Research

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.

Playing Lego with Terpene Biosynthesis

by Laura Drummond

The smell of orange, lemon and grapefruit, the fresh scent of pine trees during a walk in the forest. The taste of mint in toothpastes, the camphor in pain-relief sprays and even the bitter notes of hops in certain types of beer. Terpenes are more present in our lives than we account for, and yet most of us do not know them by name.

 Terpenes are a class of organic compounds, produced by many different types of organisms, but mostly by plants. They are responsible for severalvolatile aroma compounds that we know, but are also involved in the formation larger molecules likecarotenoids and cholesterol, as well as some very important pharmaceuticals like the anti-malarial drug artemisinin and the anti-cancer medicine taxol.

 When it comes to their biosynthesis, terpenoids always form from two universal precursors: IPP (isopentenyl pyrophosphate) and DMAPP (dimethylallyl pyrophosphate), which are isomers from each other. These two molecules have 5 carbon atomseach, and therefore moleculesdownstream normally have a multiple of 5 carbon atoms in their structures. Terpene biosynthesis is modular, with precursors of fixed size and an almost constant count of carbon atoms, which increases in blocks of five as molecules get bigger

Biosynthesis of terpenoids. The pathways have been conceptually separated into four modules. Image:  Vavitsas et al 2018  (CC BY 4.0)

Biosynthesis of terpenoids. The pathways have been conceptually separated into four modules. Image: Vavitsas et al 2018 (CC BY 4.0)

Isopentenyl pyrophosphate (IPP), the universal precursor of Terpenes, and the different precursor molecules that can be formed using a newly discovered methyltransferase.

Isopentenyl pyrophosphate (IPP), the universal precursor of Terpenes, and the different precursor molecules that can be formed using a newly discovered methyltransferase.

In our recent paper, published in ACS synthetic biology, we found a way to challenge this ‘multiples of 5’ rule. We discovered an enzyme, hidden in the genome of Streptomyces monomycini, which is able to add one or two methyl groups (CH3) to the universal precursor of terpenes IPP, creating precursors with 6 or 7 carbon atoms in their structure. The discovery brings an additional piece for the biosynthetical pathway of these compounds, which is highly modular and resembles a game of lego. We also demonstrated the formation of larger molecules, with added methyl groups, showing that natural enzymes from the pathway can accept the different versions of IPP, taking advantage of their promiscuity.

 The findings open new possibilities for the biosynthesis of compounds so far unknown, by the addition of a new piece to the lego-like terpene biosynthetical pathway.

 

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 Laura Drummond is a PhD student at the Industrial Biotechnology Department of DECHEMA Research Institute in Frankfurt, Germany. She has a BSc in Biological Sciences from the University of Sao Paulo and a MSc in Entomology from the Luiz de Queiroz College of Agriculture in Brazil.

Twitter:  @drumm34

Linkedin: https://www.linkedin.com/in/laura-drummond-dechema/

Extreme makeover yeast edition: de novo synthesis of five chromosomes

Source: Rainis Venta, Creative Commons Attribution-Share Alike 3.0

Source: Rainis Venta, Creative Commons Attribution-Share Alike 3.0

– What I cannot create, I do not understand.

This sentence taken from Richard Feynman’s board at the time of his death essentially captures, if applied to a biological frame, the holy grail of synthetic biology; the understanding of life to such an extent that living systems can be rationally designed to gain and perform specified functions. Synthetic biology is perceived by many as an extension of metabolic engineering, where organisms are modified with end goal to produce a product. This is however part of the story, and the real strength of synthetic biology is the understanding via synthesis, the decomposition of a system to its building blocks and recombination of parts from the bottom up to elucidate the biological complexity.

The construction of a bacterial minimal genome, namely the reduction of the genetic content of Mycoplasma genitalium to 473 genes (1), was hailed as a landmark about a year ago. Last week, a series of seven research articles were published, describing the advancements in the construction of yeast synthetic chromosomes—and as the articles were published in Science, I use this journal’s citation format throughout this blog.

This work is a result of a large consortium, Synthetic Yeast 2.0 , which aims to design and implement a fully synthetic genome of Saccharomyces cerevisiae. The project has existed for several years, and started with the construction of a chromosome’s arms (2), and the first reported synthetic yeast chromosome (3). The recent publications report the synthesis of five more yeast chromosomes, totaling the synthetic sequences to more than one third of the full genome (410). The workflow started from the generation of 700-2000 bp DNA blocks, which are in vitro or in yeast assembled into 10 kb ‘chunks’. A number of chunks are chemically ligated to 30-60 kb ‘megachunks’, which are inserted sequentially into the genome.

