Engineered security systems for environmental synthetic biology

Lead Research Organisation: Imperial College London
Department Name: Dept of Bioengineering

Abstract

This research seeks to provide crucial parts needed to advance the exciting new topic of synthetic biology in order to safely and securely realise biotechnology applications for health, environment and energy needs. Since 2000, the new field of synthetic biology has advanced the existing science of genetic modification by applying principles of engineering; design, simulation and testing. This approach has allowed us to predictably create exciting new technologies by modifying safe microbes to perform new tasks including computation, multi-input environmental sensing and efficient production of medicinal drugs and bioenergy. While research success in synthetic biology has been rapid, the transfer of this work into real applications used by society has been limited; we can build excellent cell biosensors for explosives, arsenic and other environmental pollutants but none have actually been put to use. Largely this is because synthetic biology creates genetically modified organisms (GMOs) and these are a regulatory hazard to deploy. The major concern is that engineered DNA of these GMOs will be transferred to natural microbes by a process called horizontal gene transfer (HGT) and that this will disrupt natural ecosystems. This concern is particularly apt when we work with bacteria such as E. coli, as in these organisms synthetic biologists typically engineer their systems to be maintained on DNA circles called plasmids, which are easy to work with but also rapidly shared among bacteria and usually confer resistance to antibiotics. To overcome this hurdle and allow safe application of synthetic biology in the environment, this research aims to use synthetic biology techniques to construct a new kind of plasmid system that is secured to the intended cell, does not confer antibiotic resistance and resists horizontal gene transfer into natural bacteria. The development of this will include an assay to measure HGT and a model simulation to predict the key factors involved.

Technical Summary

Synthetic biology applies the principles of engineering to biology and in doing so has advanced the pace of development in biotechnology, particularly in microbial systems. By-and-large most designs utilise plasmids in the engineering process and also for hosting the synthetic DNA constructs in the final application. While this is understandable from a technical perspective, it is undesirable from a safety aspect, as plasmids are inherently mobile DNA elements that are prone to spreading through diverse microbial populations through horizontal gene transfer (HGT), especially when they encode antibiotic resistance markers. For synthetic biology applications to be considered safe and gain regulatory approval a design-change is needed. For this reason, this proposal aims to provide crucial new security tools for synthetic biology. By designing, modelling and implementing several new 'security devices' using a parts-based approach, we will create multiple, redundant mutual-dependencies between modular plasmids and engineered E. coli cells and produce a kit that will be suitable for synthetic biology to be applied to real-world applications such as environmental biosensing. This will be a crucial step in allowing synthetic biology applications to be put to use in defence, energy, environment and health situations. The devices of our engineered system will be based on toxin-antitoxin (TA) systems (including those known to work in both gram-positive and gram-negative bacteria), as well as on auxotrophic selection systems and recently described tunable conditional origins of replication (CORs). To help assess our success and to inform our designs we will also develop a fluorescence-based assay to detect and measure HGT events between engineered bacteria and use measurements from this assay to derive a probabilistic model of HGT in such conditions.

Planned Impact

Synthetic biology is expected this decade to move from the demonstration of new technologies to being a major part of applied bioscience research and commercial biotechnology. In particular, synthetic biology promises innovative new solutions in the defence and security sector. Microbes can be reprogrammed to be low-cost intelligent biosensors that perform logic functions in order to report on environmental pathogens and pollutants. Microbes can also be reengineered to provide high-value chemical compounds such as fuels, therapeutics and novel biomaterials. In all of these cases, synthetic biology requires genetic modification (GM) of microbes, typically using engineered plasmids and bacteria such as E. coli. Because of this, very few demonstrated applications of synthetic biology have been actually used in the real-world. The safety and regulation concerns of introducing GM bacteria, especially those that encode antibiotic resistance, are the major barrier to synthetic biology yielding significant products.

The project proposed here intends to have a major impact on the current reality of synthetic biology, which is that there are many good ideas - biosensors, biofuels, biomaterials - but very few real applications. Without confronting the issues of GM microbes the hype of this subject will not be justified and the many security and defence projects, as well as energy and health projects that the UK government will fund, will not stand a chance of realisation. The research of this project will look at, and address one of the chief concerns of GM microbes, which is the transfer of the synthetic DNA (typically plasmids) from the engineered cells to the millions of natural microbes in our ecosystem. By assaying this transfer and then using synthetic biology to design new devices and systems to prevent it, the research of this project seeks to generate a new set of tools that will have the impact of making synthetic biology secure. This impact will be significant - it will accelerate the likelihood of synthetic biology being applied in the real world and will also improve the security of existing biotechnologies. In that respect, this research will benefit almost all current and future synthetic biology projects that utilise microbial systems and plasmids.

