Engineering orthogonal split inteins as scalable tools for synthetic biology and biomanufacturing
Lead Research Organisation:
University of Edinburgh
Department Name: Sch of Biological Sciences
Abstract
An important goal of synthetic biology is the rational design and predictable implementation of synthetic gene circuits using standardised interchangeable parts to program cellular behaviour. However, most of the circuits constructed so far are small-scale systems that have been constructed by costly and inefficient 'trial-and-error' methods with limited parts. A hard truth behind this is that the engineering of complex circuits in living cells is currently limited by the availability of well-characterised and orthogonal genetic regulatory building blocks in addition to the lack of robust wiring methods. Moreover, versatile post-translational regulation tools such as split inteins represent an untapped/underexplored invaluable resource for synthetic biologists.
This project aims to develop new enabling tools for scalable gene circuit design via engineering novel orthogonal split intein mediated gene regulation control strategies, including engineering a library of modular and orthogonal genetic logic gates and switches to significantly expand the currently limited toolbox of genetic control components. The outcome will transform the present state of gene network engineering by directly addressing a key bottleneck in the field. I will also develop new efficient automation tools for gene circuit design and diagnosis to achieve rapid, predictable design of large-scale genetic logic circuits with advanced cellular information processing capacity. Such tools have the potential to significantly increase the efficiency and scale, and shorten development time of a typical gene circuit design project from the present several years to a few months. Further, the engineered post translational tools will be applied to develop groundbreaking biotechnology applications enabled by orthogonal split-intein mediated protein ligation including manufacturing novel anti-reflection coating biomaterials and modular versatile multipecific antibodies at a later stage of this fellowship program.
The successful outcome will lead to a transformative enabling technology for synthetic biology and open up a wide range of innovative protein-based biomanufacturing applications in the biotechnology and therapeutic sectors.
This project aims to develop new enabling tools for scalable gene circuit design via engineering novel orthogonal split intein mediated gene regulation control strategies, including engineering a library of modular and orthogonal genetic logic gates and switches to significantly expand the currently limited toolbox of genetic control components. The outcome will transform the present state of gene network engineering by directly addressing a key bottleneck in the field. I will also develop new efficient automation tools for gene circuit design and diagnosis to achieve rapid, predictable design of large-scale genetic logic circuits with advanced cellular information processing capacity. Such tools have the potential to significantly increase the efficiency and scale, and shorten development time of a typical gene circuit design project from the present several years to a few months. Further, the engineered post translational tools will be applied to develop groundbreaking biotechnology applications enabled by orthogonal split-intein mediated protein ligation including manufacturing novel anti-reflection coating biomaterials and modular versatile multipecific antibodies at a later stage of this fellowship program.
The successful outcome will lead to a transformative enabling technology for synthetic biology and open up a wide range of innovative protein-based biomanufacturing applications in the biotechnology and therapeutic sectors.
Planned Impact
The proposed research falls into the BBSRC/EPSRC strategic priority areas of Synthetic Biology (one of the eight great technologies identified by UK government) and Developing New Enabling Tools for Biosciences, which hold great promise for applications in sectors such as industrial biotechnology, environment and healthcare, and are strategically important sectors for the economic future of the UK.
This proposed study aims to develop a new generation of synthetic biology tools for enabling programmable complex gene expression control and innovative biomanufacturing capacity in model E. coli chassis microorganism. The engineered scalable split intein tools will significantly expand the currently limited toolkit available for synthetic biologists to advance the engineering of large complex genetic control systems. The outcome will transform the present state of gene network engineering by directly addressing a key bottleneck in the field. The engineered tools will be applied to develop innovative biotechnology applications enabled by orthogonal split intein mediated protein ligation including manufacturing novel anti-reflection coating biomaterials and at a later stage modular versatile bispecific/multispecifc antibodies. The developed novel nanoscale coating approach has the potential to coat large area surfaces, and complex shaped structures at a significantly lower cost compared to nanolithography in addition being environment friendly and sustainable, and has a wide range of applications in both defence and civil areas. The new modular manufacturing approach will enable rapidly producing a large number of multivalent antibodies, leading to reduced costs and shortened development time, for many applications in disease diagnostics and therapeutics.
