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What is the life of cell-free genetic circuits?

What is the life of cell-free genetic circuits?


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Can genetic circuits be tested in a cell-free environment? What would be their life if we keep them at room temperature?

The circuits which are composed of DNA-based components are commonly referred to as genetic circuits. By testing, I meant to say to test its behavior i.e. applying input signals (perhaps proteins) to see if it generates an output signal (another protein, may be fluorescent protein).

By life, I meant to say, for how long such a circuit (containing strings of dna) can survive at normal room temperature.


Cell-free synthetic biology: Engineering in an open world

Cell-free synthetic biology emerges as a powerful and flexible enabling technology that can engineer biological parts and systems for life science applications without using living cells. It provides simpler and faster engineering solutions with an unprecedented freedom of design in an open environment than cell system. This review focuses on recent developments of cell-free synthetic biology on biological engineering fields at molecular and cellular levels, including protein engineering, metabolic engineering, and artificial cell engineering. In cell-free protein engineering, the direct control of reaction conditions in cell-free system allows for easy synthesis of complex proteins, toxic proteins, membrane proteins, and novel proteins with unnatural amino acids. Cell-free systems offer the ability to design metabolic pathways towards the production of desired products. Buildup of artificial cells based on cell-free systems will improve our understanding of life and use them for environmental and biomedical applications.


Ever since it was discovered that the level of calcium ions inside a cell can oscillate (Woods et al., 1986), biologists have been intrigued by the periodic nature of many cellular signals. While we are slowly starting to grasp the many and varied roles that these periodic oscillations play in cellular communication, an open question remains: how are networks of genes able to generate sustained oscillations? Now, in eLife, Sebastian Maerkl and co-workers – including Henrike Niederholtmeyer and Zachary Sun as joint first authors – report that they have used a synthetic biology approach to reveal how simple gene circuits can produce robust oscillations in cells (Niederholtmeyer et al., 2015).

Initially, it was argued that periodic oscillations in the level of calcium ions and other cellular components had no role in signaling, but decades of research has revealed that periodic signals are better at relaying information than non-periodic signals (Rapp, 1987 Behar and Hoffman, 2010 Purvis and Lahav, 2013 Levine et al., 2013). Both types of signal can encode information in the size (amplitude) of the signal, but the frequency and phase of periodic signals can also encode information. As a result, periodic signals may be able to regulate complex cell processes more precisely than non-periodic signals. Importantly, recent advances in single-cell analysis and optogenetics have resulted in numerous in-depth studies that reveal how critical events, such as the determination of cell fate and multicellular communication, are controlled by periodic signals (the review by Sonnen and Auleha, 2014 describes other examples).

Mathematical analysis shows that an essential element of an oscillating circuit is an inhibitory feedback loop: if the activity of one gene in such a feedback loop increases, it activates other genes in the circuit that ultimately inhibit it (Rapp, 1987 Novak and Tyson, 2008 Purcell et al., 2010). This feedback loop needs to have an in-built time delay to enable the activities of the genes in the circuit to fluctuate in regular cycles.

The rise of synthetic biology has made it possible to design and construct synthetic networks in living cells that perform a specific role. In an early example of this, researchers at Princeton reported that they had constructed an oscillatory gene network in E. coli based on a cyclic network of three genes called the repressilator (Elowitz and Leibler, 2000 Figure 1). Theory predicts that the repressilator and other ring oscillators that have an odd number of genes (nodes) should be capable of producing sustained oscillations. However, since designing, building and testing new gene networks in living cells is a lengthy process, ring oscillators with more than three nodes have not been reported.

Synthetic gene networks containing three, four and five genes.

The genes in each circuit (top) are translated into protein products, with each protein product repressing the activity of another gene in the network (as indicated by the arrows). Theory predicts that cyclic networks of genes display oscillatory behavior when the number of nodes in the network is odd. Niederholtmeyer et al. found that a circuit consisting of three genes gave rise to well-defined oscillations with a period of up to 8 hr, and that a circuit containing five genes oscillated with a period of 19 hours. In contrast, and in line with theoretical predictions, a network consisting of four nodes did not oscillate: instead it reached a steady state where the activity of all the genes was constant over time.

Now Maerkl and co-workers – who are based at the École Polytechnique Fédérale de Lausanne and the California Institute of Technology – have made ring architectures containing three, four and five genes (Figure 1). They built their prototype genetic circuits in a cell-free system by combining microfluidic flow reactors with extracts from E. coli bacteria (Niederholtmeyer et al, 2013 Noireaux et al., 2003). The major advantage of this approach is that it significantly decreases the time taken for each design-build-test cycle because it removes the need for various laborious tasks, such as molecular cloning and collecting measurements from individual cells.

