Difference between revisions of "Part:BBa K4044011"
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Thus we get two new promoters dependant on Gal4 that can be used in procaryotic organisms that we consider to be a significant part improvement. Our further step is to try other Anderson promoters to create a library of bacterial Gal4 dependant promoters that there could be more chance to select the best fitting element for different chassies.
 
Thus we get two new promoters dependant on Gal4 that can be used in procaryotic organisms that we consider to be a significant part improvement. Our further step is to try other Anderson promoters to create a library of bacterial Gal4 dependant promoters that there could be more chance to select the best fitting element for different chassies.
  
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[[File:T--LMSU--ResBaseline.png|450px|thumb|left|Measurement of fluorescence intencity of YFP under Gal4-dependent promoters]]
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[[File:T--LMSU--ResBaseline.png|445px|thumb|left|Measurement of fluorescence intencity of YFP under Gal4-dependent promoters]]
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[[File:T--LMSU--ResPromoters.png|450px|thumb|right|Observed inhibited relative activity of Gal4-dependent promoters in presence of QPAS1-Gal4 protein]]
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[[File:T--LMSU--ResPromoters.png|445px|thumb|right|Observed inhibited relative activity of Gal4-dependent promoters in presence of QPAS1-Gal4 protein]]
  
  

Revision as of 01:41, 22 October 2021


p119_G4-n11 (Pj23119-based partially modified Gal4 dependant promoter)

The role of this part is genetical regulating systems based on Gal4 as there are no other Gal4 dependant promoters presented in bacteria. When binding to promoter Gal4 domain inhibits transcription activity completely. The promoter contains the sequence of CGG-N11-CCG between GACA-box and TATA-box, which can be recognized by Gal4 protein. Eleven nucleotides in the core location are pj23119 promoter nucleotides. When binding to promoter Gal4 domain inhibits transcription activity completely. This promoter was tested to be active in E. coli and comparable with Anderson promoter pj23119 baseline transcriptional activity. Promoters were tested in construction containing YFP under promoter that is tested with strong RBS used (BBa_J34801). The results of QPAS1-Gal4 inhibition show a significant decrease of fluorescence and result in a close to zero level of transcription.

We tested both base activity of these promoters and the inhibition efficiency of Gal4 with them to get a proper characterisation of these parts. The baseline activity was tested by YFP output with strong RBS used (BBa_J34801). We compared the relative activity of modified promoters with the original J23119 promoter. As a negative control also, original promoter was taken. Thus we get two new promoters dependant on Gal4 that can be used in procaryotic organisms that we consider to be a significant part improvement. Our further step is to try other Anderson promoters to create a library of bacterial Gal4 dependant promoters that there could be more chance to select the best fitting element for different chassies.

Measurement of fluorescence intencity of YFP under Gal4-dependent promoters
Observed inhibited relative activity of Gal4-dependent promoters in presence of QPAS1-Gal4 protein


We have also shown with a computer simulation that HTH domains of Gal4 binds exactly to CGG-N11-CCG site of the promoter. It confirms that system with Gal4-Q-PAS1 chimeric protein works correctly.

Gal4 HTH-domain binding to 5’-CGG-N11-CCG-3' promoter region of DNA
Molecular dynamics of Gal4 HTH-domain binding to 5’-CGG-N11-CCG-3' promoter region of DNA

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]