Difference between revisions of "Part:BBa K165095"
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<partinfo>BBa_K165095 short</partinfo> | <partinfo>BBa_K165095 short</partinfo> | ||
| − | This | + | This device 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. |
The SpeI site is of no consequence, as the utility of parts on pRS vectors is to integrate into the yeast genome. | The SpeI site is of no consequence, as the utility of parts on pRS vectors is to integrate into the yeast genome. | ||
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To build a (heretofore untested) limiter, this part should be in a yeast strain with the following constructs: | 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<br> | + | '''A)''' [[Part:BBa_K165080]] pGAL1 + Untagged LexA activator on pRS305<br> |
| − | G) [[Part:BBa_K165078]] LexA activable reporter on pRS306<br> | + | '''G)''' [[Part:BBa_K165078]] LexA activable reporter on pRS306<br> |
Into mating type a. Knock out the Gal2 gene for a tunable level of induction.<br> | Into mating type a. Knock out the Gal2 gene for a tunable level of induction.<br> | ||
| − | R) [[Part:BBa_K165090]] Gli1 bs + LexA bs + mCYC + LexA repressor (mCherryx2 tagged) on pRS306<br> | + | '''R)''' [[Part:BBa_K165090]] Gli1 bs + LexA bs + mCYC + LexA repressor (mCherryx2 tagged) on pRS306<br> |
| − | Alpha) [[Part:BBa_K165095]] Gli1 bs + LexA bs + mCYC + Zif268-HIV repressor (mCherryx2 tagged) on pRS303 <br> | + | '''Alpha)''' [[Part:BBa_K165095]] Gli1 bs + LexA bs + mCYC + Zif268-HIV repressor (mCherryx2 tagged) on pRS303 <br> |
| − | Tau) [[Part:BBa_K165096]] Zif268-HIV bs + MET25+ Gli1 repressor (CFPx2 tagged) on pRS304* <br> | + | '''Tau)''' [[Part:BBa_K165096]] Zif268-HIV bs + MET25+ Gli1 repressor (CFPx2 tagged) on pRS304* <br> |
Into mating type alpha. Knock out the Gal2 gene for a tunable level of induction.<br> | Into mating type alpha. Knock out the Gal2 gene for a tunable level of induction.<br> | ||
Mate the two types, select on SD-His-Trp-Leu-Ura | Mate the two types, select on SD-His-Trp-Leu-Ura | ||
| − | Inputs: | + | '''Inputs:''' |
Set the threshold by tuning [Met] between 0 and 500 uM | Set the threshold by tuning [Met] between 0 and 500 uM | ||
Set the level of induction (A) with Galactose between 0-3% | Set the level of induction (A) with Galactose between 0-3% | ||
| − | Outputs expected: | + | '''Outputs expected:'''<br> |
| − | 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. | + | ''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> |
| − | + | ''Superthreshold [A]:'' mCherry in the nucleus from Alpha and R induction. YFP in cytosol levels out at threshold limit despite rising induction. | |
| − | Superthreshold: mCherry in the nucleus from Alpha and R induction. YFP in cytosol levels out at threshold limit despite rising induction. | + | |
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Latest revision as of 05:42, 30 October 2008
Gli1 bs + LexA bs + mCYC + Zif268-HIV repressor (mCherryx2 tagged) on pRS303
This device 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.
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
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 534
- 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 534
- 21INCOMPATIBLE 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 - 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 534
- 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 534
Illegal AgeI site found at 3464 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 119
Illegal SapI.rc site found at 1492
Illegal SapI.rc site found at 2085