Using this strategy Niederholtmeyer, Sun et al. were able to confirm the prediction that oscillators with three or five nodes are able to generate oscillations, whereas oscillators with four nodes are not. The period of the five-node oscillator is about twice as long as the three-node oscillator, indicating that cells can tune the periodicity of signals by increasing the complexity of their genetic circuits. Next, the researchers transferred their prototyped designs to living E. coli cells and showed that the oscillation period in cells matched the oscillation period in the cell-free systems. This is an important result as it shows that cell-free systems can be used to accurately capture the behavior of cells, which paves the way for researchers to use synthetic biology approaches in cell-free systems to explore the complex regulatory mechanisms that operate inside cells (van Roekel et al., 2015). The latest work should also greatly speed up the construction of complex new gene networks in bacteria, which could have applications in biofuel production, medical diagnosis and experiments to explore the ways that cells process information.


Visualising genetic circuits in space and time, with paper-based cell-free translation

We are a pair of scientists at Medical Research Council Laboratory of Molecular Biology (MRC LMB), who are passionate about helping students learn about modern science.

Synthetic biology is particularly interesting to us as we both work at the forefront of this field and appreciate how biology has transformed into more of an engineering discipline, where we learn about life by building biological systems. The same principle, i.e., learning biology by doing it, is very efficient for studying complex concepts in schools. However, performing modern synthetic biology experiments in the classroom is an expensive activity, due to the reagents, media, bacteria and lab instruments needed, not to mention the paperwork burden of dealing with genetically modified organisms.

We believe that teaching modern science can be accessible, cheap and straightforward. We are not alone in this and there are significant developments that have been done by Amino Labs, Cell-free tech and Biobits, which pursue the same goal as us: to make cutting-edge science accessible and affordable. We chose to work with the cell-free transcription-translation system (TXTL) as it is cheap to make, there is no need for safety regulations and they are highly customizable: the only thing you need is a genetic construct.

Our aim is to teach students the principles of genetic control, the foundation of synthetic biology. The first thing that struck us was the ease with which children study electric circuits by directly connecting electrical parts in chains and experimenting with them. We wanted to reiterate this logic for biology. Luckily, the major principles of genetic regulation have already been established with electrical engineering in mind the only puzzle piece missing: to connect them physically on a breadboard.

Figure A: Cell-free transcription-translation system (TXTL) using filter paper

The TXTL is meant to be a magic mixture that produces practically any genetic part (such as Green Fluorescent Protein or T7 RNA polymerase). As a material support where the reaction is contained, we chose a filter paper. The idea was to turn these pieces of paper into functional modules by expressing proteins in them. Therefore, connecting paper pieces later will let expressed proteins move from one paper piece to another with the water flow (Fig.A).

In the end, a protein expressed in one module can affect the reaction in the other. This experimental setup simplifies studying gene circuitry, as triggers and products of the circuit are physically separated and therefore theoretically it should be easier to deal with this kind of system as opposed to a black box mixture in the tube. Also, the possibilities are practically endless as this system is highly customizable and pieces could be connected in any way that should help children to experiment with material in an unconstrained manner.

Figure B: comparison of activity between commercial mixture (Promega T7 high yield S30) and inhouse E.coli mixture.

The project started with the production of highly active TXTL E.coli mixtures. To help other laboratories that have access to only basic equipment, we used a cheap and easy protocol for preparing cell-extracts, so that our work is easily reproducible. We have prepared cell extracts either traditionally with a French press (Emulsiflex) and high-speed centrifuge, or using a cheaper and more streamlined approach by using ultrasound cell-lysis and a cooled table top centrifuge. Independent of the protocol we used for the preparation of E.coli lysate, activity was on par with the commercial mixture (Promega T7 high yield S30) (Fig.B).

Figure c: TXTL mixtures showing more active in solution than on paper.

The challenges began when we tried to run the TXTL reaction on paper: the cell-free mixtures are always active in the solution, their paper-based counterpart only gives a low signal which could only be visualized with expensive instrumentation and thus could not be used in any low-resource environments (Fig.C).

For now, we have found a viable alternative that is suitable for outreach: as opposed to lyophilizing TXTL on the paper, we freeze-dry TXTL in the tube. Surprisingly, the reaction mix was as active as the original one, and according to previous reports the reaction components retain their activity for weeks, and even months. Thus, we aim to use this ‘halfway’ TXTL product in the upcoming summer outreach. However, the battle is not over yet we have now turned our attention to other support materials such as agarose, that does not interfere with TXTL, is cheap, could be freeze-dried and be cast in any form.