The technology may lead to applications in a range of industrial biotechnological areas that would benefit from the programmable advanced control of gene expression and cell behaviour, such as the life sciences research tools and reagents, microbe-based biologics bioproduction, in vivo bioprocess control and optimisation.
Hence the developed tools and technology will have wide impact in the synthetic biology and bioengineering communities by generating new scalable circuit design tools and methods, novel expanded sets of orthogonal regulatory components and innovative biomanufacuturing methods to assist the maturation of the emerging field of synthetic biology. The project will contribute to maintaining UK leadership in this strategic area and the national economical growth.
The work will be of extreme importance to researchers working at the interface of biology and engineering, and those in biotechnology industry. Potential patentable circuit designs and tools could be generated that might be of great interest to biotechnological sector and we will patent promising designs and technologies as they become available.
The project successful outcome will lead to a transformative enabling technology for synthetic biology and open up a wide range of innovative protein-based biomanufacturing applications in the biotechnology and biomedicine sectors.
This proposed study aims to develop a new generation of synthetic biology tools for enabling programmable complex gene expression control and innovative biomanufacturing capacity in model E. coli chassis microorganism. The engineered scalable split intein tools will significantly expand the currently limited toolkit available for synthetic biologists to advance the engineering of large complex genetic control systems. The outcome will transform the present state of gene network engineering by directly addressing a key bottleneck in the field. The engineered tools will be applied to develop innovative biotechnology applications enabled by orthogonal split intein mediated protein ligation including manufacturing novel anti-reflection coating biomaterials and at a later stage modular versatile bispecific/multispecifc antibodies. The developed novel nanoscale coating approach has the potential to coat large area surfaces, and complex shaped structures at a significantly lower cost compared to nanolithography in addition being environment friendly and sustainable, and has a wide range of applications in both defence and civil areas. The new modular manufacturing approach will enable rapidly producing a large number of multivalent antibodies, leading to reduced costs and shortened development time, for many applications in disease diagnostics and therapeutics.
The technology may lead to applications in a range of industrial biotechnological areas that would benefit from the programmable advanced control of gene expression and cell behaviour, such as the life sciences research tools and reagents, microbe-based biologics bioproduction, in vivo bioprocess control and optimisation.
Hence the developed tools and technology will have wide impact in the synthetic biology and bioengineering communities by generating new scalable circuit design tools and methods, novel expanded sets of orthogonal regulatory components and innovative biomanufacuturing methods to assist the maturation of the emerging field of synthetic biology. The project will contribute to maintaining UK leadership in this strategic area and the national economical growth.
The work will be of extreme importance to researchers working at the interface of biology and engineering, and those in biotechnology industry. Potential patentable circuit designs and tools could be generated that might be of great interest to biotechnological sector and we will patent promising designs and technologies as they become available.
The project successful outcome will lead to a transformative enabling technology for synthetic biology and open up a wide range of innovative protein-based biomanufacturing applications in the biotechnology and biomedicine sectors.
People |
ORCID iD |
| Baojun Wang (Principal Investigator / Fellow) |
Publications
Belkin S
(2022)
Sense and sensibility: of synthetic biology and the redesign of bioreporter circuits.
in Microbial biotechnology
Chen SY
(2022)
De Novo Design of the ArsR Regulated Pars Promoter Enables a Highly Sensitive Whole-Cell Biosensor for Arsenic Contamination.
in Analytical chemistry
Chen XJ
(2021)
Rational Design and Characterization of Nitric Oxide Biosensors in E. coli Nissle 1917 and Mini SimCells.
in ACS synthetic biology
Fa-Arun J
(2023)
Tail-Engineered Phage P2 Enables Delivery of Antimicrobials into Multiple Gut Pathogens.
in ACS synthetic biology
Gale GAR
(2019)
Emerging Species and Genome Editing Tools: Future Prospects in Cyanobacterial Synthetic Biology.
