cell-free

Optimization of cell-free biosensors for synthetic biology

By Amir Pandi and Olivier Borkowski

Cell-free synthetic biology recently became a branch of synthetic biology with dedicated research groups and conferences. Cell-free systems present a great potential for synthetic biology, allowing for quick in vitro transcription-translation from circular or linear DNA. The most common cell-free systems nowadays are lysate-based cell-free systems which are made by combining cell extract plus reaction buffers. These systems were initially used for fundamental discoveries in molecular biology (study of the genetic code and translation process) and later on to produce recombinant protein. In the past few years, cell-free attracted synthetic biologists’ attention as a platform for high-throughput characterization and prototyping of natural and synthetic biological circuitry. As advantages of cell-free systems we can be list: Non-GMO hosts, absence of growth dependent challenges, lower level of noise, less susceptibility to toxicity, simple cloning as genes can be cloned separately or possibility of using linear DNA (PCR product), high adjustability by varying the concentration of DNA parts or buffer elements.

However, there are still obstacles to use cell-free systems in synthetic biology. A major challenge is inefficient repression behavior. Since many bacterial regulatory elements rely on repression (i.e. most of transcription factors building blocks used in synthetic gene circuits), cell-free synthetic biology has been further developed for metabolic engineering applications than gene circuits development.

Composition and functioning of biosensors in transcription-translation cell-free systems .  (a)  A Cell-free biosensor is composed of the cell-free reaction mix (cell lysate and reaction buffers) plus the DNA.  (b)  The addition of the chemical (inducer) produces GFP. In this case, the inducer de-represses the promoter.

Composition and functioning of biosensors in transcription-translation cell-free systems. (a) A Cell-free biosensor is composed of the cell-free reaction mix (cell lysate and reaction buffers) plus the DNA. (b) The addition of the chemical (inducer) produces GFP. In this case, the inducer de-represses the promoter.


In a recent study published in ACS synthetic biology, we explored different optimization strategies to improve repression in a cell-free system. We designed a simple biosensor responding to D-psicose: psiR, a transcription factor (TF) actuates the expression of gfp from ppsiA promoter.

Sampling a wide range of concentrations for both plasmids expressing TF and GFP reporter is crucial. By trying random concentrations, you will likely not be able to see any GFP production and give up on the experiment. Initially, we only measured a very weak signal with the maximum concentration of the TF and low concentration of reporter DNA. At its best, our first experiment, based on variation of the 2 plasmid concentrations, led to an inefficient cell-free biosensor (very low fold change in the signal).

Optimization strategies applied to improve the fold change of a cell-free biosensor functioning through a transcriptional repressor. (a)  Doping,  (b)  Preincubation, and  (c)  reinitiation of (two-step) reaction. Adapted from  Pandi et al. 2019,  ACS synthetic biology  .

Optimization strategies applied to improve the fold change of a cell-free biosensor functioning through a transcriptional repressor. (a) Doping, (b) Preincubation, and (c) reinitiation of (two-step) reaction. Adapted from Pandi et al. 2019, ACS synthetic biology.

Then, we applied three strategies to overcome the issue of our low fold change.

The first strategy is using a TF-doped extract: the lysate is prepared from cells harboring a constitutive TF-expressing vector so the lysate already contains TF proteins. The cell-free reaction starts with the TF ready to repress its cognate promoter in the absence of inducer. Adding the inducer derepresses the promoter and produces GFP.

The second strategy is using preincubation: first, the cell-free reaction is performed only with the TF plasmid to produce the TF protein (preincubation). Then the reporter plasmid and the inducer are added to the mix before the reaction runs out resources to produce protein. The biosensor efficiency depends on the preincubation time modifying the balance between the amount of expressed TF (increases over time) and the available resources for GFP production (decreases over time). After 8 hours of preincubation, the repression of the promoter is at its highest level but there are not enough resources left for GFP production. Gene expression in cell-free drastically diminishes after 8-10 hours.

The third strategy is using the reinitiation of the cell-free reaction (two-step reaction): first, we preincubated the TF for 8 hours. Then we added the reporter plasmid plus fresh cell-free mix (lysate plus buffers) to reinitiate the cell-free reaction. We saw an improvement in the biosensor efficiency when either 15 or 30 µl were added with the reporter DNA.

Eventually, we compared the unoptimized biosensor as well as two different optimized biosensors to monitor the enzymatic production of D-psicose from fructose. With the optimized biosensors, we were able to quantify D-psicose production. The same preincubation or reinitiation approaches can be used to monitor the prototyping of multi-enzyme pathways in a faster and more efficient design-build-test cycle. Our strategies can be applied to optimize cell-free biosensors and gene circuits that mostly function through repressors and so generalize the use of cell-free systems in synthetic biology. 