Manufacturing of therapeutics

Another active area in CFS research is the biomanufacturing of therapeutics and other protein-based reagents. Natural biological systems have evolved a remarkable capacity to synthesize a variety of molecules ranging from metabolites to biopolymers. Cell-free protein expression systems allow the incorporation of such reactions into a highly controlled process that allows production of molecules as needed and in the field. Our primary focus here will be on a subset of biopolymers, namely therapeutic proteins. The ongoing work in this field rests on decades of research that have led to the productive and practical systems currently available [28, 29, 36,37,38, 40]. Recent advances in high-throughput preparation techniques [40, 45] and in the development of systems that can use more economical energy sources [64, 65] have made CFS highly accessible. Meanwhile, significant strides are being made towards resolving various protein folding issues and shortcomings in post-translational modifications [66] associated with traditional CFS. Recent advances have showcased the potential for scaling up cell-free reactions, with some having demonstrated reaction volumes reaching 100 liters [67, 68] to 1000 liters [69]. Cell-free expression has been used as a platform for the production of a wide range of potential therapeutics, some of which have been summarized in Table 1. A number of these products have been validated in animal models [49, 76].

Two primary modes of CFS have been pursued. The first, used by commercial efforts such as Sutro [94], focuses on large, centralized production. This approach leverages the advantages of synthesis outside of the cell for biomanufacturing. For these applications, CFS not only allow for rapid production, but also significantly speed up the drug development process [95]. Remarkably, Sutro has reportedly increased their cell-free production to an incredible 1000 liters [69], showcasing the scalability of centralized cell-free production. The second mode uses FD-CF systems to de-centralize biomanufacturing capacity for small-batch production of therapeutics, with applications in global health and emergency response [49, 73, 96, 97]. Using this mode of production, we have recently demonstrated the proof-of-concept capacity to manufacture over 50 therapeutics and lab reagents, including proteins (e.g., vaccines, antibodies, and antimicrobial peptides) and small molecules [49], with applications outside of the laboratory setting.

Cell-free biomanufacturing is particularly well-suited for vaccine production due to its potential for rapid scale-up in response to public health emergencies. Successful cell-free expression of a number of recombinant vaccines (e.g., botulinum, diphtheria, anthrax) has been demonstrated [49, 86,87,88,89,90, 98], with some having been validated in animal models, such as mice [49, 90]. Considering the low dose requirements (microgram range) for many of these therapeutics, commercialization of CFS-derived vaccines will likely see rapid growth in the coming years. Production of antibodies has also been an area of focus for the cell-free community [20, 49, 51, 74,75,76,77,78,79,80, 99, 100]. Due to their compact size and relatively high expression levels in CFS, single-domain antibodies have garnered particular attention and seem strategically well-placed to serve the emerging needs in personalized medicine, i.e., for therapeutics and diagnostics.

Antibiotic resistance has been recognized as a major threat to global health, resulting in approximately two million illnesses and 23,000 deaths in the US alone every year [101]. Accordingly, cell-free production of antimicrobial compounds, including antimicrobial peptides and small molecule drugs, has become the focus of some groups [49, 93]. A number of labs have also demonstrated the power of CFS to express phages [56, 102,103,104]. The upward trend in the reported antibiotic resistance cases has led to a resurgence in viewing phage therapy as a potentially viable alternative to current antibiotic regimens [101, 105]. The use of phages has also been evaluated as an effective treatment strategy for a number of plant diseases, with some phages now being commercially available for mass consumption [106]. CFS-based production of these non-traditional antimicrobials could play a significant role in battling the antibiotic resistance crisis and could also help improve food security around the globe.

Below, we will highlight some of the areas in which CFS have shown great potential for enhancing current methods of therapeutics development and manufacturing. These advances are rapidly transforming CFS into an integral part of the manufacturing ecosystem.

Membrane proteins

While approximately 70% of all drugs act on membrane proteins[107], working with these proteins is notoriously difficult due to their enrichment in hydrophobic surfaces. Cell-based expression of membrane proteins is often fraught with challenges, such as toxicity caused by their membrane incorporation or their incompatibility with the host’s physiology [108]. Recently, cell-free approaches have been used to tackle this challenging category of proteins, the coding sequences of which comprise 20–30% of all known genes [107]. When compared to current cell-based methods, CFS can be a powerful tool in the production of soluble active membrane proteins [109]. The ability to integrate steps that can tackle the challenging aspects of membrane protein synthesis is particularly valuable. For instance, previous efforts in cell-based systems have demonstrated that membrane mimics can be successfully used to synthesize and stabilize a wide range of membrane proteins such as G-protein-coupled receptors [110, 111], the epidermal growth factor receptor [71], hepatitis C virus membrane proteins [112], and an ATP synthase [109, 113]. These mimics include surfactants, liposomes, and nanodiscs [114,115,116] and can be added directly to CFS co-translationally or post-translationally. There is also evidence suggesting that functioning single-span membrane proteins can be synthesized simply in the presence of an oil–water interface (e.g., through the use of emulsions) [117].

