Engineering multi-input gene regulation for applications in Synthetic Biology

Abstract

Synthetic biology offers insight into molecular biology through the design and implementation of synthetic gene networks. One challenge in this effort is implementing transcriptional logic gates that enable synthetic gene networks to make decisions based on multiple inputs. However, the ability to implement transcriptional logic gates is inhibited by a lack of parts available to build them. In this work, we explore strategies for facilitating multi-input gene regulation in prokaryotes. That is, we develop methods for making the expression of a reporter gene dependent on two or more inputs in Escherichia coli. We first demonstrate how fragmentation of T7 RNA Polymerase (T7 RNAP) creates a multi-fragment transcription complex that facilitates AND transcriptional logic. We find split T7 RNAP to be functional in vivo and that both fragments of the split protein must be present for transcription from the T7 Promoter, PT7, to occur. We also find that the specificity of the split protein can be modified to create split protein mutants with orthogonal specificity. In addition to split T7 RNAP, we test the AND transcriptional logic made possible by co-expressing multiple chimeric LacI/GalR transcriptional repressors. We find that each chimeric repressor regulates the operator site of its DNA binding domain (DBD) according to the ligand sensed by its ligand binding domain(LBD). By co-expressing multiple chimeric repressors, we find each repressor independently regulates its DBD's operator. As a result, the number of inputs at a promoter relates directly to the number of species of chimeric repressors with the same DBD. Further, by modifying the DBD we find that we can create chimeras with orthogonal specificities that facilitate an orthogonal open reading frame. We find expression of our chimeric repressors en mass facilitates regulation such as a four-input transcriptional AND gate or two orthogonal transcriptional AND gates. Split T7 RNAP and the coexpression of chimeric LacI/GalR repressors both demonstrate strategies for multi-input gene regulation in prokaryotes. This work also suggest strategies for the engineering of additional components for use in synthetic gene networks.