Difference between revisions of "Part:BBa K4044010"

Latest revision as of 03:14, 22 October 2021

p119_G4 (Pj23119-based Gal4 dependant bacterial promoter)

The role of this part is genetical regulating systems based on Gal4 as there are no other Gal4 dependent 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 presented of the consensus sequence of Gal4 binding locus of Saccharomyces cerevisiae [1]. 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 dependent 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 dependent promoters that there could be more chance to select the best fitting element for different chassies.

Measurement of fluorescence intensity 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

1. Structural Basis for Dimerization in DNA Recognition by Gal4. Structure, 2008

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Unknown
21
INCOMPATIBLE WITH RFC[21]
Unknown
23
INCOMPATIBLE WITH RFC[23]
Unknown
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Unknown

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K4044010 short</partinfo>
-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 middle are presented of the consensus sequence of Gal4 binding locus of Saccharomyces cerevisiae. 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.
+The role of this part is genetical regulating systems based on Gal4 as there are no other Gal4 dependent 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 presented of the consensus sequence of Gal4 binding locus of ''Saccharomyces cerevisiae'' [1]. 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.
-fluorescence graf*
+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 dependent 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 dependent promoters that there could be more chance to select the best fitting element for different chassies.
-We also have shown with a computer simulation that HTH domains of Gal4 binds exactly to CGG-N11-CCG site of the promoter.
+[[File:T--LMSU--ResBaseline.png|430px|thumb|left|Measurement of fluorescence intensity of YFP under Gal4-dependent promoters]]
+[[File:T--LMSU--ResPromoters.png|430px|thumb|right|Observed inhibited relative activity of Gal4-dependent promoters in presence of QPAS1-Gal4 protein]]
-modeling of QOAS-Gal4 with DNA*
+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.
+[[File:T--LMSU--Modeling-Gal4-DNA-binding.png|400px|thumb|center|Gal4 HTH-domain binding to 5’-CGG-N11-CCG-3' promoter region of DNA]]
+[[File:T--LMSU--Modeling-otherGal4-DNA.gif|400px|thumb|center|Molecular dynamics of Gal4 HTH-domain binding to 5’-CGG-N11-CCG-3' promoter region of DNA]]
+. Structural Basis for Dimerization in DNA Recognition by Gal4. Structure, 2008
 <!-- Add more about the biology of this part here