Macromolecular production

Molecular research has highlighted the importance of protein–protein interactions and the resulting complexes that these interactions can generate. Whether it is for the biophysical study of these complexes or as vehicles for new therapeutic delivery (e.g., virus-like scaffolds for vaccines), there is a growing need for developing robust tools aimed at synthesis of such complexes. As in the case of membrane proteins, CFS have also demonstrated higher yields, compared to in vivo strategies, in the production of macromolecular assemblies such as virus-like particles (VLPs) [109]. Groundbreaking work by the Swartz group, demonstrating the cell-free expression of hepatitis B core antigen VLP (2 subunits) [91] in an E. coli-based cell-free system, opened the door to other researchers expressing a variety of macromolecular assemblies including the E. coli RNA polymerase (5 subunits) [118] and an ATP synthase (25 subunits) [113]. Earlier work with reticulocyte lysate had also demonstrated cell-free expression of the human T-cell receptor (7 subunits) [119]. Remarkably, a number of bacteriophages have now also been successfully expressed in CFS, including the T4 phage, which structurally contains 1500 proteins from 50 genes [56, 102,103,104] (Fig. 3).

Multi-subunit protein complex synthesis in CFS. Various groups have demonstrated the production of increasingly intricate protein complexes. These have included the hepatitis B core antigen (HBc) VLP (2 subunits) [91], the E. coli RNA polymerase (5 subunits) [118], the human T-cell receptor (7 subunits) [119], an ATP synthase (25 subunits) [113], and the T4 phage (1500 subunits) [102,103,104]

Non-identical subunits of a protein complex are often referred to as hetero subunits. In some instances, such hetero subunits require co-translation to yield active complexes [120]. Thus, the ability of CFS to concurrently translate multiple mRNAs facilitates the production of active complexes composed of a number of different subunits [121]. Some CFS such as E. coli-based preparations are generally not capable of producing proteins that contain disulfide bonds, which are critical to numerous pharmaceutically relevant proteins (e.g., antibodies and many cytokines) [121]. However, recent efforts have augmented these systems to enable the production of complex proteins requiring multiple disulfide bonds [85, 99, 122], expanding the range of therapies that can be made in CFS.

Modification of proteins and codon tables

Effectiveness of many protein-based therapeutics hinges upon precise control over natural or non-natural modification of their peptide sequences. One of the most compelling uses of such modifications is in the development of antibody−drug conjugates (ADCs), which are quickly gaining favor as a new class of therapeutics against cancer. Classic conjugation techniques result in a heterogeneous mixture of labeled antibodies due to their reliance on arbitrary conjugation to multiple amino acid side chains. Recent studies, however, suggest that pharmacologic properties of ADCs could be improved through site-specific conjugation. Non-natural amino acids provide an efficient avenue for such site-specific conjugation [123]. To date, co-translational incorporation of over 100 different non-natural amino acids has been demonstrated in vivo [124], allowing for a wide range of modifications [125,126,127,128,129]. Many of these modifications have been demonstrated in the cell-free context for a variety of applications, including orientation-controlled immobilization [92, 98] and site-specific functionalization (e.g., phosphorylation [130], PEGylation [131], or drug conjugation [81]) [132,133,134].

CFS platforms circumvent some of the cell-based toxicity and permeability limitations and offer greater control and versatility in making protein modifications [109, 135]. Incorporation of non-natural amino acids in cell-based approaches has typically relied on repurposing stop codons to minimize the negative impacts of recoding on cell-viability [109]. In a cell-free system, however, the entire codon table can in theory be reprogrammed, allowing not only for the incorporation of non-natural amino acids, but also for the creation of entirely novel codon tables.

Taken to its extreme, the latter could help with the protection of intellectual property. DNA sequences could be obfuscated such that they are rendered non-functional outside of their specialized cell-free context. This obfuscated code would make proprietary designs difficult to copy. Codon obfuscation could also pose serious challenges for the detection of DNA sequences that may be employed by malevolent entities. For example, DNA synthesis companies would have a much more difficult time screening against DNA sequences that could be used for nefarious activities (e.g., bioterrorism). Recent work has shown that the size of the codon table can also be expanded by augmenting the four-letter genetic alphabet with unnatural base pairs [136, 137]. Thus, proteins made in CFS could—at least in theory—hold an unlimited number of non-natural amino acids.

CFS can also be employed for making naturally occurring modifications to proteins. An example of these is the grafting of sugars (i.e., glycans) referred to as glycosylation. Successful production of many therapeutics is often contingent upon highly efficient glycosylation, as lack of proper glycosylation can reduce the efficacy and circulation half-life of many therapeutic proteins [138]. Some CFS (e.g., insect, Chinese hamster ovary, and human K562 extract-based systems) are inherently capable of glycosylation. However, their repertoire of glycan structures tends to be limited to those naturally synthesized by their lysates’ source cell type. Additionally, glycosylation in these systems often requires recapitulation of the source cell’s protein trafficking mechanisms [109]. Thus, creation of synthetic glycosylation pathways in CFS has become an area of focus in recent years [135, 139]. Success in this domain will likely serve as a key catalyst in bringing cell-free-produced vaccines and other therapeutics to the masses. Figure 4 outlines some of the possible protein modifications in CFS.

