Reliable gene expression and assembly for synthetic biological devices in E. coli through customized promoter insulator elements and automated DNA assembly
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Building reliable genetic devices in synthetic biology is still a major challenge despite the various advances that have been made in the field since its inception. In principle, genetic devices with matching input and output expression levels can be assembled from well-characterized genetic parts. In practice, a priori genetic circuit design continues to be difficult in synthetic biology due to the lack of foundational work in this area. Currently, a successful genetic device is typically created by manually building and testing many combinatorial variants of the target device and then picking the best one. While this process is slow and error-prone, as synthetic genetic devices grow in complexity, this approach also becomes unmanageable and impractical. Fluctuations in genetic context have been identified as a major cause of rational genetic circuit design failures. Promoter elements often behave unpredictably as they are moved from the context in which they were originally characterized. Thus, the ordered location of parts in a synthetic device impacts expected performance. Synthetic spacer DNA sequences have been reported to successfully buffer promoters from their neighboring DNA sequence but design rules for these sequences are lacking. I address this problem with a novel method based on a randomized insulator library. I have developed a high-throughput, flow cytometry-based screen that randomly samples from a library of 4^36 potential insulators created in a single cloning step. This method provides precise control over genetic circuit expression. I further show that insulating the promoters in a genetic NOT-gate improves circuit performance and nearly eliminates the effect of the order in which the promoters are organized in the device. This foundational work will help improve the design of reliable genetic devices in E. coli. Finally, automated DNA assembly using liquid-handling robots can help increase the speed at which combinatorial synthetic device variants are assembled. However, these systems require significant investment in optimizing the handling parameters for handling very small volumes of the various liquids in DNA assembly protocols. I have optimized and validated these liquid-handling parameters on the Tecan EVO liquid handling robotic platform. These materials have been made available to the larger community.