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Cell-free synbio: a technology whose time has come

By former Community Editor Steven Burgess

It’s like instant noodles – just add water’ exclaims Dr. Keith Pardee, now an assistant professor at the University of Toronto. He is describing a small black object developed during his postdoc in the Collins lab. This unassuming device is one of the most advanced biosensors ever built – it is able to detect the presence of the Zika virus. This is achieved by using an RNA toehold switch which provides the molecular precision required to identify and outbreaks and help guide efforts to combat virus’ spread. But things get really interesting when you look at the transformative technology behind the sensor.

 

Rather than use the common approaches of polymerase chain reaction (PCR) or a genetically modified bacteria to detect the virus, Keith decided to use a ‘cell free’ system. The principle is simple: grow up bacteria, smash open the cells, and use the contents to perform reactions in a test tube or on paper (for a guide on how to make extracts see this JoVE article). This has a number of advantages over the alternatives: unlike PCR, the assay can be used in remote locations without the need for expensive lab equipment, thereby allowing instantaneous field testing, and unlike genetically modified bacteria, cell-free systems avoid concerns about uncontrolled escape of genetically modified organisms into the environment.

Once realized, the simplicity of the approach provides a host of possibilities. Keith followed up the Zika work by demonstrating the potential of cell-free systems for on demand biomanufacturing: producing antimicrobial peptides, vaccines, antibody conjugates and small molecules all in a test tube.  He explains [we aim] to use cell-free synthetic biology to extend the reach of molecular tools beyond their current range to address needs into low and middle-income countries“.

The success of these approaches has seen initiatives such as the Bakubung Workshop Report highlighting cell-free systems as a key component of capacity building in efforts to grow the African bioeconomy, sidestepping the need for investment in expensive facilities and biocontainment procedures and the potential to transform research. Additionally, development projects such as OpenDiagnostics have adopted the technology to develop low-cost, open-source crop, livestock and environmental biosensors.

 

Turbo charging the design-build-test cycle

The use of cell-extracts in biology goes back a long way. The first experiments were carried out in the 1960s, but ever since the dawn of synthetic biology the benefits of cell free systems for applied goals have also been getting scientists excited.

It is not just low cost applications that have been gaining traction, the potential advantages of cell free systems have also caught the attention of tech firms such as Sutro biopharma and Synvitrobio which are using them for the production of high value proteins that would otherwise be difficult to generate using conventional cell culture systems – such as those that are toxic.

At Imperial College, Dr. Richard Kelwick has been exploring ways cell-free systems may contribute to speeding up the design-build test cycle during his postdoc in Prof. Paul Freemont’s lab. He has been working on an improved cell-free system for B. subtilis, a bacterium which is often used in industrial fermentation for the production of products ranging from antibodies to washing powders. “We think that cell-free systems can help researchers and companies to innovate” explains Richard. In a recent publication he demonstrated the applicability of the technology for quickly screening DNA regulatory  elements and a model enzyme – getting results in hours rather than days.  

 

An Idea whose Time Has Come

There is a growing sense that the use of cell-free systems is an idea whose time has come; 2017 has seen the 1st European Congress on Cell Free Synthetic Biology, call for papers to be included in a special issue in Synthetic Biology, workshops aimed at using cell free systems to bring biology to engineers.

 

Practical Synthetic Biology Symposium, Pretoria, 24 February 2017. Source: https://www.openplant.org/global-cha

There are, of course, some limitations to these methods. Batch variability is an ongoing concern in the field, though experienced cell-free biotechnologists can minimise these effects. Cost is also a potential issue, Richard Kelwick explains “small scale reactions (e.g. for prototyping) have been quoted as low as 10-30 cents per reaction [not including sample preparation]” but bringing costs down much further may involve using picoliter volumes on a high-throughput phenotyping platform.

Dr. Richard Kelwick, Courtesy of Richard Kelwick.

These issues have slowly been addressed through years of hard slog through modification of energy production systems to removing proteases (reviewed in an excellent bioRxiv article), and standardization of preparation methods. Richard also pointed out the choice of strain is important, he explains “[systems] may benefit from the unique biochemistries of specific organisms – whether that is the presence of specific enzymes, or cofactor recycling systems.” For example he found that by simply switching strains of B. subtilis used in his cell free system he was able to improve performance ~70 fold. As a result, the Freemont group has already begun to explore (test) the potential of cell extracts, from diverse organisms that have unique biochemistries in the hope of building more robust cell-free systems.

The result of these developments is that you can now buy extracts such as Mytxtl or PURExpress to perform cell free reactions without the need of preparing your own. So will we see cell-free systems commonly used anytime soon? In everyday labs costs will come down, and the performance will continue to be improved through better strains and development of standardized protocols. In regards to applied research Keith explains “the challenges going forward include streamlining patient sample preparation and validation of the tools under field conditions,” and he is currently looking to begin field trials of the Zika sensor in Latin America.

The technology is there, all it takes is your imagination to decide what to do with it.
Note by the author: I want to thank Dr. Keith Pardee and Dr. Richard Kelwick for their input and email correspondence following presentations organised by the University of Cambridge Synthetic Biology Research Initiative on the 16th of March 2017.

Note by the editor: You can follow Steven on Twitter @sjb015

 

Discussion
  1. Interesting post. I’d be keen to know more about why people are pushing for cell free *now* i.e. why has its ‘time come’? Seeing as people have been working with cell free systems for decades, what are we witnessing here? The broadening of the range of purchasable lab products? An increase in the number of people looking to do tests but with no interest in the organism?Any and all insights gratefully received.

  2. Hi Dominic, thanks for the questions. Others may have some different ideas, but I think there are a few factors why it is coming into vogue – the main one is the system has improved a lot, making it much easier to use.

    Second is that cell free is seen as a way of increasing exposure to synthetic biology concepts – it is a very physical experience – working with parts. So seen as an educational tool for undergrads etc. It is also easier to use so long as you have a -80 – no need to grow bacteria. Related to this results can be obtained much quicker.

    Finally on the biosensor front, cell free is probably a better option for two reasons. There is no risk of accidental release of GMOs, and they are probably quicker to develop.

  3. That is an interesting point. It is not something I have seen anyone talk about.

    I have no grasp over whether it is something to be concerned about or not, or how easy it would be to properly test.

    I guess a responsible thing to do would be ensure there is nothing in the cell free system that could be harmful (such as antibiotic resistance), or as your report mentions introducing ‘kill switches’ to plan for a worst case scenario.

    Perhaps a relevant parallel to the discussion would be seeing what is being done regarding ‘RNA sprays’ (https://www.newscientist.com/article/2117460-gene-silencing-spray-lets-us-modify-plants-without-changing-dna/)?

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