Protein modifications in CFS. Possible protein modifications include but are not limited to glycosylation, disulfide-bond formation, acetylation [140], phosphorylation [141], and PEGylation [131] (which may be accomplished through the use of non-natural amino acids). Non-natural amino acids can also be used for the conjugation of a wide range of compounds such as drugs (e.g., through click chemistry) [81] or fluorescent molecules [142]. Figure adapted from Pagel et al. [143]

Directed evolution

Directed evolution is a powerful tool for aptamer and protein engineering that uses iterative rounds of mutagenesis and selection to modify or tune specific bimolecular properties (e.g., an enzyme’s substrate activity). Utility of aptamers or proteins, in a given context, with respect to their corresponding nucleotide sequences is often described as a fitness landscape. Directed evolution provides a massively parallel method for searching through a fitness landscape to find optimal variants and their corresponding genotypes [144]. This generally requires one-to-one mapping of phenotype to genotype. Although cells have a built-in capacity for such mapping due to their compartmentalized nature, using cells to conduct directed evolution can impose limits on the size of candidate libraries screened, and restricts the type of solvents, buffers, and temperatures that can be sampled [145]. As a result, cell-free directed evolution platforms have gained favor [145], starting with the first truly cell-free systems published in the late 90s [146, 147]. More recently, connecting phenotype to genotype has been accomplished through artificial compartmentalization (e.g., using emulsion, microbeads, and liposomes) [145, 148,149,150,151]. Applications have included the design and optimization of Fab antibody fragments [77, 152], membrane proteins [151], and, as we will discuss below, enzyme discovery [52].


Abstract

Synthetic regulatory networks with prescribed functions are engineered by assembling a reduced set of functional elements. We could also assemble them computationally if the mathematical models of those functional elements were predictive enough in different genetic contexts. Only after achieving this will we have libraries of models of biological parts able to provide predictive dynamical behaviors for most circuits constructed with them. We thus need tools that can automatically explore different genetic contexts, in addition to being able to use such libraries to design novel circuits with targeted dynamics. We have implemented a new tool, AutoBioCAD, aimed at the automated design of gene regulatory circuits. AutoBioCAD loads a library of models of genetic elements and implements evolutionary design strategies to produce (i) nucleotide sequences encoding circuits with targeted dynamics that can then be tested experimentally and (ii) circuit models for testing regulation principles in natural systems, providing a new tool for synthetic biology. AutoBioCAD can be used to model and design genetic circuits with dynamic behavior, thanks to the incorporation of stochastic effects, robustness, qualitative dynamics, multiobjective optimization, or degenerate nucleotide sequences, all facilitating the link with biological part/circuit engineering.


Building blocks of life, it's all in the genes

By Deborah Smith

What is essential for life? Not much, it seems. It was revealed this week that some bacteria - tiny though they be - carry around a lot of extra genetic baggage.

Only 12 per cent of the DNA of a common freshwater microbe was necessary for its survival, researchers discovered, after knocking out the other 88 per cent. Not unexpectedly, these vital bits of the genetic code included genes, and other segments of DNA that switch genes on and off.

'ɻut there were many surprises,'' a Stanford University biologist, Dr Lucy Shapiro, says. 'ɿor example, we found 91 essential DNA segments where we have no idea what they do.''

Whittling down the genetic blueprint of an organism to its most basic essentials is part of a new quest to understand life better, and then redesign it.

The big promise is the ability to construct new, artificial lifeforms that can solve many of the world's problems: to clean up oil spills and other pollutants, and to produce foods, fuel, plastics and drugs.

Formal engineering principles are the key to this controversial quest, known as synthetic biology, which is still in its infancy, Dr Jim Haseloff, an Australian plant expert at the University of Cambridge, says.

''The field is in a situation similar to mechanical engineering in the early 1800s and microelectronics in the early 1950s,'' he says.

Two centuries ago, mechanical engineers constructed each steam engine individually. But the development of standardised parts, such as screw threads in the 1830s, and modular construction, led to the mass production of engines from blueprints.

Electrical engineering followed a similar pathway, with the development of key components, such as transistors and integrated circuits, leading to today's international electronics industry. 'ɺnd now you have the same kind of trajectory occurring in biology,'' Dr Haseloff says.

Like the early engine builders, biologists have become very good at creating their own particular genetically modified organisms by adding one or two genes to a plant or microbe.

But a revolution in design of new organisms is needed if the world is to escape its reliance on non-renewable sources of energy and materials, Dr Haseloff, who recently gave a talk at the University of Sydney, says.