Source:  Flickr  CC BY-NC 2.0

Source: Flickr CC BY-NC 2.0

The new chromosomes contain slight modifications from the native ones (4). The tRNA genes were removed from their original loci, and relocated to a specialized neochromosome. Repeating sequences were also removed, the TAG stop codon was replaced with TAA—leaving TAG open for repurposing, such as incorporation of a non-canonical amino acid. The design took place in BioStudio, an open-source computational framework that allows working in genome-scale (4).

Maybe the most important intervention is the inclusion of recombination sites every 10 kb and after every non-essential open reading frame. Consequently, one can implement SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution) and produce numerous strains with rearranged genetic loci, thus acquiring a powerful tool for directed evolution and functional analysis experiments.

What comes next? According to Jeff Boeke, the consortium expects to have a fully synthesized genome by the end of the year (11). However, having obtained all synthetic chromosomes does not automatically mean that a fully ‘synthetic’ yeast strain will become immediately available;  although strains incorporating one heterologous chromosome do not display a particular phenotype, a strain with three of its chromosomes replaced had a growth defect (5). Issues arising from chromosome interactions and telomere function already show, while more unknown challenges are sure to appear.

The long-term goal of this colossal undertaking is to allow research labs and organizations to routinely use custom-made organisms. This dream seems to come closer and closer, but I do not think it will happen within the next few years. The cost for synthesizing the whole S. cerevisiae genome is estimated to 1-1.5 M dollars, without taking into account salaries and maintenance (4).  But I believe that the scientific insights obtained by this project will affect synthetic biology in multiple ways. And then, why not expand to more organisms? The recent proposal to synthesize a human genome gained a lot of publicity (and controversy).  A minimal or synthetic plant genome [see Marchantia polymorpha, a small liverwort (12), and Physcomitrella patens, a moss where homologous recombination happens with the same efficiency as in yeast (13)] will be leap forwards in understanding and harnessing photosynthesis…

Disclaimer: This post originally appeared at PLOS synbio (link) and is reproduced with permision

References

  1.    C. A. Hutchison et al., Design and synthesis of a minimal bacterial genome. Science. 351, 6253–6253 (2016).
  2.     J. S. Dymond et al., Synthetic chromosome arms function in yeast and generate phenotypic diversity by design. Nature. 477, 471–476 (2011).
  3.     N. Annaluru et al., Total Synthesis of a Functional Designer Eukaryotic Chromosome. Science. 344, 55–58 (2014).
  4.     S. M. Richardson et al., Design of a synthetic yeast genome. Science. 355, 1040–1044 (2017).
  5.     L. A. Mitchell et al., Synthesis, debugging, and effects of synthetic chromosome consolidation: synVI and beyond. Science. 355 (2017), doi:10.1126/science.aaf4831.
  6.     G. Mercy et al., 3D organization of synthetic and scrambled chromosomes. Science. 355 (2017), doi:10.1126/science.aaf4597.
  7.     Y. Wu et al., Bug mapping and fitness testing of chemically synthesized chromosome X. Science. 355 (2017), doi:10.1126/science.aaf4706.
  8.     W. Zhang et al., Engineering the ribosomal DNA in a megabase synthetic chromosome. Science. 355 (2017), doi:10.1126/science.aaf3981.
  9.     Y. Shen et al., Deep functional analysis of synII, a 770-kilobase synthetic yeast chromosome. Science. 355 (2017), doi:10.1126/science.aaf4791.
  10.   Z.-X. Xie et al., “Perfect” designer chromosome V and behavior of a ring derivative. Science. 355 (2017), doi:10.1126/science.aaf4704.
  11.   A. Maxmen, Synthetic yeast chromosomes help probe mysteries of evolution. Nature (2017), doi:10.1038/nature.2017.21615.
  12.   C. R. Boehm, B. Pollak, N. Purswani, N. Patron, J. Haseloff, Synthetic Botany. Cold Spring Harb. Perspect. Biol., a023887 (2017).
  13.   B. C. King et al., In vivo assembly of DNA-fragments in the moss, Physcomitrella patens. Sci. Rep. 6, 25030 (2016).

Synthetic metabolic route allows optimal use of xylose for bio-production of chemicals

Scientists from Toulouse, France, genetically modified Escherichia coli in order to assimilate (d)-xylose and direct it towards commercially interesting compounds in a novel manner.

