"Exploiting the syntegron technology platform for assembly and optimisation of complex genetic ensembles"

Previously we developed an innovative, versatile technology for enzymatically assembling and dynamically rearranging DNA modules. This "Syntegron" platform enables rapid assembly of multiple standardized DNA modules into large assemblies such as metabolic pathways and to exchange individual parts of assemblies (e.g., regulatory elements) to allow variation and optimization. We propose to build on these core technologies to develop a comprehensive experimental and computational toolkit enabling the rapid generation of important biomolecules:
Objective 1: Develop Syntegron "parts" enabling the discovery and production of diverse, valuable biomolecules. An important source for drug leads has been plant natural products but many of these drugs are produced in miniscule amounts in their native hosts, making the drugs expensive and environmentally taxing to harvest. Organic chemistry methodologies have been widely used to synthesize pharmaceuticals but many natural pharmaceutically-relevant scaffolds cannot be achieved by these methods. An alternative is the use of enzymes to enable the production of drug candidates from inexpensive, green starting materials. The goal of this research is to synthesize a variety of natural product variants e.g. terpenes using the Syntegron technology platform constructed in the first phase of funding, and to manufacture these natural product variants for drug discovery applications. We will 1) Take advantage of the recently described phenomena of plant metabolic gene clusters to identify genes encoding enzymes involved in plant secondary metabolite pathways via the development of novel bioinformatic and data mining tools. 2) Use the Syntegron platform to assemble the synthetic metabolic pathways for natural products e.g. the triterpenes and enzymes that will decorate them with functional groups to synthesize a variety of natural product variants. 3) Develop new Syntegron host strains for metabolic engineering 4) Engineer in vivo biosensors for natural products and their variants and use these sensors in high throughput screens allowing dynamic optimization of metabolic pathways.
Objective 2: Develop analytical tools, methods, and conceptual insights enabling the optimisation of diverse multigenic functions using Syntegron technology. Cell-free technology is useful for decoupling intracellular biochemical transformations from confounding experimental factors including cellular toxicity and mass transfer limitations. This has proven useful for quantitatively characterizing fundamental synthetic biology "parts". The relationship between cell-free parts characterization and performance in vivo remains unclear, and remains a fundamentally important scientific question. We propose to extend cell-free approaches to the high-throughput characterization of multi-genic assemblies constructed using the Syntegron platform, and to compare them with performance in vivo. The development of the Syntegron platform into a true technology for the synthetic biology community requires the development of novel computational modelling frameworks and quantitative tools for analysing and performing Syntegron-based directed evolution. We will therefore 1) develop a quantitative computational modelling framework describing Syntegron-based diversification and selection. 2) Optimise Syntegron-based directed evolution using in silico simulations paired with experiments. The ultimate test of the combined experimental and computational Syntegron platform will be to perform, analyse, and ultimately guide Syntegron-based directed evolution of a model metabolic pathway. We will initially pair in silico simulation with computational optimization to explore the influence of tuneable experimental parameters on the predicted chances of (a) generating sufficient genetic diversity to sample many potentially functional syntegron configurations, and (b) successfully selecting for variants that exhibit optimised biosynthesis.

This project will provide new scientific advances and state of the art techniques in: a) Tools for rapid strain improvement and pathway engineering. b) Synthetic engineered organisms for efficient production of high-value products. c) A novel synthetic biology toolbox of genes, regulatory elements and vectors which will be made freely available to the Synthetic Biology community for future research via Open Wetware.
We will fully engage with the Knowledge Transfer Networks and invite a member of the Bioscience for Business KTN to an annual Project Management. The impact on academic beneficiaries will be realized by communicating the outputs from this project through regular publication in high-impact peer-reviewed journals, at conference talks.
The strength of the Syntegron Platform is that it is a generic technology for Synthetic Biology applications. We will seek interest from companies in application areas such as, Pharmaceuticals, Industrial chemicals and Agriculture via research collaborations and access to IP and know-how via licensing deals. We anticipate that the technology developed here could form the basis for a start-up company. To do so, all PI's will disclose any technology to their institution's intellectual property offices, which will file patents on the technologies. These will then be proactively licensed to either large companies or to small, venture-backed start-ups.
An exciting aspect of Synthetic Biology is that it encourages collaboration and multidisciplinary approaches to research, and it is important that the community develops people who want to engage with these diverse ideas and technologies. This project will provide an excellent training experience for the PDRAs employed on the grant and associated PhD studentships. The multidisciplinary collaborations will help them expand their scientific knowledge and experience. Each of the partner institutions have extensive staff development programmes which the PDRAs will be encouraged to make use of.
The PIs are actively involved in a number of outreach programmes, and have delivered talks to diverse audiences including schools, youth organizations such as the cub scouts, lawyers, political parties, journalists and the general public about our research. We plan to continue these outreach activities and look for new opportunities including participating in activities with the SAW trust. The Science Art and Writing (SAW) Trust (see www.sawtrust.org) established in 2005 has a strong track record for taking real research topics into communities in accessible and inclusive ways. Exploring topics through practical science, creative writing and visual arts enables individuals with diverse learning styles and interests to participate in activities that inform on directions of scientific investigations and their relevance to society. The SAW Trust, working with the Syntegron consortium over 3 years would provide training to scientists from each of the 6 member labs in the design of novel projects working with local artists and writers that are delivered locally to engage communities with research into engineering biological systems.
Sponsored by the Joint BioEnergy Institute (JBEI) and the Synthetic Biology Engineering Research Center, Jay Keasling and colleagues sponsor approximately 10 high school students each year at JBEI to learn basic synthetic biology. These students come from economically disadvantaged families in Richmond and Oakland, CA. The students learn about synthetic biology in a laboratory setting over a 10-week period. In addition, they get help with college preparation, including their college essay. After five years of this program, nearly all students choose to go to college. This year a student from our first class graduated from the University of California, Berkeley, the premier public education institution in the US. We will continue to work with these students through this funding.