Beyond research, this project will also impact on both educational training and the private commercial sector. The members of this project play an active role in the training and education of the next generation through synthetic biology teaching at Imperial and through the annual iGEM undergraduate competition. The results of the project described here will feed into both. The UK has had remarkable success in teaching parts-based synthetic biology, producing many world-class undergraduate projects, so investment in further research here in the UK is critical to retaining the best students in the country and building a successful UK biotechnology industry.

The private sector is actively involved in synthetic biology and this project will impact on commercial biotechnology by creating a secure, safer alternative to out-dated plasmids currently in use and we expect that will aid acceptance by UK regulators. We will seek to form collaborations with UK and international partners through an established synthetic biology industry club that we run at Imperial College.

In addition to this we also plan to work with our CSynBI partners based at LSE BIOS who are exploring the social, political, economic and ethical dimensions of synthetic biology and have requested funds in order to do this. We will look into the steps required for regulatory approval of typical synthetic biology applications - such as environmental biosensors - and assess how our project can answer the needs of the public. This will be greatly aided by a one-day workshop we intend to run looking at the regulation of synthetic biology applications.

Publications


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Wright O (2015) GeneGuard: A modular plasmid system designed for biosafety. in ACS synthetic biology

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Wright O (2013) Building-in biosafety for synthetic biology. in Microbiology (Reading, England)

 
Description Our GeneGuard system was developed with DSTL to provide a multipoint-safety mechanism to engineer bacteria cells with DNA programs that are secured to their intended cells. This allows engineered bacteria to be more-safely placed in real-world scenarios without their DNA programs unintentionally spreading in the environment. This specifically answered recommendations from major UK/USA workshops for researchers, regulators and NGOs that outlined future best practices. Our work produced a landmark review paper regarding engineering biosafety in synthetic biology and also a major research paper describing the development and use of the GeneGuard system which is now available as a toolkit to be shared among other users and with companies. The grant also funded an important international workshop on regulation around environmental use of synthetic biology that has informed many high-level international discussions since.

The key research of the grant was to investigate potential 'locks' that can be used to tie radically redesigned plasmids to engineered E.coli strains that are used in biotechnology. We developed 3 different lock mechanisms: auxotrophy, conditional replication and toxin-antitoxin paris, and in each case produced two different options. By developing these different lock modules we were able to build a final set of several different GeneGuard plasmids that each lock the plasmid to the host cell by 3 independent mechanisms. Our testing showed that the 3-lock plasmids worked as efficiently as normal plasmids for running biosensors, but were far less likely (zero evidence in our tests) to be unintentionally transferred from their host cells to other surrounding natural cells by horizontal gene transfer.
Exploitation Route The GeneGuard system can be used by other academic groups and is available via Addgene.org (international) and through the SEVA collection (EU region). It can be put to use for decreasing the likelihood of genetic pollution when microbes are used for applications e.g. in remediating polluted environments, in biosensors that will be used outside the lab and in engineered cells used for microbiome therapies.
Sectors Agriculture, Food and Drink,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
 
Description This project on enhanced security of engineered microbes, that had three significant outputs: (a) a research paper describing a innovative 3-lock mechanism ('GeneGuard') that secures engineered plasmids exclusively to their intended host (Wright et al. ACS Synthetic Biology 2014), (b) a timely review paper covering strategies and issues with biosafety and security in synthetic biology (Wright et al. Microbiology 2013), and (c) an international workshop on the Synthetic Biology Biosafety that brought together government, industry, research and NGO representatives. These last two outputs have since become instrumental references in issues regarding the safety of synthetic biology and both are being used by the UN's Secretariat of the Convention on Biological Diversity to determine whether international GM regulations should be modified in the light of synthetic biology advances. The former output, the published GeneGuard system, has been integrated into the Standard European Vector Architecture (SEVA) system shared by the top European synthetic biology labs, and is currently being assessed by a leading US microbiome therapy company.
First Year Of Impact 2014
Sector Communities and Social Services/Policy,Environment,Healthcare,Manufacturing, including Industrial Biotechology
Impact Types Economic,Policy & public services