INCORPORATION OF BACTERIAL QUORUM SENSING IN SYNTHETIC BIOLOGY

Abstract

The global objective of this research is to develop a synthetic biology toolkit consisting of molecules, cells, and devices that provide flexible, yet selective targeting, sensing, and switching capabilities, that in turn guide biological behavior in user-specified manner. We employ bacteria as "smart" programmable devices. We envision creating bacteria that autonomously move to specific areas, synthesize a drug, deliver the drug, and move on to new sites. "Targeting" endows bacterial cells the means to dock onto specific surfaces with antibody-antigen specificity. Sensing and switching capability allows bacteria to sense and, after making a "decision", respond by synthesizing and delivering cargo to molecular scale features displayed on target surfaces. Relevant surface features may include an overexpressed receptor on a tumor cell, glucagon-like peptide-1 receptor on pancreatic beta cells, or even other bacterial cells resident in a recalcitrant biofilm. Towards the realization of this goal, we employed an antibody-binding protein G display strategy to complex target-binding antibodies with bacteria. We characterized the assembly and efficacy of this complex by binding to well-defined surfaces decorated with specific antigens. For sensing and switching we made use of the genetic circuitry of bacterial quorum sensing (QS) that coordinates multicellular responses. In particular, we hypothesized the creation of a biological "switch" that would take action only after a certain threshold "feature" density had been detected. Specifically, in our most significant demonstration we designed and implemented QS based sensing and actuating based on the surface density of cancer-indicating EGFR receptors displayed on epithelial cancer cell lines. Because recent reports have demonstrated bacterial placement of a molecular "cargo" or "payload" in unrelated studies involving vaccination or direct attack on bacterial pathogens, we turned to developing innovative RNA-based drug syntheses concepts for eventual use in cancer therapy. That is, we designed RNA interfering (RNAi) technology to arrest the progression of the eukaryotic cell cycle by silencing gateway genes that serve to guide cell division and proliferation. Thus, our strategy serves to inhibit cell growth and promote cell death - actions that could find utility in treating metastatic cancer. Through a different lens, this same concept, the molecularly "programmed" manipulation of cell cycle status and cell growth via synthetic biology can serve to promote recombinant protein production in an industrially relevant eukaryotic insect cell line. In summary, we envision the exploitation of bacterial cells as programmable smart devices that can target, dock and deliver cargoes that are synthesized and delivered only after a set of predetermined parameters are met. We also envision a new biological "switch" that is based on the area-based density of a molecular feature - this will dramatically expand the capabilities and reach of synthetic biology. Our concepts embrace the notion that the individual cell may be the product of synthetic biology, as opposed to a synthesized molecule which is the prevailing product of choice.