Team:Berkeley/Project

Traditionally, many sensing systems have been borrowed from nature. Biologists find and characterize ligand-sensing proteins in natural organisms, and then apply these proteins in their synthetic constructs. However, when researchers want to detect a ligand that does not have a receptor on the existing list of characterized sensors, using this method of finding a specific sensor is difficult and time-consuming, when it is not impossible. Moreover, as synthetic biology moves realizes more and more unnatural functions, we will want to be able to design cells that can detect molecules that life has never before responded to. This is something we cannot achieve using natural receptors.

Here at Berkeley we were motivated to construct a modular biosensing system that could be readily adapted to the ligand of choice. We wanted to build a system that could be modified through the exchange of binding domains. This would provide a standardized method of building biosensors with both natural binding domains that evolution has optimized and engineered domains that can respond to molecules outside the range of natural receptors.

Dimerization of ToxR has been found to activate the ctx promoter in Vibrio cholerae by binding directly to the DNA. DiRita et. al. (1991). The periplasmic domain detects changes in the environment and facilitates the dimerization of the cytoplasmic domain, which activates transcription. These domains are all on one peptide, so signals can be quickly relayed from the periplasm to the cytoplasm. This unique aspect of ToxR makes it a good part for building a biosensor. We aim to make a biosensor by utilizing this dimerization-dependent transcriptional activation feature of the ToxR system.

We hypothesized that we could create a biosensor if we attach ligand dependent dimerizing proteins to the cytoplasmic domain of ToxR. The fused protein will dimerize only in the presense of a particular ligand. This will cause ToxR to dimerize and activate the ctx promoter. By putting GFP after the ctx promoter, we can then detect if the ctx promoter is activated.

Our initial step was to validate that a ToxR based two-hybrid system could be expressed in E. Coli. We first truncated ToxR to eliminate the periplasmic domain because it is only the dimerization of the cytoplasmic domain that controls transcription. Next, we attached the constitutively dimerizing proteins IILK to these ToxR truncates such that the constitutively dimerizing proteins were located in the periplasm. IILK is a leucine zipper that is well characterized to be a constitutive dimer. However, the initial ToxR chimeras under the pBad promoter were toxic to the cell and could not be expressed. Even at low concentrations of arabinose, there was high toxicity and cells did not grow well.

In order to resolve the toxicity of ToxR, we constructed a new design principle. Stress is a natural regulatory cue in bacteria that dictates changes in behavior or expression levels. We decided to look into the E. coli genome for any type of stress based down regulatory elements. Toxic over expression of ToxR would be down regulated with this negative feedback system. With this method, we are able to express ToxR chimeras at optimal levels without it being toxic to the cell.