Difference between revisions of "Part:BBa K165078"

 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K165078 short</partinfo>
 
<partinfo>BBa_K165078 short</partinfo>
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This activator reporter consists of the LexA binding sites on the minimal mCYC promoter driving expression of YFP.  It is the G construct in the limiter system, modeling an endogenous gene of interest for our proof-of-concept.
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[[Image:Glyph_labeled2.png|center|400px]]
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===Usage and Biology===
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To build a (heretofore untested) limiter, this part should be in a yeast strain with the following constructs:
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'''A)'''    [[Part:BBa_K165080]] pGAL1 + Untagged LexA activator on pRS305<br>
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'''G)'''    [[Part:BBa_K165078]] LexA activable reporter on pRS306<br>
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  Into mating type a.  Knock out the Gal2 gene for a tunable level of induction.<br>
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'''R)'''    [[Part:BBa_K165090]] Gli1 bs + LexA bs + mCYC + LexA repressor (mCherryx2 tagged) on pRS306<br>
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'''Alpha)''' [[Part:BBa_K165095]] Gli1 bs + LexA bs + mCYC + Zif268-HIV repressor (mCherryx2 tagged) on pRS303 <br>
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'''Tau)'''  [[Part:BBa_K165096]] Zif268-HIV bs + MET25+ Gli1 repressor (CFPx2 tagged) on pRS304* <br>
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  Into mating type alpha. Knock out the Gal2 gene for a tunable level of induction.<br>
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Mate the two types, select on SD-His-Trp-Leu-Ura
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'''Inputs:'''
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Set the threshold by tuning [Met] between 0 and 500 uM
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Set the level of induction (A) with Galactose between 0-3%
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'''Outputs expected:'''<br>
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''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.<br>
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''Superthreshold [A]:'' mCherry in the nucleus from Alpha and R induction.  YFP in cytosol levels out at threshold limit despite rising induction.
  
 
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Revision as of 05:41, 30 October 2008

LexA activable reporter on pRS306

This activator reporter consists of the LexA binding sites on the minimal mCYC promoter driving expression of YFP. It is the G construct in the limiter system, modeling an endogenous gene of interest for our proof-of-concept.

Glyph labeled2.png


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.

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Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1082
    Illegal BsaI.rc site found at 1832