Part:BBa_K165095

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

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 2321
  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
  Illegal AgeI site found at 3464
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 119
  Illegal SapI.rc site found at 1492
  Illegal SapI.rc site found at 2085