Engineering Scalable Combinational Logic in Escherichia coli Using Zinc Finger Proteins

Permission to make digital or hard copies of all or part of this work for personal or classroom use is granted without fee provided that copies are not made or distributed for profit or commercial advantage and that copies bear this notice and the full citation on the first page. To copy otherwise, to republish, to post on servers or to redistribute to lists, requires prior specific permission. Available to synthetic biologists are a wide range of genetic devices. Many of these devices are able to either sense or alter local conditions. The ability to sense a multitude of inputs combined with diverse outputs could enable engineered organisms that interact with their environment in new and complex ways. Currently the complexity of such systems has been limited by our ability to integrate several inputs into a desired output. Simple combinational logic functions, containing 1 to 3 logic gates, have been constructed in Escherichia coli, but more complex logic networks are needed to fully exploit the opportunities presented by these sensors and actuators. Use of genetic logic gates is constrained by the specific molecular interactions that are used to implement each gate. These interactions involve diffusible molecules that can move within the cytoplasm of the cell and therefore are not spatially separated from other gates. To make larger logic blocks, sets of gates that use unique molecular interactions with minimal crosstalk are required. Zinc finger proteins (ZFPs) can be used to predictably create a large number of unique protein-DNA interactions. These proteins can then be used to build transcriptional activators or repressors in E. coli, but these methods are not well defined. Attempts at using ZFPs to make one-hybrid transcriptional activators have failed to give a fold activation of higher than 2. ZFP repressors based on steric hindrance of RNA polymerase performed better with fold repressions values of up to 300. The positional dependence of the ZFP operator site within the promoter was investigated, and both position and dissociation constant were found to play important roles in determining the level of repression. ZFP based repressors without cooperativity cannot be used to create logic gates. A new inverter topology using both ZFP based repressors and sRNA was designed. This topology uses a reference promoter to set the switching threshold of the gate. There are no cooperative interactions in this topology, but the maximum slope of the transfer function is similar to a …