Short bios

Amir Pandi:

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I am a PhD student in synthetic biology at Micalis institute, INRA, University of Paris-Saclay. With a bachelor's in the cell and molecular biology from the University of Tehran and a master of systems and synthetic biology from Paris-Saclay, I also participated in iGEM competition as a member (2016), as an advisor (2017), and a mentor (2019). In my PhD, I have been working on the development of biosensors and analog metabolic circuits in whole-cell and cell-free systems.

 

Olivier Borkowski:

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I am a Research Associate at Genoscope, located in Paris area. My research focus is on the relationship between protein production and host physiology. I work both with living cells and cell-free to understand the mechanisms behind the optimization of protein production and resource competition. Currently I am using approaches coupling cell-free technology and machine learning to optimize metabolic pathways.

1st European Congress on Cell-Free Synthetic Biology

Cell-free synbio community, taken from the  eccsb.epfl.ch  webpage

Cell-free synbio community, taken from the eccsb.epfl.ch webpage

 

Last March the 1st European Congress on Cell-Free Synthetic Biology took place at the Congressi Stefano Franscini (CSF), a Swiss Federal Institute of Technology of Zurich (ETH, Zurich) division, situated at Monte Verità (south of Switzerland). Top scientists working in the field of cell-free synthetic biology shared their most recent research with a large audience of PhDs, postdocs and PIs. The conference was divided in eight major sessions, four junior researchers’ sessions and a keynote talk, with a broad range of cell-free synbio, from genetic circuits to metabolic engineering.

Richard Murray, opening talk

Richard Murray, opening talk

Richard Murray from Caltech opened the first session, with the inspiring talk ‘Towards genetically-programmed artificial cells in multi-cellular machines’. During his talk he set the basis that will lead us to artificial cells in 10-15 years. He also explained which, he believes, are the main challenges to accomplish such ambitious goal. He mentioned five: i) How artificial should they be?, ii) Which source of power do they require? iii) Can they propagate information? iv) How to integrate multiple systems? v) Which source of motely force could they use? He also highlighted the need of model-based design workflows and more open-source research. Sebastian Maerkl (EPFL) followed the session with his interesting research on microfluidics platforms for the rapid implementation and characterization of genetic circuits. He showed that such an in vitro system resembles quite well the in vivo environment. Finally, Friedrich Simmel (TU Munich) showed that RNA-circuits are capable to perform complicated operations.

After the coffee break, we had the first junior researchers’ session. The talks were given by David Foschepoth (TU Delf), Alice Banks (U. of Newcastle) and Henrike Niderhiltmeyer (UCSD). They presented different systems for the maintenance of genetic networks or minimal genomes, going from platforms fueled solely with PURE systems, to automated platforms for the design and characterization of genetic circuits, and even synthetic shells, with some primordial organelle-like organization.

The second session introduced us to the possibility of generating minimal cells, completely enzyme-free. The first speaker, Erik Winfree (Caltech), with the talk ‘Enzyme-free nucleic acid dynamical systems’ presented the richness of DNA strand displacement for the implementation of basic autonomous and programmable molecular systems, capable to interact with and control their environment. Yannick Rondelez (U.Tokyo), went to the basics and explained the principles of circuits based on DNA strand displacement with his PEN DNA-toolbox. The last talk was given by Georg Seeling (U. Washington), presenting a clear application of DNA strand displacement for disease diagnostic, a fast and reliable technique, cost-efficient when compare with gene expression diagnostic methods. In my opinion, DNA strand displacement seems like a suitable option to program minimal cells, capable of basic tasks with the main advantage that such components are easy to program and characterize.

For the third session, Jørgen Kjems (Aarhus U) presented the versatility of DNA origami. From pores, to compartmentalization, and even direct drug delivery; these were some of the examples of the usefulness of DNA origami towards the assembly of minimal cells. The last talk of the day was given by Paul Freemont (Imperial College). In his talk, he showed that cell-free expression systems (TX-TL) do not only need to rely on E. coli machinery, but it is also possible to get TX-TL systems from other organisms such as Bacillus subtilis or Streptomyces venezuelae, customizing the expression systems upon need.

On the second day, session number 4 covered some recent advances on protein design and the usefulness of cell-free systems for the characterization of such protein entities. Bruno Correia (EPFL) presented his work towards the increase of a structural repertory that later will lead us to the design of functional proteins. Also, Tanja Kortemme, shared her computational pipeline to employ protein-protein interfaces as a scaffold to engineer new functions. On the other hand, Tom de Greef (TU Eidhoven), went back to DNA-based networks and showed how promising are this systems to transform intricate signaling networks into minimalistic circuits.

