ToxR Chimeras

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. We aim make a biosensor by taking this concept one step forward by utilizing this dimerization-dependent transcriptional activation feature of the ToxR system.

We predict that we can 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 affirm that a ToxR based two-hybrid system could be expressed in E. Coli. We truncated ToxR in various locations to eliminate the periplasmic domain. It is dimerization of the cytoplasmic domain that controls transcription. We attached lambdaRep and MukF, constitutively dimerizing proteins, to these truncates of ToxR. Thus the constitutively dimerizing proteins were located in the periplasm.

The initial ToxR chimeras under the pBad promoter were toxic to the cell and could not be expressed. This is likely because of the stress that overexpression of these transmembrane proteins cause on the cell membrane. 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. Colonies were green and tecan fluorescence data prove that constitutively dimerizing proteins, lambdaRep and MukF, caused ToxR to dimerize and activate transcription of GFP.

Estrogen Receptor

Once we had successfully expressed a ToxR proof of principle chimera we were ready to prove that this system could be inducible, and thus a more useful biosensor. We set out to find a ligand dependent homodimer and after an extensive literature search decided on using the Estrogen Receptor. Estrogen in the environment poses numerous hazardous risks and thus the ability to sense it cheaply and effectively would have great implications.

The Estrogen Receptor is made up of 5 domains: A/B, C, D, E, and F. Domain A/B is a transcriptional factor when activated, domain C is a DNA recognition domain that is also involved in dimerization. Domain E is the Ligand Binding Domain (LBD), and when it binds to estrogen homodimerizes to another Estrogen Receptor. Domain D is most likely involved in conferring conformational change to domain C when estrogen binds. Domain F inhibits dimerization and/or binding of estrogen and is thus undesireable for our project. We began by making two different ToxR chimeras with two Estrogen Receptor truncations: ER∆F (no F domain) and the ER-LBD alone. Both of these ER truncations were attached to the 3' end of ToxR and tested under various Estrogen levels. However, these truncations were non-responsive so we made 8 more truncations that spanned the range between Domain A and Domain C.

Preliminary data suggests that truncations 5, 6, and 8 all showed some sort of ToxR dimerization and resulting fluoresence, however, none seemed to be inducible in response to Estrogen. We believe that there may be issues with expression of the larger chimeras and not enough binding area for the smaller truncations. However, we believe we are getting very close to successfully expressing a chimera that will be inducible.