Development of microfluidic tools and methods for the biochemical characterization of scFv and Cas9 affinity reagents and their applications in molecular detection

Recent progress in protein engineering, empowered by the rapidly evolving fields of synthetic biology and DNA synthesis, has enabled the generation of various custom-designed proteins. Their thorough analysis has led to applications in areas including medicine, biotechnology and basic research. However, despite increasing throughput in DNA synthesis, characterization of engineered proteins remains difficult. One of the main reasons is the lack of cost effective and scalable characterization technologies. High-throughput microfluidic platforms have the potential to overcome such hurdles. This thesis describes the development of various state-of-the-art methods in the fields of cell-free synthetic biology and high-throughput microfluidics, vastly improving the speed and cost for probing not only protein-protein, but also DNA-RNA-protein interactions. These methods and biochemical characterizations in turn allowed us to develop a cell-free bead-based scFv biosensor and apply Cas9 in a label-free single molecule detection method.

First, we used an integrated microfluidic platform coupled with an \textit{in vitro} cell-free transcription-translation (TXTL) system to characterize a small single chain variable fragment (scFv) library. The constructed scFvs retained their antigen specificity when expressed in commercially available TXTL. The high-throughput of the microfluidic platform allowed simultaneous screening and selection of scFvs with defined specificity and affinity, obviating tedious and time-consuming cell-handling and protein purification procedures. Owing to their versatility, scFvs have found applications in different biological domains such as structural and cell biology as well as diagnostic and therapeutic applications. Here, based on the scFv screening platform that was developed, we combined high affinity scFv binders with additional genetically-encoded functionalities. This approach allowed the construction of a cell-free bead-based biosensor with an integrated reporter gene construct for signal enhancement.

The microfluidic platform was used not only for protein-protein interaction profiling but also for studying complex protein machineries. We were able to reconstruct an \textit{in vitro} CRISPR-Cas9 system and determine absolute affinity values to a library of DNA targets. Better understanding of the dependencies for proper functionality of the \textit{de novo} synthesized system found application in a label-free single molecular detection method enabling effective charge estimation of the tested protein complexes. In addition to the \textit{in vitro} approaches used for biomolecular interaction characterization, we also developed a microfluidic biodisplay which senses small molecules.

Synthetic biologists have designed, synthesized, and fully characterized various types of gene encoded parts and devices that can be utilized to create new functions. However, their \textit{in vivo} characterization is often hampered by the lack of cheap biosafety technologies. The device described here allowed detection of low arsenite concentration in tap water using genetically modified \textit{E. coli} cells. Moreover, it enables environmental monitoring. To sum up, all these examples underscore how cell-free synthetic biology together with high-throughput microfluidic technology can facilitate the application of various protein engineering approaches.