylla (the ʻresurrection plantʼ), nematodes, brine shrimps and tardigrades to withstand desiccation. Trehalose is thought to prevent damage to membranes and proteins during the drying process, but the mechanism by which this occurs is not fully understood.
Although trehalose is not the panacea for preserving biomolecules and living organisms that it was thought to be In the early 1990s, its potential as a stabiliser of air-dried enzymes and nucleic acids has been proven and commercialised (4). Given appropriate strains and conditions, cultures of microorganisms, including E. coli, can also be preserved in a similar fashion (5), with or without subsequent freeze-drying (6). There is consequently good reason to believe that the essential TX- TL components of cell free extracts (tRNAs, enzymes, and other proteins) could be stabilised using trehalose.
This feasibility study will use commercially-prepared cell-free TX-TL reagents. If successful, we will consider, in the future, the application of the same storage technology to cell-free reagents, produced using DIY extraction methods similar to those described by Sun et al (1). This should result in a further significant reduction in the cost and therefore the accessibility of TX-TL systems.
(1) Sun, Z. Z. et al (2013) Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology. Journal of visualised experiments 79, e50762.
(2) Pardee, K. et al (2016) Rapid, low-cost detection of Zika virus using programmable biomolecular components. Cell 165, 1255–1266.
(3) Pardee, K. et al (2016) Portable, on-demand biomolecular manufacturing. Cell 167, 248–259.
(4) Colaço, C. et al (1992) Extraordinary stability of enzymes dried in trehalose: Simplified molecular biology. Nature Biotechnology 10, 1007–1011.
(5) Mateczun, A. J. and Peruski, L. F. (2003) Viable dried bacteria produced by drying in the presence of trehalose and divalent cation. US Patent US 6610531.
(6) Leslie, S. B. et al (1995) Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. Applied and environmental microbiology 61, 3592–3597.
Who We Are
Susana Sauret-Gueto email@example.com
Research Associate and Lab Manager, Jim Haseloff group, Department of Plant Sciences, University of Cambridge
Susana has extensive experience in imaging fluorescent markers in plants as well as experience with tissue culture work with Marchantia. She is developing foundational technologies, protocols and workflows for the engineering of the model plant Marchantia polymorpha. As research manager in the Cambridge OpenPlant Lab, she oversees instrumentation, including robots and digital microscopes, and pursues and coordinates several projects in the lab on Marchantia.
Colette Matthewman Colette.Matthewman@jic.ac.uk
Project Manager, Metabolic Biology, John Innes Centre, Norwich
Colette has a research background in the plant sciences and a keen interest in education and outreach activities. She has a growing experience working with schools and the general public in a variety of formats, including school workshops and developing interactive festival exhibits. She is already leading an OpenPlant Fund project to integrate synthetic biology relevant resources activities into secondary schools and has links to a number of teachers and educational experts in Norfolk through her work.
Fernán Federici Noe http://federicilab.org firstname.lastname@example.org
Pontificia Universidad Católica de Chile and OpenPlant Synthetic Biology Centre, University of Cambridge, Cambridge, UK
Fernán is a synthetic biologist and microscopist interested in the development of open source tools for bioengineering and the translation of cutting-edge technology into teaching resources. He has been involved in the development of open source teaching resources in Chile and the UK. He has also been part of the organisation of educational initiatives such as TECNOx (http:// tecnox.exp.dc.uba.ar) and advising high school teams such as the BioBuilderChile team (http:// biobuilderchile.weebly.com).
Dean Madden D.R.Madden@reading.ac.uk
Director, National Centre for Biotechnology Education (NCBE), University of Reading, UK.
Dean is a former UK secondary school teacher who has worked at the University of Reading for 26 years and has been responsible for developing most of the NCBEʼs educational materials, which are aimed predominantly at post-16 biology students in UK schools. These materials include trehalose-stabilised DNA, restriction enzymes and DNA ligase with protocols for their use in gel electrophoresis, DNA assembly and bacterial transformation.
Drying the cell-free TX-TL reagents
The NCBE at the University of Reading has 14 yearsʼ experience of stabilising nucleic acids and DNA-modifiying enzymes (restriction enzymes and DNA ligase) at room temperature using trehalose.
Testing the dried TX-TL reagents
Fernán and his group at the Universidad Católica de Chile in Santiago will test the dried components by fluorometric analysis and with a low-cost open-source imaging station that has been developed by them. This uses inexpensive, safe, 470 nm (blue) LEDs. Fernán will use DNA reactions with fluorescent proteins already proven to work on cell-free systems. Control reactions will be used for comparison.
Communication between project partners
The time zone difference between the UK and Chile is merely three hours, so this will facilitate communication between partners in the project, either by eMail or Skype.