Lego blocks in a child's play box provided the first inspiration. In recent years, scientists have created a library of thousands of standardised, biological building blocks. These small DNA spare parts can be used by any researchers to assemble new microbes that can, for example, operate at higher temperatures, soak up carbon dioxide or latch onto heavy metals.

''It is a simple idea. But it is also a profound change in the way you do things - akin to moving from biology into engineering,'' Dr Haseloff says.

The latest, more sophisticated phase involves construction of complex genetic circuits, made of interacting genes and proteins, to control cell behaviour.

Natural circuits, for example, direct cells to grow, divide, produce a signal, or turn into a different type of cell. Synthetic circuits might be able to be integrated with natural circuits to ''rewire'' cells and get a plant or microbe to produce the fuel, medicine or material that is desired.

Dr Haseloff is particularly interested in how to modify the shape of plants, by altering the number of cells that proliferate or change type during development, to increase the amount of certain tissues, such as fruit.

He points to the remarkable differences between the ancient, small tomatoes of Peru and the large, red juicy ones we enjoy today, achieved by traditional breeding. ''The question is whether you can rationally engineer and produce those changes [with synthetic circuits].''

Medical researchers have already begun to use synthetic biology techniques in the laboratory, to try to tackle infectious disease and cancer, researchers report this week in the journal, Science.

For example, bacteria have been engineered to produce a protein that allows them to invade cancer cells, and to then produce a small piece of RNA that turns down the effect of a gene in the cell that stimulates colon cancer.

Concerns about synthetic biology focus on bioterror - the potential for development of new deadly microbes as weapons - and bioerror - their escape into the environment or unforseen harm to human health.

Some environmental groups have called for a moratorium on the science, but many researchers say the technology is an extension of genetic modification techniques widely used now. 'ɺnd we already have a very efficient framework of regulation and governance for these kinds of manipulations,'' Dr Haseloff says.


What is the life of cell-free genetic circuits? - Biology

Store, Exchange, and Interact with Synthetic Biological Data

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As synthetic biology techniques become more powerful, researchers are anticipating a future in which the design of biological circuits will be similar to the design of integrated circuits in electronics. Cello is a framework that describes what is essentially a programming language to design computational circuits in living cells. The circuits generated on plasmids expressed in Escherichia coli required careful insulation from their genetic context, but primarily functioned as specified. The circuits could, for example, regulate cellular functions in response to multiple environmental signals. Such a strategy can facilitate the development of more complex circuits by genetic engineering.

Cello converts electronic design specifications of combinational logic to complete DNA sequences encoding transcriptional logic circuits that can be executed in bacterial cells. A database of transcriptional repressors characterized in the Voigt lab provide genetic NOT gates and NOR gates that can be composed into any logic function.

The automated workflow and in-house genetic gates will make circuit design more reproducible and broadly accessible to biological engineering labs.

Generates a Boolean circuit as a Directed Acyclic graph from an HDL specification (Verilog).

Uses a database of second-generation genetic logic gates whose transfer functions are insulated from promoter context.

Allows users to upload custom UCF (User Constraint file) files.

Searches for the optimal assignment of transcriptional repressors to NOT/NOR gates by signal matching with the experimentally measured transfer functions.

Offers a wide range of assignment algorithms including Simulated annealing (default), Hill climbing, Breadth first search, Random permutations,etc.

Generates histograms for predicted gate REU for each row of the truth table for the best genetic circuit assignment.

Generates multiple plasmid versions using the Eugene language for constrained combinatorial enumeration of transcriptional unit orders and orientations.

Allows external applications to connect to Cello using a REST API.

Life Technologies (a Thermo Fisher brand): A114510

Office of Naval Research: Multidisciplinary University Research Initiative grant N00014-13-1-0074

Siebel Scholarship: Class of 2015

Air Force Office of Scientific Research: National Defense Science and Engineering Graduate fellowship FA9550-11-C-0028


Synthetic biologists extend functional life of cancer fighting circuitry in microbes

Bioengineers at the University of California San Diego have developed a method to significantly extend the life of gene circuits used to instruct microbes to do things such as produce and deliver drugs, break down chemicals and serve as environmental sensors.

Most of the circuits that synthetic biologists insert into microbes break or vanish entirely from the microbes after a certain period of time -- typically days to weeks -- because of various mutations. But in the September 6, 2019 issue of the journal Science, the UC San Diego researchers demonstrated that they can keep genetic circuits going for much longer.

The key to this approach is the researchers' ability to completely replace one genetic-circuit-carrying sub-population with another, in order to reset the mutation clock, while keeping the circuit running.

"We've shown that we can stabilize genetic circuits without getting into the business of fighting evolution," said UC San Diego bioengineering and biology professor Jeff Hasty, the corresponding author on the study. "Once we stopped fighting evolution at the level of individual cells, we showed we could keep a metabolically-expensive genetic circuit going as long as we want."

