A platform for rapid and precise DNA module rearrangements in Synthetic Biology

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci

Abstract

Recently, a new field of science has emerged called Synthetic Biology, which aims to apply engineering principles (for example, the use of modular components, and a "design-build-test-modify" approach to improvement) to the development of biological systems for useful purposes. One major target in Synthetic Biology is the creation of genetically modified microorganisms, to produce valuable chemical substances economically, in high yield and with low environmental impact, or to carry out beneficial chemical transformations such as neutralization of pollutants in waste water. To create these organisms, it is often necessary to introduce a set of new genes (encoded in DNA sequence) and assemble them in specified positions within the organism's long intrinsic DNA sequence ('genome'). The genetic techniques currently available for this 'assembly' task are still quite primitive and inadequate, and gene assembly is considered to be a serious bottleneck in the work leading to the development of useful microorganisms. The first main aim of our proposed research programme is to establish a sophisticated new methodology for this gene assembly process which will achieve a step-change in the speed and efficiency of creating new microorganism strains. For this purpose we will adapt a remarkable group of bacterial enzymes called the serine integrases, whose natural task is to carry out this kind of genetic rearrangement but which have hitherto been underused as tools for Synthetic Biology. We will design rapid, robust and efficient ways of making gene cassettes that can be slotted in (using serine integrases) to any one of a number of different specified positions ('landing pads') in genome DNA. By doing this we can assemble collections of genes to order within a particular microorganism. Furthermore we can choose where to place the genes in the genome and in what order, and replace any individual parts with different versions. This permits much easier optimization of complex genetic systems than is currently possible. Using our new methods we intend to engineer microbial cells to make next-generation biofuels, to make chemicals for the plastics industry by microbial fermentation instead of by using fossil fuel, and to synthesise new antibiotics.
A second major target in Synthetic Biology is to make 'smart cells' that can respond in clever ways to external signals (for example, light, high temperature, or a chemical in their environment), or that can 'remember' if they have been exposed to a particular signal and how many times. These smart cells could thus be switched on to perform a useful function only when we need it, or could be programmed to carry out an ordered series of tasks, rather like the wash-rinse-spin-dry cycles of a washing machine. The serine integrase-based tools that we will create for gene assembly lend themselves to the construction of simple yet highly effective intracellular devices for detecting and counting signals. So a second part of our programme is to show the way to the design and construction of these memory devices, and prove that they can work in the way we envisage.

Technical Summary

We aim to establish a versatile, widely applicable site-specific recombinase-based platform to facilitate rapid DNA assembly and rearrangement. Bacteriophage-derived serine integrases will be used to establish a platform of DNA assembly tools that can be used to apply engineering principles to a wide range of microorganisms. Our platform will rely on the high efficiency, specificity and especially unidirectionality of the bacteriophage serine integrases. Our first key objective is to assemble a collection of up to 20 fully orthogonal serine integrase systems, along with their recombination sites (attP, attB, attL, attR) and recombination directionality factors (RDFs), the presence or absence of which specifies attL x attR or attP x attB recombination, respectively. Our approach will be to characterize these systems (a) from published examples that have been shown to have recombination activity, and (b) by identifying new temperate phages with serine integrases from environmental samples. Using these systems we will establish methodologies for precise assembly and substitution of genes and their regulatory components in vivo and/or in vitro, and to facilitate pathway assembly in industrially important microorganisms including E. coli, Streptomyces, Aspergillus and yeast species. Specifically, we aim to engineer a model pathway for carotenoid biosynthesis in E. coli, and, in collaboration with industrial partners, pathways for production of ethanol, polymer intermediates, and antibiotics. Pathway design will be supported by predictive mathematical modelling. We will also demonstrate the promise and potential of these systems for construction of genetic memory devices, including "binary" counting circuits based on integrase-mediated inversion, that can be coupled to make devices to count up to large numbers (>1000).