Because we will send the dried TX-TL reagents by airmail without refrigeration, it will be possible to use a standard four-day courier service to Chile for testing there. Inexpensive USB temperature dataloggers will allow the internal temperature of the packages to be monitored in transit. (The NCBE has packaging, tested over almost 30 years, to maintain trehalose-preserved materials in a dry state, so humidity should not be a concern. Excessive temperatures, however, could denature the product in transit, so temperature monitoring will be essential.)
The work would have the following main components [those responsible for implementation are shown in square brackets]:
1. A literature search to ensure that the product does not run the danger of IP litigation (an initial search suggests that the patents related to air-drying of biological reagents using trehalose have expired or been invalidated). [NCBE]
2. Preservation of a commercially-produced TX-TL reagent system e.g., NEB PURExpress (including tests of dosage of trehalose, buffers and other components, vial size, drying conditions etc). Pre-screening of the dried reagents using qualitative methods. [NCBE]
3. Monitoring of the dried TX-TL reagent system in transit to Chile (data to be recovered from the USB datalogger on receipt in Chile). [Universidad Católica de Chile]
4. Testing of the dried TX-TL reagent system [Universidad Católica de Chile].
5. Stability trials of the dried reagents (using heat to simulate longer-term storage, as is commonplace in the pharmaceutical industry). [Heat treatment - NCBE; Testing after heat treatment and shipping - Universidad Católica de Chile]
6. Additional trials and expert advice [Department of Plant Sciences, University of Cambridge and John Innes Centre, Norwich]
Benefits and outcomes
Potential benefits and outcomes include:
TX-TL reagents that can be shipped and stored inexpensively at ambient temperature:- This will widen the range of institutions that can use TX-TL technology and the uses to which it can be put, particularly where storage at –80 °C proves to be difficult or impossible. This could, for example, facilitate in the longer term the development of cell-free medical and other diagnostic tests to be used in the field.
Dried TX-TL reagents could also become a game-changer in secondary education, enabling fundamental biological concepts to be learnt practically that are currently studied in theory only. In tertiary and higher education, the technology could provide a straightforward, inexpensive platform for safe in vitro testing of DNA constructs without the practical, legal and safety concerns that arise from the use of GMOs, especially in the UK and more widely within the European Union.
Assay methods of TX-TL reagents that are particularly suited to educational use:- Quantitative assessment of the efficacy of synthetic biology constructs has often proved difficult in educational contexts, largely because of a lack of suitable instrumentation and standardised protocols. Fernán Federici and his team have developed an open-source imaging station for fluorescent proteins that could be used more widely. Even if this is not used it should be possible to provide a semi-quantitative colour test strip such as that suggested by MYcoarray in its technical documentation (http:// www.mycroarray.com/mytxtl/MYtxtl+custom+invitro+protein+expression+kit+TXTL.html).
Further research using inexpensive DIY TX-TL reagents rather than commercial products:- If the trehalose preservation method is successful, we plan to investigate the possibility of preserving similar cell-free preparations produced inexpensively from cell lysates (Sun et al, 2013).
Final GBP 1000
Clearly, if the trehalose preservation method works, there is significant scope for additional development. The project partners, using their contacts with schools, higher education and research, would make this technology available for others to trial with a view to facilitating future development.
The costs below include VAT and delivery, where appropriate.
Material costs for drying and shipping the TX-TL reagents:
● Trehalose dihydrate (SigmaAldrich) 100 g GBP 190.50
● PURExpress In Vitro Protein Synthesis Kit #E6800 (NEB) 100 reactions GBP 2353.20*
● Laboratory consumables e.g., tips, microcentrifuge tubes GBP 50.00
● Impermeable foil sachets x 20 (Technology packaging) GBP 30.00
● Silica gel sachets x 40 GBP 4.00
● Postage to Chile (UK ParcelForce 4-day service) 400 g x 5 packages GBP 300.00
● Packaging, 5 boxes, etc GBP 7.20
● USB dataloggers x 2 for monitoring temperature of the packages in transit (Mindsets online, Middlesex University) GBP 50.58
Return train travel Reading to Cambridge for meetings GBP 65 x 3 = GBP 195.00
Subtotal: GBP 3180.48
Note: This sum does not include inflation which, due to the fall in the relative value of Sterling, which could be 4% in the UK in 2017, so the costs may be nearer GBP 3300.
Test reactions to be performed in Chile:
● 1 kit of 96 cell free extracts from MYtxtl GBP 500.00
● V bottom 96 well plates and lids GBP 200.00
Subtotal: GBP 700
TOTAL REQUESTED: GBP 4000
*This cost represents the maximum sum likely to be required. We could use 50% NEB PURExpress and 50% MYcoarray MYtxtl. If necessary, we could reduce costs by reducing the number of dried tests. In any event, in the first instance, it would be prudent to buy smaller quantities of these products until we are certain that the preservation method is successful.