Photo-controlled gene expression in E.coli and characterization of a synthetic dual-mode promoter in S.cerevisiae.

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Mostafizur Mazumder, Bill Kim, Jordan Ang, G. Andrew Woolley, David McMillen

University of Toronto, Canada

Synthetic biology, an emerging field of research combining molecular biology and engineering to design and characterize novel biological networks, is constantly in need of new biological “parts” for its increasingly complex devices. To suit the quantitative nature of the field, it is imperative that these parts be robust and well characterized. One class of parts consists of elements involved in transcriptional control, and here we present two examples: an optically-controllable protein and a novel engineered promoter that can be separately activated and repressed. (1) Synthetic biology to date has worked with a limited set of chemically inducible and repressible promoters to design its networks. Placing gene expression under optical control is thus an attractive prospect: it provides a simple method of altering the internal behaviour of cells without the need for time-varying extracellular inducers; and it offers a new set of control mechanisms to be used in complex synthetic network designs. The Woolley group has engineered a fused protein system that displays light-dependant DNA-binding activity in vitro. By incorporating the target DNA sequence into a modified promoter, we have designed and characterized a promoter system that can be regulated using blue light in E.Coli.(2) Yeast is an attractive model organism for the design and testing of biological “parts” for synthetic biology, and parts with multiple functions are helpful in tuning complex networks. In particular, there is a need for “dual-mode” promoters, able to be activated by one input and repressed by another, allowing specific types of feedback (integral controller) to be achieved. We have designed and characterized a synthetic promoter, which contains androgen receptor binding elements upstream and lac operator site downstream of minimal promoter cytochrome C. The promoter regulation was measured by expressing green fluorescent protein in yeast.