 

Early, this July I attended the Synthetic and Systems Biology School in Taormina, Italy. Apart from the lectures and poster sessions, selected talks from the participants were also presented. Amongst others, I distinctly remember two postdoctoral researchers, Ceren and Débora, who enthusiastically described their work about producing interesting chemicals in E. coli. I was therefore very pleased when I saw their paper published in ACS Synthetic Biology a few weeks ago.

     
  
 
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      Native (blue) and synthetic (green) (D)-xylose assimilation pathway. The chemicals of interest (pink) and the first steps of glycolysis are also noted. The green numbers represent the heterologous enzymes that were used. Figure adapted with permission from Cam et al., 2015. Copyright American Chemical Society (2015).   

 

Native (blue) and synthetic (green) (D)-xylose assimilation pathway. The chemicals of interest (pink) and the first steps of glycolysis are also noted. The green numbers represent the heterologous enzymes that were used. Figure adapted with permission from Cam et al., 2015. Copyright American Chemical Society (2015).   

This article narrates a nice metabolic engineering approach to introduce a novel (D)-xylose assimilation metabolic route in E.coli. Naturally, E.coli phosphorylates (D)-xylose at C5 and incorporates it into the main metabolism. What the designed pathway does is assimilate the (D)-xylose through C1 phosphorylation, bypassing the pentose phosphate pathway and its native regulation. The sugars can now be introduced in the glycolysis pathway and thus be used for organism growth, while the desired chemical products are produced in a more targeted manner by expressing the respective biosynthetic enzymes. The theoretical product yields were computed and compared to the native metabolism and other engineering approaches, the novel enzymes that need to be expressed were identified and characterised, and microarray experiments and metabolite analysis were carried out to study the organism’s response to the new pathway. Finally, selected chemicals were produced by dedicated strains that performed impressively in terms of yields and product concentration.

So what's missing? It would be informative to see how the strains behave and what titres can be achieved in large batch-fermentation experiments. There is a small loss of growth rate of the modified strains. Moreover, it would be interesting to optimise the strains further and to test how generic this approach is by transferring the pathway to other organisms, such as baker’s yeast. Overall, however, this research paper is an easy-to follow biotechnology story that begins with the motivation and design and reaches the desired outcome of increased product formation.


There are two take-home messages that I would like to address after reading this article. The first comes from the use of (D)-xylose as a substrate. The rationale behind it is that xylose constitutes a large proportion of the unused cellulose and hemicellulose biomass that are byproducts of bio-refinery. This is a prime example of synthetic biology employed in sustainability efforts, where a waste product is converted into commercially interesting compounds. Those formed products are currently available in industrial scale as fossil fuel byproducts and their production using E.coli is an environmentally friendlier alternative. However, the bio-sustainability argument has some pitfalls and needs to be employed carefully. A fermentation production is itself an energy consuming process. Also, the use of sugars should not directly compete with or use up agriculture resources. Nevertheless, it is my opinion that the industry needs to disengage fast from oil as a feedstock, and synthetic biology is a powerful tool that can provide novel alternatives.

A second point is the use of systems biology together with synthetic biology. Taking into account that a system cannot be simply described by the addition of its components but also requires the interactions between those parts, likewise a bioengineering approach cannot rely on the simple expression of a heterologous pathway and the optimisation of the participating enzymes. This principle is illustrated in this article, where metabolic modelling and microarray analysis that provide invaluable insights during the pathway design and the strain optimisation steps respectively. Interdisciplinary research is also present in this work, as computational science, bioinformatics, and analytical chemistry are employed together with molecular biology and biochemistry. It becomes more and more obvious that single-focus and narrow approaches are getting obsolete. The new generation of scientists needs to speak the language of and understand collaborators coming from different backgrounds. Synthetic biology, which in principle combines parts to obtain new properties and novel functions, cannot fall behind in combining different disciplines in a way that facilitates research and strengthens innovation and creativity.

 

 

Research Paper: 

Yvan Cam, Ceren Alkim, Debora Trichez, Vincent Trebosc, Amélie Vax, François Bartolo, Philippe Besse, Jean Marie François, and Thomas Walther (2015) Engineering of a Synthetic Metabolic Pathway for the Assimilation of (d)-Xylose into Value-Added Chemicals. ACS Synthetic Biology DOI: 10.1021/acssynbio.5b00103

 

Written by:

Konstantinos Vavitsas

Konstantinos is a PhD student at the University of Copenhagen, working on the photosynthetic production of high-value compounds. 

Edited by: Devang Mehta

 

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