The circuit the UC San Diego researchers used in the Science study is one that this team, and others, are actively using to develop new kinds of cancer therapies.

"As synthetic biologists our goal is to develop gene circuits that will enable us to harness microorganisms for a wide range of applications. However, the reality today is that the gene circuits we insert into microbes are prone to fail due to evolution. Whether it be days, weeks, or months, even with the best circuit-stabilization approaches, it's just a matter of time. And once you lose functionality in your genetic circuit, there is nothing to do but start over," said Michael Liao, a UC San Diego bioengineering PhD student and the first author on the Science paper. "Our work shows there is another path forward, not just in theory, but in practice. We've shown that it's possible to keep circuit-busting mutations at bay. We found a way to keep hitting reset on the mutation clock."

If the team's method can be optimized for living systems, the implications could be significant for many fields, including cancer therapy, bioremediation, and bioproduction of useful proteins and chemical components.

Rock Paper Scissors

To actually build a "reset button" for the mutation clock, the researchers focused on dynamics between strains of microbes, rather than trying to hold selective pressures at bay at the level of individual cells. The researchers demonstrated their community-level engineering system using three sub-populations of E. coli with a "rock-paper-scissors" power dynamic. This means that the "rock" strain can kill the "scissors" strain but will be killed by the "paper" strain.

Most published work tends to focus on stabilization strategies that act at the level of single cells. While some of these approaches may be sufficient in a given therapeutic context, evolution dictates that single cell approaches will naturally tend to stop working at some point. However, since the rock-paper-scissors (RPS) stabilization acts at a community level, it can also be coupled with any of the systems that act on a single cell level to drastically extend their lifespan.

Making Cancer Drugs and Delivering them to Tumors

In 2016 in Nature, UC San Diego researchers led by Hasty, along with colleagues at MIT, described a "synchronized lysis circuit" that could be used to deliver cancer-killing drugs that are produced by bacteria that accumulate in and around tumors. This led the UC San Diego group to focus on the synchronized lysis platform for the experiments published in Science.

These coordinated explosions only occur once a predetermined density of cells has been reached, thanks to "quorum sensing" functionality also baked into the genetic circuitry. After the explosion, the approximately 10% of the bacterial population that did not explode starts growing again. When the population density once again reaches the predetermined density (more "quorum sensing"), another drug-delivering explosion is triggered and the process encoded by the researchers' synchronized lysis circuit restarts.

The challenge, however, is that this cancer killing genetic circuit -- and other genetic circuits created by synthetic biologists -- eventually stop working in the bacteria. The culprit. Mutations driven by the process of evolution.

"The fact that some bugs naturally grow in tumors and we can engineer them to produce and deliver therapies in the body is a game-changer for synthetic biology," said Hasty. "But we have to find ways to keep the genetic circuits running. There is still work to do, but we're showing that we can swap populations and keep the circuit running. This is a big step forward for synthetic biology."

Biomedical Research Advances

One of the research teams working to further advance and implement the synchronized lysis circuit is run by Tal Danino, now a professor at Columbia University, who also published seminal work on the development of quorum sensing for synthetic biology as part of his Ph.D. at UC San Diego.

"Tal recently showed that synchronized lysis technology can be used to deliver an immunotherapy to tumors in mice. To my knowledge, they are the first to show that bacterial drug production and delivery within a treated tumor can modify the immune system to attack untreated tumors. The results are fascinating. They also highlight how important it is for us to figure out how to keep the lysis circuit running as long as possible," said Hasty.

The current approach is not limited to a three-strain system. Individual sup-populations of microbes, for example, could each be programmed to produce different drugs, offering the potential of precise combination drug therapies to treat cancer, for example.

The researchers studied the dynamics of the populations using microfluidic devices that allow for controlled interactions between the different sub-populations. They also demonstrated the system is robust when tested in larger wells.

One next step will be to combine the approach with standard stabilizing approaches and demonstrate the system works in live animal models.

"We are converging on an extremely stable drug delivery platform with wide applicability for bacterial therapies," said Hasty.

Hasty, Din, and Danino are co-founders of GenCirq, a company which seeks to transfer this and related work to the clinic.


The Cell: A Molecular Approach. 2nd edition.

Cells are divided into two main classes, initially defined by whether they contain a nucleus. Prokaryotic cells (bacteria) lack a nuclear envelope eukaryotic cells have a nucleus in which the genetic material is separated from the cytoplasm. Prokaryotic cells are generally smaller and simpler than eukaryotic cells in addition to the absence of a nucleus, their genomes are less complex and they do not contain cytoplasmic organelles or a cytoskeleton (Table 1.1). In spite of these differences, the same basic molecular mechanisms govern the lives of both prokaryotes and eukaryotes, indicating that all present-day cells are descended from a single primordial ancestor. How did this first cell develop? And how did the complexity and diversity exhibited by present-day cells evolve?

