Parts and methods in synthetic biology: An investigation of light induced gene regulation in E. coli and the design and characterization of a dual mode promoter in S. cerevisiae

Cellular processes are astonishing and complex but precise in regulation with great modularity which makes us wonder "how do they work?" or if it is possible to redesign them for our specific need. Synthetic biology is a field of research that combines molecular biology and engineering aspect to understand cellular processes as well as to redesign "biological devices" (e.g. feedback networks) or "parts"(e.g. promoters) for constructing controlled biological system. For this, synthetic biology needs resources such as libraries of biological parts and methods to develop those parts, and to validate them in targeted organisms. Here, one of the projects was to validate and develop an in vivo system for light-responsive transcriptional gene regulation. We implemented a blue light-responsive reporter system with conventional E. coli promoter design methods to validate DNA binding activity of six in vitro characterized variants of fusion photoactive yellow protein (PYP). The in vivo system design involved placing blue light induced PYP binding DNA sequences such that PYP would inhibit access of RNA polymerase to the promoter upon light exposure. Comparison of results of in vivo DNA binding activity via fluorescence of these six fusion PYP with the Kd value of in vitro DNA binding activity showed weak correlation. Development of this kind of in vivo screening method would narrow down the search for better light responsive DNA binding fusion PYP variants. The other project in this work was to develop a "dual-mode" promoter to enrich the synthetic biology library, with the motivation for the design arising from our ongoing effort to design and implement a feedback controller in S. cerevisiae. To design this promoter we started with the minimal cytochrome C promoter in yeast to incorporate five steroid hormone responsive elements and one lac operator site, respectively upstream and downstream of the TATA box. This allows activation through the testosterone-responsive androgen receptor, and repression through the LacI repressor. Exposure to varying concentrations of testosterone and IPTG demonstrated the functionality of the promoter's activating and repressing modes, and the ability to combine these effects to tune the promoter's output curve over a wide range.