Creating Components for Synthetic Gene Networks

The ability to implement multi-input gene regulation has been a limiting factor in the construction of synthetic gene networks. This limitation becomes more prescient as researchers seek to go beyond simple synthetic gene networks. In this study we develop methods for multi-input transcriptional activation and repression. For multi-input transcriptional activation, we split T7 RNA polymerase (RNAP) to create a two input transcriptional AND gate whereby gene expression is observed if and only both fragments of the split protein are present. We then modify the specificity of split T7 RNAP to create functionally orthogonal split T7 ORFs. For multi-input  transcriptional repression, we co-express multiple lacI/galR chimeric repressors whereby each chimeric repressor regulates an operator site dictated by its DNA binding domain according to inducer sensed by its ligand binding domain. Co-expressing chimeric repressors enmasse creates multi-input regulatory networks such as a four input transcriptional AND gate or even two functionally orthogonal transcriptional AND gates. Altogether our work demonstrates strategies for engineering multi-input transcriptional regulation. These tools will enable the construction of the next generation of synthetic gene networks.