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

Project: Research project (funded)Research

Project Details


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).

Layman's description

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 stepchange 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.

Key findings

We have initiated a pipeline for the discovery and characterisation of novel integrases and their attachment sites.
Effective start/end date11/04/1331/03/19