Integrating Gene Synthesis and MITOMI for Rapid Protein Engineering

The research described within this thesis is primarily motivated by two fields of science with reversed, yet complementary, approaches to addressing the same essential objective: manipulating and understanding the complex program of life. Whereas synthetic biology has to do with the bottom-up construction of living systems from a library of âpartsâ, systems biology takes a top-down, reverse-engineering analysis of the same processes within functioning cells to elucidate the crucial elements and interactions. Both perspectives have led to important contributions in learning how biological systems operate. Discoveries from these studies can have far-reaching implications, notably in medicine, manufacturing, energy, and the environment. One exciting outcome of the research efforts within synthetic biology is the ability to design genetic constructs encoding proteins with novel, optimized functions. Although advances have been made in predicting how a proteinâs amino acid sequence relates to its higher order structural form and behavior, experimental methods for protein engineering remain invaluable for evaluating computationally derived designs. Unfortunately, the design-build-test cycle for rational protein development is commonly a time, labor and resource intensive endeavor. This is primarily due to limitations in the ability to generate libraries of gene variants and bottlenecks in cell-based expression and quantitative screening of protein products. This thesis describes the development of a solid-phase gene assembly technique and its integration with microfluidic protein analysis to accelerate the prototyping of novel protein designs. The gene assembly method utilizes commodity-scale, chemically manufactured DNA, and operates without the need for ligase or restriction enzymes. As a bench top process amenable to scale up, the modularity of the assembly process allows the efficient and economical generation of expression-ready gene variants. We directly coupled this gene assembly pipeline to a microfluidic device to enable high-throughput, on-chip, cell-free protein expression, purification, and characterization. By circumventing molecular cloning and cell-based steps, the lag time between protein design and quantitative analysis was dramatically reduced. As a proof of concept, this protein engineering platform was applied towards the construction of over 400 artificial, engineered variants of C2H2 zinc finger (ZF) proteins. ZF protein domains are the most prevalent form of transcription factor (TF) in humans, and are found throughout the tree of life. They provide a convenient structure for refactoring due to their relatively small size and composability, and are an ideal model for exploring the biophysics of TF-DNA specificity. The ability to engineer ZF proteins makes them useful as programmable, DNA-targeting units for applications in biotechnology and synthetic biology. We demonstrate that although ZFPs can be readily engineered to recognize a particular DNA target, engineering the precise binding energy landscape remains a challenge. Additionally, we show that ZF-DNA binding affinity can be tuned independently of sequence specificity. Together, these results demonstrate the versatility of the coupled gene assembly and microfluidic analysis platform as a new tool for rational protein engineering.