Difference between revisions of "Part:BBa K165095"

Revision as of 05:29, 30 October 2008

Gli1 bs + LexA bs + mCYC + Zif268-HIV repressor (mCherryx2 tagged) on pRS303

This devise constitutes the alpha element in one instance of our limiter construct. It is placed in a mutually inhibitory relationship with the tau element. Under the control of the same promoter as the gene of interest, alpha serves as a readout of its level of induction. In a superthreshold state, it represses tau, allowing R to be expressed and repress G back the threshold level.

To build a (heretofore untested) limiter, this part should be in a yeast strain with the following constructs:

A: Part:BBa_K165080 pGAL1 + Untagged LexA activator on pRS305 G: Part:BBa_K165078 LexA activable reporter on pRS306

  Into mating type a.  Knock out the Gal2 gene for a tunable level of induction.

R: Part:BBa_K165090 Gli1 bs + LexA bs + mCYC + LexA repressor (mCherryx2 tagged) on pRS306 Alpha: Part:BBa K165095 Gli1 bs + LexA bs + mCYC + Zif268-HIV repressor (mCherryx2 tagged) on pRS303 Tau: Part:BBa_K165096 Zif268-HIV bs + MET25+ Gli1 repressor (CFPx2 tagged) on pRS304*

  Into mating type alpha. Knock out the Gal2 gene for a tunable level of induction.

Mate the two types, select on SD-His-Trp-Leu-Ura

Inputs: Set the threshold by tuning [Met] between 0 and 500 uM Set the level of induction (A) with Galactose between 0-3%

Outputs expected: Subthreshold: CFP in the nucleus from Tau, YFP in the cytosol proportionate to subthreshold induction from G being activated by A and not repressed by R.

Superthreshold: mCherry in the nucleus from Alpha and R induction. YFP in cytosol levels out at threshold limit despite rising induction.

Sequence and Features