Knight:Evolving Reshmaverters/Promoter library design: Difference between revisions
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BBa_R2001 agtttattcTTGACAtggtcatattacggtgaGATACT<font style="background-color: yellow">cccacgcgcgtggg</font> | BBa_R2001 agtttattcTTGACAtggtcatattacggtgaGATACT<font style="background-color: yellow">cccacgcgcgtggg</font> | ||
BBa_R2002 agtttattcTTGACAtggt<font style="background-color: yellow">cccacgcgcgtggG</font>ATACT<font style="background-color: yellow">cccacgcgcgtggg</font> | BBa_R2002 agtttattcTTGACAtggt<font style="background-color: yellow">cccacgcgcgtggG</font>ATACT<font style="background-color: yellow">cccacgcgcgtggg</font> | ||
==Brainstorming== | |||
How could these promoters be redesigned to be repressible? | |||
'''Comments welcome''' | |||
*Repressor sites tend to fall between the -35 and -10 regions and/or downstream of the -10 (around the +1). (Not upstream of the -35). <cite>Collado-Vides-MicrobioRev-1991 Gralla-Cell-1991</cite> | |||
*Binding of the repressor dimer to the promoter may lead to DNA bending rendering binding of more dimers unfavorable. Perhaps a single binding site is preferable. | |||
*The TG sequence at -16 is causing the promoters to be too strong. The RNA polymerase is "winning" the competition for binding with the repressor. Perhaps this dinucleotide should be removed. <cite>Burr-NAR-2000 Voskuil-MolMicrobiol-1995</cite> | |||
*A high k<sub>ON</sub> (rate of complex formation between RNA polymerase and promoter) correlates inversely with repressibility. <cite>Lanzer-PNAS-1988</cite> Perhaps, a -35 consensus sequence is responsible for high k<sub>ON</sub>? | |||
==Relevant work== | ==Relevant work== | ||
| Line 23: | Line 33: | ||
#Lanzer-PNAS-1988 pmid=3057497 | #Lanzer-PNAS-1988 pmid=3057497 | ||
#Besse-EMBO-1986 pmid=3015603 | #Besse-EMBO-1986 pmid=3015603 | ||
#Gralla-Cell-1991 pmid=1868543 | |||
#Collado-Vides-MicrobioRev-1991 pmid=1943993 | |||
#Burr-NAR-2000 pmid=10756184 | |||
#Voskuil-MolMicrobiol-1995 pmid=7494476 | |||
</biblio> | </biblio> | ||
Revision as of 17:36, 18 April 2006
Existing promoters
The following promoters have been tested and are not repressible under my testing conditions.
Heterodimers:
-35 -10
Promoter1 cacgtgtgcgtgggTTGACAcgtgtgcgtgggaagtcGATACTgagcaca
Promoter2* TTGACAcgtgtgcgtgggaagtcGATACTtagattcacgtgtgcgtggg
Promoter3 cacgtgtgcgtgggTTGACAcgtgtgcgtgggaagtcGATACTtagattcacgtgtgcgtggg
Promoter4 cacgtgtgcgtgggTTGACAcacgtgtgcgtgggaatGATACTgagcaca
Promoter5 TTGACAcacgtgtgcgtgggaatGATACTtagattcacgtgtgcgtggg
Promoter6 cacgtgtgcgtgggTTGACAcacgtgtgcgtgggaatGATACTtagattcacgtgtgcgtggg
Homodimers:
-35 -10
BBa_R2000 agtttattcTTGACAtggtcccacgcgcgtggGATACTacgtcag
BBa_R2001 agtttattcTTGACAtggtcatattacggtgaGATACTcccacgcgcgtggg
BBa_R2002 agtttattcTTGACAtggtcccacgcgcgtggGATACTcccacgcgcgtggg
Brainstorming
How could these promoters be redesigned to be repressible?
Comments welcome
- Repressor sites tend to fall between the -35 and -10 regions and/or downstream of the -10 (around the +1). (Not upstream of the -35). [1, 2]
- Binding of the repressor dimer to the promoter may lead to DNA bending rendering binding of more dimers unfavorable. Perhaps a single binding site is preferable.
- The TG sequence at -16 is causing the promoters to be too strong. The RNA polymerase is "winning" the competition for binding with the repressor. Perhaps this dinucleotide should be removed. [3, 4]
- A high kON (rate of complex formation between RNA polymerase and promoter) correlates inversely with repressibility. [5] Perhaps, a -35 consensus sequence is responsible for high kON?
Relevant work
- Collado-Vides J, Magasanik B, and Gralla JD. Control site location and transcriptional regulation in Escherichia coli. Microbiol Rev. 1991 Sep;55(3):371-94. DOI:10.1128/mr.55.3.371-394.1991 |
- Gralla JD. Transcriptional control--lessons from an E. coli promoter data base. Cell. 1991 Aug 9;66(3):415-8. DOI:10.1016/0092-8674(81)90001-5 |
- Burr T, Mitchell J, Kolb A, Minchin S, and Busby S. DNA sequence elements located immediately upstream of the -10 hexamer in Escherichia coli promoters: a systematic study. Nucleic Acids Res. 2000 May 1;28(9):1864-70. DOI:10.1093/nar/28.9.1864 |
- Voskuil MI, Voepel K, and Chambliss GH. The -16 region, a vital sequence for the utilization of a promoter in Bacillus subtilis and Escherichia coli. Mol Microbiol. 1995 Jul;17(2):271-9. DOI:10.1111/j.1365-2958.1995.mmi_17020271.x |
- Lanzer M and Bujard H. Promoters largely determine the efficiency of repressor action. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8973-7. DOI:10.1073/pnas.85.23.8973 |
- Lutz R and Bujard H. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res. 1997 Mar 15;25(6):1203-10. DOI:10.1093/nar/25.6.1203 |
- Besse M, von Wilcken-Bergmann B, and Müller-Hill B. Synthetic lac operator mediates repression through lac repressor when introduced upstream and downstream from lac promoter. EMBO J. 1986 Jun;5(6):1377-81. DOI:10.1002/j.1460-2075.1986.tb04370.x |
