Enabling synthetic biology with an expanded library of engineered orthogonal genetic logic gates and switches

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 and interchangeable parts to program cellular behaviour. However, unlike electronic digital circuits, the components in a biological circuit are not connected by wires with physical insulation, and the flow of biological information has to depend on their specific chemical interactions to avoid cross talk. As a result, the same genetic part may not be used twice in one integrated system to prevent the potential unintended interactions between them. Therefore, orthogonal parts and modules are necessary for the compatibility and scalable design of large gene circuits comprising many components. Orthogonality implies that the newly added parts and modules should not cross-talk with those present in the engineered biological systems as well as the host genetic background.

Most of the gene circuits constructed so far are small scale systems that have been constructed by costly and inefficient 'trial-and-error' methods with very limited parts. For example, it has taken almost 12 years to progress from the first 3-gene toggle switch to the so far largest constructed 11-gene 4-input AND logic gate in a single cell. A hard truth behind this slowness is that the engineering of complex circuits in living cells is currently limited by the availability of well-characterised and orthogonal (non cross-talk) genetic regulatory building blocks. Hence, an urgent need in synthetic biology is to expand the currently limited toolbox of biological parts with many functional orthogonal elements to scale up our capacity for building large and complex circuits.

Nevertheless, it remains a big foundational challenge to expand the range of available orthogonal components in the synthetic biology toolbox. This project aims to address this challenge by developing two novel scalable tools to engineer an expanded library of versatile orthogonal genetic building blocks. In particular, we will build a library of modular and orthogonal genetic NAND and NOR logic gates; these are universal logic gates and their combinations can be used to accomplish any arbitrary complex Boolean logic operations, providing a powerful scalable method for cellular process control. Further, we will create multi-layer genetic programs from different permutations of these engineered logic gates to demonstrate the potential for composing high-order signal processing and transcriptional control functions in a single cell. For example, the engineered genetic programs will be used to implement a high level logic computing device - 1 bit full adder that intake three chemical inputs in specified logic manners to produce two optical outputs. In addition, we will demonstrate that large complex transcriptional control programs can be implemented in a microbial cell factory to precisely and rapidly tune gene expression profiles within the biosynthesis pathway of a high value chemical (violacein).

The engineered scalable tools from this study will increase significantly the number of orthogonal control elements, gates and wires in the limited toolbox of synthetic biology, leading to large-scale complex genetic control programs attainable to program advanced behaviours in cells. The successful outcome will lead to a number of applications expected in the biotechnology industry (high gain), and will be of enormous benefit to researchers not only in the synthetic biology and but also in bioengineering communities and those in the biotechnology industry.

Technical Summary

The project aims to engineer an expanded library of versatile orthogonal genetic building blocks to enable advanced cellular signal processing capability needed to fulfil the potential of synthetic biology. To achieve this, we will focus on repurposing two new scalable genetic tools, split inteins and CRISPR RNA-guided gene regulation, to engineer libraries of orthogonal genetic logic gates and switches. We will also build multi-layered genetic programs from these orthogonal blocks to implement large-scale complex transcriptional control functions in a single cell. First, an expanded library of orthogonal NAND, AND gates will be built based on trans-splicing inteins of transcriptional repressors and activators. A number of candidate orthogonal transcription activators, repressors and split inteins will be mined and characterised from both reported literature and bioinformatic search. By splitting a transcriptional repressor or activator, with each split intein controlled by a separate promoter, a NAND or AND gate is formed. Next, a library of XOR gates will be generated via intein-split orthogonal sigma and anti-sigma factors. We will then apply model-guided construction to engineer the largest ever logic computing circuit- 1-bit full adder that comprises 5 genetic logic gates wired in 3 layers. Second, orthogonal NOR gates and cascaded programs will be engineered via CRISPR-dCas9 regulated looping of bacterial enhancer-dependent promoters. A library of orthogonal NOR gates with two distinct input small guide RNAs will be obtained via directing catalytic inactive dCas9 protein to the unbound loop regions of synthetic s54-dependent promoters, leading to interruption of transcription required DNA looping induced by enhancer binding proteins. Next, complex programs comprising multi-layered NOR gates will be designed to precisely and rapidly tune gene expression profiles in the violacein pathway in response to external chemical inputs under various logic combinations.

Planned Impact

The proposed research falls into the BBSRC strategic priority areas of Synthetic Biology and Developing New Technologies for the Biosciences, which hold great promise for applications in sectors such as industrial biotechnology, the environment and healthcare, and are strategically important sectors for the economic future of the UK.

As referred in the UK Synthetic Biology Roadmap, a recent assessment performed by BCC Research Inc. predicts the scale of global market of synthetic biology, will grow from $1.6 bn in 2011 up to $10.8 bn by the year 2016. To achieve this economic goal and its growth momentum, new enabling tools and technologies have to be developed to facilitate the design and fabrication of large scale genetic programs with complex functions in a modular and scalable manner.

This project aims to develop a new generation of synthetic biology tools for enabling programmable and scalable complex gene expression and signal processing control capacity in model E. coli chassis microorganism. The engineered scalable tools will significantly expand the currently limited toolkit available for synthetic biologists and bioengineers to advance the engineering of large genetic control systems with complex functions. The successful outcome will transform the present state of gene network engineering by directly addressing a key bottleneck in the field.

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, and cell-based therapy sectors.