Planned Impact

The first to benefit from the outputs of our research programme will be others working in the field of Synthetic Biology, both in the academic and industrial sectors. Our SIDR platform will provide new methodologies for metabolic engineering, allowing the rapid construction, testing and optimization of novel metabolic pathways for the production of high-value industrial and platform chemicals in microbial cell factories. We envisage that we will thereby dramatically enhance the speed and economic efficiency of research and development in this field, allowing industrial researchers to progress towards full-scale production much faster than is currently possible with existing gene assembly techniques, and to be able to afford much more advanced assembly projects than was hitherto feasible. Similarly, the platform will enable academic researchers to construct experimental systems and achieve their research aims much more quickly and effectively than before. BBSRC has identified Synthetic Biology as a field which could supply substantial economic benefits for the UK, and our technology will enable rapid progress of those seeking to realize these benefits. This programme will therefore have a directly beneficial effect on the competitiveness of the UK economy within a relatively short timescale (years rather than decades), by stimulating activity in the Synthetic Biology sector, as well as supporting global advances in economic activity in this area.
To achieve imaximum impact we will investigate the possibility of setting up a spin-out company using IP derived from this project, in order to optimize production and dissemination of the materials we generate through the SIDR platform.
The specific projects that we plan to undertake together with our industrial partners should lead directly to improved biosystems for production of ethanol, polymer precursors, and antibiotics, benefiting the Industrial Biotechnology sector. As the field of Synthetic Biology progresses, it is expected that intervention in cellular processes to achieve the desired outcomes will become more and more complex and sophisticated, and the biosensors and counting systems that we aim to develop in this programme are likely to become essential tools to reach the required levels of control. The economic benefits of these systems are likely to be slightly further in the future than the benefits of our pathway assembly tools, but ultimately may be very substantial, opening up new ways to control and actuate useful biological processes.
The project will have an impact on training in the new field of Synthetic Biology, as a significant number of people will be exposed to new enabling new technologies, and to how to apply them. Training and education will be at all levels; postdoctoral researchers, Ph.D students, undergraduates and schools. A major objective of our impact plan is to stimulate young scientists to take up biological science and in particular Microbiology as a career path by meetings, talks, iGEM projects, SAW projects etc. Outreach activities to the general public will also be undertaken with the aim of achieving a greater understanding of the potential of Synthetic Biology.
The ultimate beneficiaries will be the public UK and worldwide, who will gain access to novel, cheaper and cleaner products from industrial biosystems. By their nature, some of these products are likely to impact on the Healthcare sector; for example, pharmaceutical products and biomaterials for therapy. Biological production of certain chemicals will reduce our dependence on dwindling fossil hydrocarbon resources as feedstocks and allow for cleaner, environmentally friendly production methods. The UK public will also gain directly if we have a lead in this sector bringing economic wealth to the nation, as well as enjoying better health and new or cheaper consumer products.

Publications

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Bowyer J (2015) Development and experimental validation of a mechanistic model of in vitro DNA recombination. in Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual International Conference

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Brown WR (2014) Kinetochore assembly and heterochromatin formation occur autonomously in Schizosaccharomyces pombe. in Proceedings of the National Academy of Sciences of the United States of America

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Fogg PC (2014) New applications for phage integrases. in Journal of molecular biology

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Fogg PCM (2017) Genome Integration and Excision by a New Streptomyces Bacteriophage, ?Joe. in Applied and environmental microbiology