Table 1.1

Prokaryotic and Eukaryotic Cells.


Researchers create synthetic cells to isolate genetic circuits

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Synthetic biology allows scientists to design genetic circuits that can be placed in cells, giving them new functions such as producing drugs or other useful molecules. However, as these circuits become more complex, the genetic components can interfere with each other, making it difficult to achieve more complicated functions.

MIT researchers have now demonstrated that these circuits can be isolated within individual synthetic “cells,” preventing them from disrupting each other. The researchers can also control communication between these cells, allowing for circuits or their products to be combined at specific times.

“It’s a way of having the power of multicomponent genetic cascades, along with the ability to build walls between them so they won’t have cross-talk. They won’t interfere with each other in the way they would if they were all put into a single cell or into a beaker,” says Edward Boyden, an associate professor of biological engineering and brain and cognitive sciences at MIT. Boyden is also a member of MIT’s Media Lab and McGovern Institute for Brain Research, and an HHMI-Simons Faculty Scholar.

This approach could allow researchers to design circuits that manufacture complex products or act as sensors that respond to changes in their environment, among other applications.

Boyden is the senior author of a paper describing this technique in the Nov. 14 issue of Nature Chemistry. The paper’s lead authors are former MIT postdoc Kate Adamala, who is now an assistant professor at the University of Minnesota, and former MIT grad student Daniel Martin-Alarcon. Katriona Guthrie-Honea, a former MIT research assistant, is also an author of the paper.

Circuit control

The MIT team encapsulated their genetic circuits in droplets known as liposomes, which have a fatty membrane similar to cell membranes. These synthetic cells are not alive but are equipped with much of the cellular machinery necessary to read DNA and manufacture proteins.

By segregating circuits within their own liposomes, the researchers are able to create separate circuit subroutines that could not run in the same container at the same time, but can run in parallel to each other, communicating in controlled ways. This approach also allows scientists to repurpose the same genetic tools, including genes and transcription factors (proteins that turn genes on or off), to do different tasks within a network.

“If you separate circuits into two different liposomes, you could have one tool doing one job in one liposome, and the same tool doing a different job in the other liposome,” Martin-Alarcon says. “It expands the number of things that you can do with the same building blocks.”

This approach also enables communication between circuits from different types of organisms, such as bacteria and mammals.

As a demonstration, the researchers created a circuit that uses bacterial genetic parts to respond to a molecule known as theophylline, a drug similar to caffeine. When this molecule is present, it triggers another molecule known as doxycycline to leave the liposome and enter another set of liposomes containing a mammalian genetic circuit. In those liposomes, doxycycline activates a genetic cascade that produces luciferase, a protein that generates light.

Using a modified version of this approach, scientists could create circuits that work together to produce biological therapeutics such as antibodies, after sensing a particular molecule emitted by a brain cell or other cell.

“If you think of the bacterial circuit as encoding a computer program, and the mammalian circuit is encoding the factory, you could combine the computer code of the bacterial circuit and the factory of the mammalian circuit into a unique hybrid system,” Boyden says.

The researchers also designed liposomes that can fuse with each other in a controlled way. To do that, they programmed the cells with proteins called SNAREs, which insert themselves into the cell membrane. There, they bind to corresponding SNAREs found on surfaces of other liposomes, causing the synthetic cells to fuse. The timing of this fusion can be controlled to bring together liposomes that produce different molecules. When the cells fuse, these molecules are combined to generate a final product.

More modularity

The researchers believe this approach could be used for nearly any application that synthetic biologists are already working on. It could also allow scientists to pursue potentially useful applications that have been tried before but abandoned because the genetic circuits interfered with each other too much.

“The way that we wrote this paper was not oriented toward just one application,” Boyden says. “The basic question is: Can you make these circuits more modular? If you have everything mishmashed together in the cell, but you find out that the circuits are incompatible or toxic, then putting walls between those reactions and giving them the ability to communicate with each other could be very useful.”

Vincent Noireaux, an associate professor of physics at the University of Minnesota, described the MIT approach as “a rather novel method to learn how biological systems work.”

“Using cell-free expression has several advantages: Technically the work is reduced to cloning (nowadays fast and easy), we can link information processing to biological function like living cells do, and we work in isolation with no other gene expression occurring in the background,” says Noireaux, who was not involved in the research.

Another possible application for this approach is to help scientists explore how the earliest cells may have evolved billions of years ago. By engineering simple circuits into liposomes, researchers could study how cells might have evolved the ability to sense their environment, respond to stimuli, and reproduce.

“This system can be used to model the behavior and properties of the earliest organisms on Earth, as well as help establish the physical boundaries of Earth-type life for the search of life elsewhere in the solar system and beyond,” Adamala says.


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