Difference between revisions of "Part:BBa K1216008:Design"

(→‎Design Notes)
(→‎Design Notes)
 
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[[File:G1VariantLibrary.png|600px|left|thumb|<b>Figure 1: Sequence alignment of the additional luxR variants.</b>]]
 
[[File:G1VariantLibrary.png|600px|left|thumb|<b>Figure 1: Sequence alignment of the additional luxR variants.</b>]]
 
<p>According to initial model predictions confirmed by successive [http://2013.igem.org/Team:ETH_Zurich/Experiments_6 experimental validation] the [https://parts.igem.org/Part:BBa_K1216007 previously achieved] promoter sensitivity is too low to drive a significant response in the concentration gradient established in our system.<br>
 
<p>According to initial model predictions confirmed by successive [http://2013.igem.org/Team:ETH_Zurich/Experiments_6 experimental validation] the [https://parts.igem.org/Part:BBa_K1216007 previously achieved] promoter sensitivity is too low to drive a significant response in the concentration gradient established in our system.<br>
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We needed a collection of promoters with a set of EC<sub>50</sub> values between the [https://parts.igem.org/Part:BBa_J09855 wild type] and the resulting [ https://parts.igem.org/Part:BBa_K1216007 promoter from the first] mutagenesis screening.
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We needed a collection of promoters with a set of EC<sub>50</sub> values between the [https://parts.igem.org/Part:BBa_J09855 wild type] and the resulting [https://parts.igem.org/Part:BBa_K1216007 promoter from the first] mutagenesis screening.
 
Since Bba_K1216007 has been obtained through two random mutations of the lux box (4T>A and 16C>G), reverting one of the two, we reasoned, should result in a LuxR binding strength closer to the wild type.  
 
Since Bba_K1216007 has been obtained through two random mutations of the lux box (4T>A and 16C>G), reverting one of the two, we reasoned, should result in a LuxR binding strength closer to the wild type.  
 
To test this hypothesis we ordered oligos encompassing all the possible combinations of single position mutants (see figure 1) to experimentally characterize all the resulting P<sub>LuxR</sub> variants. </p>
 
To test this hypothesis we ordered oligos encompassing all the possible combinations of single position mutants (see figure 1) to experimentally characterize all the resulting P<sub>LuxR</sub> variants. </p>

Latest revision as of 15:21, 29 October 2013


Variant of the wild-type pLuxR promoter with lower sensitivity


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
    COMPATIBLE WITH RFC[1000]


Design Notes

Rational design and single cell analysis of additional PLuxR variants with partial sensitivity recovery based on the first PLuxR variant (G1).

Figure 1: Sequence alignment of the additional luxR variants.

According to initial model predictions confirmed by successive [http://2013.igem.org/Team:ETH_Zurich/Experiments_6 experimental validation] the previously achieved promoter sensitivity is too low to drive a significant response in the concentration gradient established in our system.
We needed a collection of promoters with a set of EC50 values between the wild type and the resulting promoter from the first mutagenesis screening. Since Bba_K1216007 has been obtained through two random mutations of the lux box (4T>A and 16C>G), reverting one of the two, we reasoned, should result in a LuxR binding strength closer to the wild type. To test this hypothesis we ordered oligos encompassing all the possible combinations of single position mutants (see figure 1) to experimentally characterize all the resulting PLuxR variants.

Source

The promoter was built through site-saturation mutagenesis of the wild-type promoter.

References