 
Description The aims of our research were to establish a set of tools for 'editing' DNA sequences analogously to the 'cut and paste' functions of a word-processing program. Enzymes called site-specific recombinases (SSRs) promote the breaking and joining of DNA strands to bring about this editing. We focused on one group of SSRs called serine integrases which have especially good properties for this type of application. We aimed to characterise about 20 of these serine integrases, together with the DNA sequences they act on, each of which acts only on its own specific sites and does not interfere with any of the other integrases. We have substantially achieved this aim (there are still some minor incompatibilities in our systems). We have demonstrated how our SSR platform can be used in several contexts in vitro and in living organisms, as we proposed in the grant application. These uses include genetic manipulation of organisms of interest, and creation of genetic memory and circuits (analogous to electronic logic gates) in living cells.
Exploitation Route As we set out to do, we have created a set of very useful tools/reagents for genetic manipulation/gene editing, and we have demonstrated how these can be used in a number of ways. We continue to supply materials to anyone who wishes to use our SSR-based systems, and we have promoted their use by our publications and presentations. Further research into development of the systems is being carried on in our labs and in other places. We are also collaborating with potential users, as noted elsewhere in this report.
Sectors Agriculture, Food and Drink,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description BBSRC DTP Studentship
Amount £75,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2013 
End 09/2017
 
Description BBSRC Project grant
Amount £465,184 (GBP)
Funding ID BB/R008593/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2018 
End 04/2021
 
Description EPSRC Studentship
Amount £75,000 (GBP)
Funding ID 66545 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 10/2013 
End 09/2016
 
Description Leverhulme Trust Programme award
Amount £574,000 (GBP)
Funding ID RP2013-K017 
Organisation The Leverhulme Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2014 
End 07/2019
 
Title Novel methods for gene assembly 
Description We have developed (and are continuing to develop) new methods for rapid efficient assembly of DNA fragments using serine integrases. 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact First version of the method is published (S.D. Colloms et al., Nucleic Acids Res. 42, e23). 
 
Description Collaboration with Ingenza Ltd 
Organisation Ingenza Ltd
Country United Kingdom 
Sector Private 
PI Contribution Discussions and experimental work to apply serine integrases to gene assembly for improved production strains of biofuels and intermediates
Collaborator Contribution Progress is being made - the grant-funded side of the collaboration is led by Prof. W.M. Stark (Glasgow University).
Impact None
Start Year 2014
 
Description Collaboration with Isomerase Ltd 
Organisation Isomerase Therapeutics
Country United Kingdom 
Sector Private 
PI Contribution Materials and discussions for antibiotic production in actinomycetes using serine integrase-mediated gene assembly
Collaborator Contribution Progress is being made - grant-funded side of the collaboration is led by Propf. M.C.M. Smith (York University).
Impact None yet
Start Year 2013
 
Description Consultancy for Flagship Pioneering Inc. 
Organisation Flagship Pioneering
Country United States 
Sector Private 
PI Contribution Marshall Stark acts as Consultant for this company on applications of site-specific recombinases.
Collaborator Contribution (not applicable)
Impact None as yet.
Start Year 2019
 
Description Art exhibition 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Prof. Stark and Dr Sean Colloms have collaborated with a resident artist in our Institute who is currently preparing exhibits based on our work.
Year(s) Of Engagement Activity 2015
 
Description Art students project 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact Dr Colloms helped Project Design students at the Glasgow School of Art to create designs for future real world products based on Synthetic Biology. An exhibition featuring these designs will go on show in Glasgow, London and Singapore
Year(s) Of Engagement Activity 2015
 
Description Article in magazine for schools 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Prof. Stark wrote an article on Synthetic Biology for a magazine, Biological Science Review, aimed at senior school students (2014).
Year(s) Of Engagement Activity 2014
 
Description Delivery the Leeds Darwin Day lecture to the British Humanist Association 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact British Humanist society invited me to deliver a lecture entitled 'Back to the Dark Ages; Evolution and antibiotic resistance'. This was a 1 hour lecture followed by 30 minutes of Q&A.
Year(s) Of Engagement Activity 2017
URL https://humanism.org.uk/2017/02/09/is-humanity-heading-back-to-the-dark-ages-professor-maggie-smith-...
 
