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.

ToxR elegantly allows Vibrio cholerae rapid sensing and the activation of pathogenic functions. Virulence factors such as those controlled by this system are often clustered in the genome within pathogenicity islands, remnants from a past horizontal gene transfer. The simplicity and orthogonality of the systems contained in these small virulence cassettes make them an ideal source for modular biosynthetic tools. Indeed, one of the reasons ToxR is so interesting is that it single handedly achieves the task of the standard two-component signal transduction pathway: it is activated in the periplasm and directly promotes transcription in the cytoplasm. The simplicity of this regulation system and the scope of this single protein make it an ideal starting point for synthetic design.

Stress is a common regulatory cue that allows organisms to adjust to variable conditions in the environment. As a result, stress responsive regulatory elements are prevalent motifs in nature. Due to the toxicity caused by the toxR chimeras in E. coli, a method of regulating toxR-IILK expression to produce the maximum amount of toxR-IILK without killing the cell was needed. We hypothesized that the E. coli genome likely contains such stress responsive regulatory systems from which regulatory feedback systems can be constructed. Expression of the toxR chimera to toxic levels by stress promoters can be downregulated by stress in a feedback loop. This maintains a high but nonlethal level of expression of the toxic gene.

In order to construct a feedback system, we analyzed microarray data of the E. coli genome for regulatory elements downregulated by stress. Moen, et. al. (2009) subjected E. coli to generalized stresses and measured the global mRNA levels of all genome elements for stress response. From their data, we identified 35 stress promoters that were downregulated under all stress conditions tested for further characterization. We isolated the region of the genome upstream of the ORF through PCR and placed the resulting pool of 35 stress promoters in front of GFP. The construct was transformed into E. coli and subjected to general stress conditions. We then measured the fluorescence level and looked for decreased fluorescence. While not all promoters responded by downregulating GFP expression, a subset did respond, with the cold condition yielding the best data. The fluorescence measurements from the Tecan plate reader were confirmed by flow cytometry.

Since a subset of the stress promoters did display the desired behavior, we proceeded to incorporate them into a stress response system with toxR. In our stress feedback system, toxic and constitutively dimerizing toxR-IILK both activates Pctx, driving GFP production, and creates cellular stress. The stress promoter responds to stress by downregulating toxR-IILK expression to a nonlethal level, allowing the cell to maintain a high level of toxR chimera expression without toxicity.

In order to ensure nonlethal expression of the toxR chimera, we created an rbs library for each of the 35 stress promoters to construct a promoter-rbs library in front of toxR-IILK. We transformed the plasmid into cells containing Pctx.GFP and grew them up to assay the efficacy of the stress responsive feedback system. In our growth assay, we looked for large, healthy colonies as well as strong fluorescence, indicators of robust growth and desired expression levels.

Dimerization of ToxR has been found to activate the ctx promoter in Vibrio cholerae by binding directly to the DNA.(insert source) Fusion proteins or ToxR-based two-hybrid systems have previously been made for the detection of periplasmic and cytoplasmic protein-protein interactions in E. coli. (insert source) These two-hybrid systems were simply made to determine if two proteins interacted. There is a periplasmic domain that detects changes in the environment and a cytoplasmic domain that 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. 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.

PICTURE OF DEAD CELLS. :(

This high toxicity is likely due to stress that overexpression of these transmembrane proteins cause on the cell membrane. To address this toxicity, we looked at microarray data and screened for stress promoters that showed downregulation upon stresses such as cold temperatures. The promoter rffGH demonstrated a negative feedback system, which allowed us to express the ToxR chimeras that were initially too toxic to be expressed.

The table to the left is fluorescence data demonstrating that IILK is dimerizing and driving ToxR to dimerize. There was just as much if not more fluorescent than our positive control. The results of this experiment demonstrate that ToxR can be expressed in E. coli. Furthermore, it demonstrates that dimerization of ToxR does activate transcription off of the ctx promoter.

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