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 information processing modules 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.
 
Description In this project, we have developed new enabling synthetic biology tools including novel bacterial CRISPR gene activation tool and an expanded library of orthogonal split inteins that could aid achieving complex gene regulation control in living cells and engineering large scale gene regulatory circuits. 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. A bacterial programming breakthrough - a new efficient programmable gene activation tool in bacteria:
Liu Y, Wan X and Wang B*, "Engineered CRISPRa enables programmable eukaryote-like gene activation in bacteria", Nature Communications, 2019, 10:3693 doi (Reported by The National, The National Tribune, PhysOrg, Science Daily, Drug Target Review, Weatherherald, BioArt, Zhihu-Regenesis, Edinburgh University News and UoE SynthSys News among others)
Brief summary: The study reports the engineering of a new eukaryote-like programmable CRISPR gene activation system in bacteria, supporting long distance and multi-input regulation with ultrahigh dynamic ranges compared to previous programmable gene activation tools available in bacteria . The study has significant economic and societal impact by providing a powerful and versatile synthetic biology tool to aid studying diverse disease related genes and to increase biochemical production yields in microbial cell factories.

2. A low-cost, easy-to-use biosensor to meet a unmet global health challenge - the arsenic contamination that affects 140 million people:
Wan X, Volpetti F, Petrova E, French C, Maerkel SJ and Wang B*, "Cascaded amplifying circuits enable ultrasensitive cellular sensors for toxic metals", Nature Chemical Biology, 2019, 15(5):540-548 doi (Reported by Yahoo News, E&T Magazine, PhysOrg, Science Daily, BioArt, Edinburgh University News and UoE Biological Sciences News among others)
Brief summary: This work reports innovative methods of improving cell-based biosensors to meet their field application requirements using cascaded transcriptional signal amplifying circuits and microbial sensor cell arrays. The low-cost, portable sensor technology has been demonstrated by detecting arsenic contamination in groundwater samples from local villages in Bangladesh to generate easy-to-interpret output patterns captured by a phone camera, leading to wide interests among media and the general public with significant societal and economic impact for improving the public health by addressing an unmet global health challenge.
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 gene regulation tools CRISPRa and orthogonal split inteins can be used to control multipe gene exprssion in metabolic pathways, and to design large scale gene regulatory circuits to control cellular behaviour. In particular, the novel CRISPRa tool generated has been patented by us for future translational research and potential commercialisation in the related sectors.
Sectors Environment,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description The related project findings have been used to address a highlighted global health challenge in developing countries - to create portable, cost effective, point-of-care biosensors for contaminants and pathogens for example in drinking water. We visited Bangladesh and India for a week between March-April 2017 to engage with local end users and governmental collaborators regarding our proposed new paper-based biosensors for arsenic groundwater contamination. Notably, two 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 Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic,Policy & public services

 
Description Programmable single cell biocomputers with scalable computing capacity
Amount £113,000 (GBP)
Organisation Microsoft Research 
Department Microsoft Research Cambridge
Sector Private
Country United Kingdom
Start 10/2017 
End 09/2021
 
Description A UK-China partnership to scale up gene circuit design 
Organisation Chinese Academy of Sciences
Department Institute of Microbiology
Country China 
Sector Learned Society 
PI Contribution We will provide a test platform and system to verify a new method to digitize genetic switching response and demonstrate the generality of the method.
Collaborator Contribution The China Partner (Prof Chunbo Lou group) at IoM of CAS will contribute their expertise and materials on a new method to digitize genetic switch response that can be used to create more digital-like genetic logic gates and assembed large scale gene circuits.
Impact The collaboration is multi-disciplinary including microbiology, synthetic biology, bioengineering, molecular biology and biochemistry. We have submitted a joint journal publication on the method proposed.
Start Year 2016
 
Description Programmable single cell biocomputers with scalable computing capacity 
Organisation Microsoft Research
Department Microsoft Research Cambridge
Country United Kingdom 
Sector Private 
PI Contribution I and my team contribute our time, expertise and extensive lab resources to the collaborative project. For example, I was invited to visit Microsoft Research Cambridge Lab in September 2017 and gave a research seminar talk and held individual meetings with their biological computation group members to exchange the research progress on the collaborative project and other areas in large.
Collaborator Contribution Microsoft research lab Cambridge has provided the studentship funding to support a four-year phd student to work on the projects, and provide a supervisor to work together with us on the project. The partner is supposed to provide some in-kind bespoke software, tools and their own expertise to the project.
Impact As part of this effort, Microsoft has funded a graduate student in Baojun's lab to engineer and compile a library of versatile orthogonal (i.e. those that won't cross-react with the cell's native genetic components) genetic building blocks. These will then be used to program advanced signal processing capabilities in live bacterial cells - something that to date has proven very challenging. Microsoft provides bespoke software tools and cutting edge modelling expertise to the team realising its ambition to design a biological computer that could contain dozens or hundreds of biological computing blocks. Dr Neil Dalchau, of the Biological Computation group at Microsoft Research Cambridge, says: "This strategic partnership will combine our strengths in computational modelling and Edinburgh's expertise in synthetic biology to push the frontier of biocomputation, scaling up the presently limited computational capacity of biological machines. The resulting benefits could be enormous with applications as varied as tools for in vivo diagnosis and treatment of diseases, and enhancing high-value biomolecule production in microbial bioreactors."
Start Year 2018