IGEM:MIT/2005/From email of 7/7 to NK

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"the cI-ToxR fusions behaved as a dimer in E. coli and as a higher order oligomer in V. cholerae. In both organisms, dimerization was independent of any environmental modulators that were tested (i.e. dimerization was essentially constitutive). Additionally, dimerization appeared to be mediated by more than one domain of the ToxR protein. And, as suggested, it apparently does a pretty good job of simply existing as a dimer on it's own as best we can tell. The hypothesis that environmental signals modulate the monomer-dimer transition was _not_ proven by our studies.

To complicate the issue, there was a publication in 1998 from Japelli and Brenner that looked at how the length of "linker" domains and expression levels affected fusion protein activity. Furthermore, I know from my studies that expression levels are important for dimerization read-out via cI. There is a window within which the reporter system works, and then if over expressed, the system appears to fail, possibly because of the complication of ToxR as a membrane protein. My memory is a little fuzzy on the details but I think the basic info is accurate.

A number of groups have been interested in using ToxR as a reporter system for dimerization and have decided not pursued this avenue. It has turned out to be rather complicated and additional proteins (TcpPH) have now been postulated to interact with ToxRS. This is a whole 'nother can of worms.

The strains and plasmids Jennifer requested are not under my jurisdiction to distribute. The AH strains were a generous gift of the Hochshield lab (as you will recognize!!), and the pHK plasmids belong to a group in Germany that we briefly collaborated with in an effort to try to sort things out (Kolmar et al 1995 EMBO preceded our joint paper).John is still the arbiter of my constructs."