Device

Part:BBa_K165095

Designed by: John Szymanski   Group: iGEM08_BrownTwo   (2008-10-29)
Revision as of 05:31, 30 October 2008 by Jszymans (Talk | contribs)

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.

Glyph labeled2.png

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


[edit]
Categories
//chassis/eukaryote/yeast
//function/regulation/transcriptional
Parameters
None