In the second round of junior talks, Jeo Rollin (NREL) gave us the first example of how cell-free systems are suitable for bioproduction. Nadanai Laohakunakorn (EPFL), gave a great talk on zinc fingers and showed that binding affinity correlates with repression strength. Yong Wu (Caltech) showed TX-TL systems could accelerate the process of design and implementation of novel biosynthetic pathways.

Session 5 was opened by Heiz Koeppl (TU Darmstadt), he gave us a great example of circuits characterization: they characterized a decoupled TX-TL system (TX-only environment), with the purpose to fully understand the dynamics of the implemented circuits. Gašper Tkačik (IST Autria) gave us a great example of how modeling can help understand a biological system. Elisa Franco (UC Riverside) closed the session with a beautiful example on DNA as a way to replace structural functions so far only acquainted by cytoskeletal proteins such as microtubules.

Vincent Noireux, TXTL platform

Vincent Noireux, TXTL platform

After lunch, we had a great talk by one of the pioneers in cell-free synthetic biology, Vincent Noireaux (U. Minnesota). He took us to a tour that covered his first TX-TL system, to the latest version that also includes CRISPR. The session continued with Roy Bar-Ziv (Weizmann) who showed how the combination of different technologies: DNA arrays, microfluidics, and TX-TL systems can be seen as artificial cells capable to be programmable at will. The day ended with Rebecca Schulman’s (Johns Hopkins) talk. There, we got to know that molecular circuits are also capable of some programming chemomechanics. In her research she works with hydrogels in combination of DNA circuits, and such circuits upon stimuli can expand, and this expansion is sequence specific.

On the third day, the first talk of the 7th session was under the charge of Yolanda Schaerli (UNIL). She uses synthetic gene networks to mimic regulatory networks, such as the one present in Drosophila during differentiation. Sven Panke talked about his research done in biocatalysis in cell-free systems: he told us about the beauty of cell-free platforms where all the resources are targeted to the production of a specific compound, rather than to cell maintenance. The session ended with James Bowie (UCLA) who made the point that cell-free biocatalysis can achieve much higher productivity that cell-based. However, there are still some limiting reactions that need to be overcome to get to that point.

In the 3rd junior researchers session, Richar Kelwick (Imperial College) went deeper into the TX-TL system from Bacillus subtilis. Maaruthy Yelleswarapu (Radbound U.) shared his research on cell-free expression platforms, claiming that the main cause of mRNA inactivation is sequence-dependent mRNA secondary structures. Lastly, Alexandar Tayar (Weizmann) gave further details into the programmable artificial cells presented by Roy Bar-Ziv.

On the last day, the 8th session started with Esther Amstad (EPFL), with an interesting talk about on-chip cell-free systems, where, by means of microfluidics devices, screening of different conditions could be tested inside isolated drops in a high throughput manner. Keith Pardee (U. Toronto) was the next on stage, and for me, one of the most exciting talks of the conference. He presented his work on cell-free synbio on paper. He generated paper-based sensors for the rapid and low-cost diagnostic of different diseases, targeting diseases that currently affect humanity, such as Zika. The final talk was delivered by Igor Medintz (US Naval Research). His talk on biocatalysis without cells had an unexpected component, at least for me; the use of quantum dots as a platform to channel reactions and substrate accumulation.

The last junior talks where given by Celine Love (MPI) and Mattheaus Schwarz-Schilling. They talked about cell-like compartments, that could sustain basic cellular functions and capable to even communicate with cells.

Keynote speaker, Petra Schwille

Keynote speaker, Petra Schwille

The final talk was given by my PI, Petra Schwille (MPI). I have to say that she nicely presented the work of many people who have been working towards the reconstitution of a minimal divisome. We are mainly working with components from E. coli division machinery, mainly MinDEC and FtsZ. We expect that a deep characterization of such components will lead us to the primordial division machinery that a minimal cell could use, protein or even DNA based.

The whole conference gave the audience an overview on the state of the art in the field of cell-free synthetic biology. The take home message of this enriching week was that in order to achieve our goal, the generation of a minimal cell, where all the components are known, easy to program and generate, will be achieved by the shared work of numerous research groups. We, as researchers in cell-free synbio need to work together, collaborate, and share our expertise in the different fields where we are currently working on.

Daniela Garcia-Soriano is a 3rd year PhD student working at the Schwille Lab, MPI-Biochemistry. She’s passionate about SynBio and minimal cells. Follow her on Twitter or connect with her on LinkedIn.