Description Glasgow Science centre meet the expert session with iGEM 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Undergraduate students participating in iGEM meet the general public at the Science centre to tell them about their project and get feedback on the public perception of synthetic biology.
Year(s) Of Engagement Activity 2013,2014,2015,2016
URL http://www.glasgowsciencecentre.org/special-events/meet-the-expert-events.html
 
Description Laboratory experience for school students (Nuffield Trust) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Prof. Stark's lab has hosted school students funded by Nuffield Trust for experience of laboratory work.
Year(s) Of Engagement Activity 2015,2016
 
Description Leadership of iGEM team (University of Glasgow) 2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact Dr Sean Colloms helped to lead a team of about 12 undergraduate students in their activities for the international iGEM competition, which culminated in attendance at a large international symposium in Boston USA. The team was awarded prizes for specific achievements and received nominations for others.
Year(s) Of Engagement Activity 2017
URL http://2017.igem.org/Competition/Results
 
Description Microbiology Society Annual conference 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact I spoke about our work on serine integrases for synthetic biology to a session on Novel tools for manipulation of genes and genomes at the Microbiology Society annual conference.
Year(s) Of Engagement Activity 2016
URL http://www.microbiologysociety.org/events/annual-conferences/index.cfm/annual-conference-2016
 
Description Pint of Science 2015 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Prof Maggie Smith explained antibiotic resistance and how Synthetic Biology can facilitate bioprospecting for new antimicrobials.
Year(s) Of Engagement Activity 2015
 
Description Radio York interview by Prof. Maggie Smith 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Prof. Maggie Smith was interviewed about her work on the Radio York Breakfast Show.
Year(s) Of Engagement Activity 2014
 
Description Schools partnership initiative (Prof. maggie Smith/York) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Prof. Smith also leads a "Small Worlds Initiative" partnership between the University of York and the Society for General Microbiology, in which undergraduate and school students collaborate to bioprospect for new antibiotics from soil microorganisms.
Year(s) Of Engagement Activity 2013,2014,2015,2016
 
Description Synthetic Biology Introductory course 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Steven Kane (PDRA on the BBSRC LOLA grant) worked with the science development officer of South Lanarkshire Council to create an introductory course to Synthetic Biology aimed a secondary school students aged 12-17. The course is currently being tested at Hamilton Grammar School.
Year(s) Of Engagement Activity 2016
 
Description Talk at 'machines on Genes' meeting, Snowmass USA, 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation on applications of serine integrases.
Year(s) Of Engagement Activity 2018
 
Description Talk at Site-specific recombination and transposition workshop, Netherlands 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk and poster at international meeting on site-specific recombination.
Year(s) Of Engagement Activity 2018
 
Description Talk at conference (Machines on Genes III, UK) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Conference talk at Machines on Genes III, Biochemical Society Harden Conference, August 2016.
Year(s) Of Engagement Activity 2016
 
Description Visit to University of Ilorin, Nigeria 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Prof. Stark and Dr Femi Olorunniji (PDRA on the sLOLA grant) visited the University of Ilorin, Nigeria (July 2015) to publicize our Synthetic Biology work and encourage collaboration between Ilorin and Glasgow Universities.
Year(s) Of Engagement Activity 2015
 
Description Visit to University of Ilorin, Nigeria 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Two grant-funded staff (Prof Stark, Dr Olorunniji) made a 1-week visit to the University of Ilorin, Nigeria, to present a short workshop to postgraduate students there on molecular biology techniques including molecular cloning, strategies for successful completion of PhD projects, etc.
Year(s) Of Engagement Activity 2017
 
Description Visit/workshop in Ilorin, Nigeria 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Dr Femi Olorunniji visited the University of Ilorin, Nigeria. He gave a short series of talks including our research, and organized a practical workshop in molecular biology techniques for postgraduate students at the University.
Year(s) Of Engagement Activity 2016
 
Description iGEM competition 2013, 2014 and 2015 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact Teams of 10 undergraduate students from University of Glasgow (led by Dr Sean Colloms) and University of York (co-led by Prof. Maggie Smith) designed Synthetic Biology-related projects, carried out experimental work, engaged with the public, made websites/wiki, and presented their work at international meetings in Boston USA. Teams won Gold/Silver medals and were nominated for category awards.
Year(s) Of Engagement Activity 2013,2014,2015