A DNA Synthesis and Construction Foundry for Synthetic Biology @ Imperial College

Synthetic biology is an emerging field that brings together biological scientists and engineers with the aim of developing new systematic ways to build and design biological systems and cells for useful purposes. Biological cells can carry out a vast array of functions when driven by instructions. These instructions are encoded by DNA much like a computer is controlled by software. By analogy, the DNA in a cell can be considered as the cell's software or operating system - containing the genetic instruction set (genome) for different types of cells and organisms. The code of DNA is composed of four complementary chemical building blocks called nucleotide bases (G, C, A and T) linked together in a sequence and these building blocks pair up specifically (G-C and A-T) such that the two interwoven strands of DNA forms a helical repeating structure. The beauty of DNA is such that a single strand can act as a template for the other strand so that it can be easily copied and replicated.

How is the DNA code decoded by the cell? The instructions encoded within DNA are decoded by two biochemical processes called transcription and translation. Transcription results in one strand of DNA being copied to produce an intermediary messenger molecule (mRNA) and translation results in the production of proteins coded for by the mRNA with each 3 nucleotide unit (codon) specifying a particular amino acid which is the basic building block for proteins. There are 20 different amino acid building blocks and depending on the sequence of the DNA, which directs which amino acids are linked together, fold into complex 3D structures that perform specific functions. Proteins are, in essence, the cellular machines that carry out all of the necessary chemical reactions for life, e.g. the conversion of food sources like sugar into energy. Over the last 20 years technological advances have resulted in the routine ability to 'read' the DNA code - deciphering, essentially, the genetic instruction sets (proteins) for every major living organism on Earth. These DNA sequences can run into many hundreds of thousands of base units written as GCATGCCCTTTAGCTA etc. They encode the basic code to make proteins for a specific organism. More recent technology advances have now resulted in our ability to chemically synthesise DNA or 'write' DNA in a test tube.

Synthetic biology aims to establish a proper systematic engineering framework that will allow researchers to design and write DNA tailored to specific human-defined applications, such that these new synthetic DNA sequences can be inserted into laboratory cells to perform specific functions. The aim of this proposal is to make the 'writing' of DNA streamlined and more automated at an industrial scale - such that tens of thousands of designed DNA constructions can be build and tested routinely as engineers would prototype a design. To do this we will establish a DNA synthesis and construction foundry that will comprise a number of robotic and lab-based instruments that will allow the design and testing of synthetic biology constructions to be carried out routinely and in a safe way under a controlled laboratory environment.

The DNA synthesis and construction foundry will establish streamlined workflows and experimental platforms that will enable DNA synthesis, assembly and verification. The foundry will have four main components:

DNA/Gene synthesis platform will establish new innovative protocols that utilise chip-based oligos for initial part synthesis including software development for parallel design of 1000s of oligos. Off-chip assembly will be performed by emulsion PCR assembly of oligos to dsDNA fragments and droplets will be characterised using in vitro transcription and/or translation assays (utilising qPCR and flow cytometry) in order to rapidly screen 1000s of draft parts ahead of selection for further assembly.

Gene and Genome assembly platform will apply standardisation combined with parallel and combinatorial assembly based on our MODAL and BASIC assembly protocols. Assembly reactions will be automated in vitro for 1-20kb, with larger chromosome assembly semi-automated in vivo. The platform will be designed to be scalable such that 1000s of 10 to 200 kb DNA constructs can be assembled per month.

Assembly verification platform will implement 3 approaches (i) high-throughput sequencing; (ii) mass-digest screening and/or colony PCR (iii) functional screening using fluorescence and/or mass spectrometry. We will establish a DNA sequencing platform to confirm large DNA sequences (genome-level construction) and use colony PCR, automated DNA digestion and automated capillary electrophoresis to confirm small-to-mid-sized DNA sequences (genes and pathways).

IT infrastructure platform will include the implementation of a LIMS (laboratory information management system) and other software to manage complex synthesis and assembly workflows. Integrated within this will be a bar coding system for archival and retrieval of physical DNA parts within the various workflows. Associated software for data analysis and integration will also be developed.

The British Government clearly recognises the importance of synthetic biology in relation to the UK's economic development. The field has been identified as one of the Government's "8 Great Technologies" and this has been endorsed by The World Economic Forum. Synthetic biology is a classic example of a knowledge-based industrial area which is leveraging the UKs science and engineering base. Effective industrial translation is, therefore, a key element in the development of new processes and industries. Standardisation is central to industrial translation and industrial processes. Consequently, an important component of this process in synthetic biology is the handling, assembly and verification of DNA components of various lengths - and in the context of various cellular hosts. The Proposal is based on the concept of developing systematic Workflow (i.e. a system comprising molecular biology hardware and software) which utilises the design-test-build paradigm to produce standardised, verified sections of DNA up to the chromosomal level. We propose to develop the Workflow so that it is a high throughput system capable of rapidly producing 1000s of sections of DNA (parts) which are fully verified. The impact of this will be to create a foundry which reliably produces DNA according to human design. When the DNA is placed in a particular, designated host the cell creates the required device, pathway or system. An important aspect of the proposed Workflow is that it will employ a high level laboratory robots and automation controlled by software, in the context of an integrated system. Because the Workflow will comprise the integration of industry-standard hardware and software, when fully implemented and tested it will be possible to replicate the system at multiple sites around the UK.

More generally, the proposed Workflow will add major value to the processing and manipulation of DNA at scale, with the ability to undertake rapid prototyping. This is particularly important in the optimisation of genetic circuit designs where the individual sections of the circuit and their order (e.g. in the plasmid) need to be optimised. Whilst it is possible to design systems, increasingly with the assistance of BioCAD tools, the rapid testing of variants of the design will continue to be important. This will be readily achieved with the proposed system. The Workflow will enable the UK to have fundamental capability in the synthesis of DNA and the associated technological platforms. The Workflow is designed to not only synthesise and assemble DNA using in-house methodology, but, also, to accept sections of DNA created, using a range of industrial methods, from third party sources (e.g. using oligo chip technology) and to readily accommodate new methodology as it develops. This strategy avoids major investment in a range of synthesis techniques, whist exploiting the major added value in the areas of assembly, construction and verification. The Workflow facility will also be available to other UK groups and companies, particularly those working on collaborative projects (e.g. in the IKC) as well as acting as a training hub. This is seen as being important for UK start-ups and SMEs for whom the Workflow will be essential, but who do not have the expertise or the resources to establish their own facility.

In summary, we believe that the Workflow which we are proposing, is strategically important for the industrial translation of synthetic biology and the development of UK industry in the field. Our strategy is to create and implement a Workflow system, which will enable the UK to have significant capability and expertise in DNA synthesis, assembly and verification now and in the longer term.