The MIT-Broad Foundry: TA-2 (Phase III)

This is for Phase III Synopsis: Two critical bottlenecks in our capacity to rationally and predictably engineer biological systems are: (i) the limited number of well-characterized genetic elements from which to build (particularly for non-model, difficult organism hosts), and (ii) quality control of genetic constructs once built. Here, we will address these limitations by developing a rapid, high-throughput emulsion-based in vitro pipeline for prototyping genetic construct performance. We will identify robust, reliable genetic elements (e.g., promoter function, terminator strength, regulatory control, gene expression, etc.) prior to putting them in hosts. Uniquely, we will combine state-of-the-art cell-free protein synthesis (CFPS) systems, droplet based microfluidics, and novel methods in single droplet sequencing for measuring transcripts and proteins. Work to be performed: Specifically, Jewett’s team will enable the aforementioned effort by contributing to the following tasks: - Demonstration of the capacity to synthesize 50μg/mL (mg/L) sfGFP, or similar reporter, in emulsions suitable for droplet based microfluidics from 5 different CFPS platforms. - Demonstration of a microfluidic system for screening expression of construct libraries using CFPS (completed in collaboration with researchers from the MIT-Broad Foundry – the microfluidics will occur within the Foundry). Deliverables: We will: - provide experimental protocols for optimized CFPS systems and optimization, and - provide technical support to the Foundry for transfer of extract-making protocols.