Part:BBa_K165090

Gli1 bs + LexA bs + mCYC + LexA repressor (mCherryx2 tagged) on pRS306

This is an example of the "R" repressor element in our genetic limiter circuit. It contains the Gli1 and LexA binding sites upstream of mCYC, regulating the expression of the LexA repressor. This is the element of the limiter that represses the gene of interest in the superthreshold state, being induced by G's inducer, the LexA activator in this case. It is repressed by the tau transcription factor when the system is subthreshold, in this case,the Gli1 repressor under control of the threshold-setting promoter.

The SpeI site is of no consequence, as the utility of parts on pRS vectors is to integrate into the yeast genome.

Usage and Biology

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

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 534
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 534
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2587
  Illegal XhoI site found at 1
  Illegal XhoI site found at 122
  Illegal XhoI site found at 284
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 534
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 534
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 119
  Illegal SapI.rc site found at 1758
  Illegal SapI.rc site found at 2351