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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.

iGEM Paris Bettencourt 2018: STAR CORES - Protein scaffolds for star-shaped AMPs

iGEM Paris Bettencourt Team (2018) group photo. Left to right: Maksim Baković, Juliette Delahaye, Annissa Améziane, Santino Nanini, Elisa Sia (Team leader), Antoine Levrier, Jake Wintermute (Secondary P.I.), Ariel Lindner (Primary P.I.), Darshak Bhatt (Team leader), Oleksandra Sorokina (Advisor). Bottom row: Anastasia Croitoru, Camille Lambert, Naina Goel. Missing from the photo are Shubham Sahu, Alexis Casas, Haotian Guo (Mentor), Ana Santos (Mentor), and Gayetri Ramachandran (Mentor)

iGEM Paris Bettencourt Team (2018) group photo. Left to right: Maksim Baković, Juliette Delahaye, Annissa Améziane, Santino Nanini, Elisa Sia (Team leader), Antoine Levrier, Jake Wintermute (Secondary P.I.), Ariel Lindner (Primary P.I.), Darshak Bhatt (Team leader), Oleksandra Sorokina (Advisor). Bottom row: Anastasia Croitoru, Camille Lambert, Naina Goel. Missing from the photo are Shubham Sahu, Alexis Casas, Haotian Guo (Mentor), Ana Santos (Mentor), and Gayetri Ramachandran (Mentor)

Microorganisms such as bacteria and yeasts are fascinating! They are both beneficial and harmful to us. Over the decades, we have been using antibiotics to kill such harmful, disease-causing bacteria. With time, over-prescription and misuse of these drugs have made bacteria resistant to them; thus, evolving into what we call “superbugs”. This is a global health crisis that we currently face where simple and treatable bacterial infections have become incurable.
Recent statistics have shown that antibiotic resistance is responsible for an estimate of 25,000 deaths per year in the European Union (EU). More importantly, it is predicted to be responsible for up to 700,000 deaths each year, which is expected to rise – overtaking cancer by 2050. Not only does it take many lives but it also has a huge economic impact. In 2009, the cost of treating multidrug-resistant bacterial infections amounted to € 1.5 million in the EU alone. Likewise, according to a CDC report in 2013 entitled, “Antibiotic Resistance Threats in the United States”, antibiotic resistance was responsible for $20 billion in direct health-care costs in the United States.

In order to fight this catastrophe, many strategies have been developed but are primarily focused on humans. Thus, the World Health Organization (WHO) has come up with a more holistic approach to deal with this problem - One health concept. This states that the dispersal of resistance genes is not only limited to human species but it also spread through animals and the environment. Given the complex interactions between different sectors, one has to expand our focus to other areas like animal farming, agricultural industries, hospitals, urban and rural sectors to curb the spread of this man-made problem.
    
In response, the iGEM Paris Bettencourt 2018 team has decided to concentrate on animal-husbandry. According to Agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail (ANSES), the pig and pork industry consumed the highest amount of farm antibiotics in the year 2015 (129 kg/PCU), making the situation worse, while the European Food and Safety Authority (EFSA) stated that it is time to Reduce, Replace, and Re-think the use of antimicrobials given to animals. Thus, our team chose to work for a possible replacement for antibiotics for reared pigs, along with engaging the public to increase awareness of the misuse of antibiotics.

After doing some intensive research, we came across a promising alternative to antibiotics which is called antimicrobial peptides (AMPs), a diverse class of naturally occurring proteins. AMPs have a broad host range and are highly efficient: since they target the bacterial membranes, resistance to AMPs evolves at a much slower rate. Despite their great potential, there are some limitations for clinical usage including their potential toxicity, susceptibility to protease degradation, and high cost of production.

Considering the prospects of using AMPs and to overcome their drawbacks, we have proposed to employ an E. coli-based cell-free expression platform to produce naturally occurring or artificially designed AMPs (Fig. 1, step 2). These AMPs have been fused to self-assembling scaffold proteins to improve their bactericidal efficiency. We started with screening the best possible AMPs and scaffold protein complexes, in terms of their bactericidal property and biocompatibility (Fig. 1, step 1), which will followed by their production in cell-free systems. Then, we would test their mechanism of action by liposome leakage assay and microscopy. In addition, we want to mathematically model the influence of the charge distribution on the efficacy of the AMPs. To achieve this aim, we have generated 12,000 variants from 5 native sequences which were suitable candidates for designing our library. Lastly, the selected AMPs fused with the scaffold proteins would be tested for their efficacy via killing kinetics experiment in which the minimum inhibitory concentration was also determined (Figure 1, Step 3). We also check for any resistance development after exposure to the controls and our experimental product. Finally, we would determine their toxicity on mammalian cell lines.

Figure 1. The three core components of the project. Step 1: Screening of the AMPs and scaffold protein complexes. Step 2: Production of the selected AMPs and scaffold protein via E. coli-based cell-free expression. Step 3: Test for the efficacy and safety of the AMPs fused with the scaffold protein produced via cell-free synthesis.

Figure 1. The three core components of the project. Step 1: Screening of the AMPs and scaffold protein complexes. Step 2: Production of the selected AMPs and scaffold protein via E. coli-based cell-free expression. Step 3: Test for the efficacy and safety of the AMPs fused with the scaffold protein produced via cell-free synthesis.

Our battle against antibiotic resistance bugs has just started, and if you would like to join us on our journey to save the planet, please do follow us on our social media portals (Facebook, Instagram, Twitter, and our YouTube channel). Feel free to message us via email or any of our social media accounts. We are open to questions, suggestions, collaborations, and monetary support.


By Nympha Elisa M. Sia and Gayetri Ramachandran - iGEM Paris Bettencourt 2018