in Microorganisms
Gale GAR
(2020)
Evaluation and Comparison of the Efficiency of Transcription Terminators in Different Cyanobacterial Species.
in Frontiers in microbiology
Hicks M
(2020)
Synthetic Biology Enables Programmable Cell-Based Biosensors.
in Chemphyschem : a European journal of chemical physics and physical chemistry
Hicks M
(2020)
Front Cover: Synthetic Biology Enables Programmable Cell-Based Biosensors (ChemPhysChem 2/2020)
in ChemPhysChem
Hicks M
(2020)
Synthetic Biology Enables Programmable Cell-Based Biosensors.
in Chemphyschem : a European journal of chemical physics and physical chemistry
| Description | In this project, we have developed a new expanded library of orthogonal split inteins that could aid achieving complex gene regulation control in living cells and engineering large scale gene regulatory circuits, as well as to assemble bespoke protein-based functional biomaterials. In addition, we have applied the developed tools to demonstrate pilot applications in environment (e.g. cell-based/cell-free biosensors) and biotechnology (e.g. metabolic pathway screening and optimisation). In particular, we would like to highlight below two representative works that resulted from this project and widely reported in public media in brief summaries. 1. Harnessing nature's protein glue could aid medicine and industry - A new protein production technique could speed up vaccine development and lead to high-tech synthetic fabrics and materials. Pinto F, Thornton E and Wang B, "An expanded library of orthogonal split inteins enables modular multi-peptide assemblies", Nature Communications, 2020, 11, 1529 (Reported by Edinburgh BioSciences News, Nature Research Bioengineering Blog and BioArtReports among others) Brief Summary: This study reports a chemical and synthetic biology enabling toolbox of 15 mutually orthogonal split inteins (protein glues), the largest reported to date, and their detailed biochemical characterisation in one single context. Moroever, we demonstrated new breakthrough applications of this toolbox in enabling complex genetic logic circuit design in living cells, and in vitro one-pot scarless assembly of large repetitive proteins with biotechnological relevance. This orthogonal split intein toolbox has the potential to be scalable tools for gene circuit design, protein engineering and biomaterial manufacturing applications. 2. DNA sponges - new tool for fine tuning cell engineering: Protein-binding DNA could be used to mop up proteins and fine-tune the control of gene expression. Wan X, Pinto F, Yu L and Wang B, "Synthetic protein-binding DNA sponge as a tool to tune gene expression and mitigate protein toxicity", Nature Communications, 2020, 11, 5961 (Featured by Nature Research Bioengineering Blog, BioArt, Zhihu-Regenesis and UoE SynthSys News among others) Brief Summary: This study reports new striking findings of using decoy protein-binding DNA sites named DNA sponge to modulate target gene expression. For the first time we showed that synthetic DNA sponges can be designed to systematically reshape the response profiles of gene circuits, lending multifaceted tuning capacities including reducing basal leakage, increasing system output amplitude and dynamic range, and mitigating host growth inhibition induced by burden-causing regulatory proteins. This work shows synthetic DNA sponge offers a simple yet generalizable route to engineer the performance of synthetic gene circuits, expanding the current toolkit for gene regulation with broad potential applications. |
| Exploitation Route | The library of genetic parts, building blocks and various synthetic biology tools generated in this project could be used in both scientific research in both academic community and industry including life sciences, biochemical production and biomedicine. For instance, the developed protein assembly tool orthogonal split inteins can be used to design large-scale gene regulatory circuits to control cellular behaviour, and to assemble bespoke functional biomatrials made of designer proteins, benefiting future translational research and potential commercialisation in the related sectors. |
| Sectors | Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| URL | https://www.ed.ac.uk/biology/news-events/news-2020/harnessing-nature-s-protein-glue-could-aid-medicin |
| Description | Notably, the synthetic biology enabling tools generated from this project have been filed for patent applications by the University of Edinburgh. The pending patented technologies could have a wide range of applications in biosensors and industrial biotechnology sectors. |
| Sector | Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Impact Types | Societal,Economic,Policy & public services |