Knight:Evolving Reshmaverters/Promoter library design

Promoter library design
In progress!

Promoter architecture
-35                   -10        +1                 ______                 ______       _ TTGACA-TATAAT- CA--- (consensus) TTGCTT-TATAAT-GATT CATAAATTTGAGAGAGGAGTT (good promoter clearance?) Kammerer-EMBO-1986 TTGACT-GATACT--CA--- (repressible, low KON?) Lanzer-PNAS-1988

TTGACCccacgcgtggg --TATAAT- CA--- (operator site in position of maximum steric interference with RNAP) Wolfe-Structure-2000, Murakami-Science-2002a, Murakami-Science-2002b TTGACA-CccacgcgTGGG AT- CA--- (operator site in position of maximum steric interference with RNAP) Wolfe-Structure-2000, Murakami-Science-2002a, Murakami-Science-2002b

Constant promoter
-35                   -10        +1                 ______                 ______       _ tttatcaaaaagagtgTTGATCccacgcgtggg atatagGATACTtagattcataaatttgagagaggagtt (promoter9) tttatcaaaaagagtgTTGACAtttttaagtcccacgcgTGGG ATtagattcataaatttgagagaggagtt (promoter8)

Questions

 * 1) Do I include too much extraneous sequence? These promoters are longer to reflect those found in papers.

Libraries
-35                   -10        +1                 ______                 ______       _ tttatcaaaaagagtgTTGNTCccacgcgtggg annnnnNATANTnnnnnncannnnnnnnnn (based on promoter9) tttatcaaaaagagtgTTGACAnnnnnnnntcccacgcgTGGG ATnnnnnncannnnnnnnnn (based on promoter8) nnnnnnnnnTTGNCAnnntcccacgcgcgtggG ATANTnnnnnnca (based on BBa_R2000)

Questions

 * 1) Are these promoters likely to be functional? Too much sequence diversity?
 * 2) I potentially don't need to vary the -35 and -10 from consensus because I can still achieve a range of promoter strengths without changing them. Hammer-TrendsBiotechnol-2006

Operator
0 site operator: cccacgcgcgtggg (14bp) -2 site operator: cccacgc gtggg (12bp) (higher affinity)

Brainstorming
How could these regulatory regions 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). Collado-Vides-MicrobioRev-1991 Gralla-Cell-1991
 * 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 regulatory regions to be too strong in the derepressed state. The RNA polymerase is "winning" the competition for binding with the repressor.  Perhaps this dinucleotide should be removed. Burr-NAR-2000 Voskuil-MolMicrobiol-1995
 * A high kON (rate of complex formation between RNA polymerase and promoter) correlates inversely with repressibility. High kON may result in RNAP outcompeting the repressor for the regulatory region binding.  High regulatory region clearance rates enable strong transcription initiation and allow for repressor binding. Lanzer-PNAS-1988
 * +1 base should be an A with 6-7 bases between end of -10 hexamer and +1. Lanzer-PNAS-1988, Kammerer-EMBO-1986
 * Use the -2 operator site because it binds the homodimer more tightly. (But it is less modular).


 * How can we design promoters with low kON but high clearance rates for improved repressibility?
 * KON and promoter occupancy by RNAP is determined by the -35 and -10 hexamers. Closer to consensus sequences means high kON and more stable RNAP-promoter complexes. Ellinger-JMB-1994
 * An AT rich region around the transcription start site may lead to better promoter clearance? Ellinger-JMB-1994, Kammerer-EMBO-1986

Existing promoters
The following promoters have been tested and are not repressible under my testing conditions.

Heterodimers: -35                   -10 Promoter1 cacgtgtgcgtggg TTGACAcgtgtgcgtggg aagtcGATACTgagcaca Promoter2*             TTGACAcgtgtgcgtggg aagtcGATACTtagattcacgtgtgcgtggg Promoter3 cacgtgtgcgtggg TTGACAcgtgtgcgtggg aagtcGATACTtagattcacgtgtgcgtggg Promoter4 <font style="background-color: yellow">cacgtgtgcgtggg TTGACA<font style="background-color: yellow">cacgtgtgcgtggg aatGATACTgagcaca Promoter5              TTGACA<font style="background-color: yellow">cacgtgtgcgtggg aatGATACTtagatt<font style="background-color: yellow">cacgtgtgcgtggg Promoter6 <font style="background-color: yellow">cacgtgtgcgtggg TTGACA<font style="background-color: yellow">cacgtgtgcgtggg aatGATACTtagatt<font style="background-color: yellow">cacgtgtgcgtggg

Homodimers: -35                  -10 BBa_R2000 agtttattcTTGACAtggt<font style="background-color: yellow">cccacgcgcgtggG ATACTacgtcag BBa_R2001 agtttattcTTGACAtggtcatattacggtgaGATACT<font style="background-color: yellow">cccacgcgcgtggg BBa_R2002 agtttattcTTGACAtggt<font style="background-color: yellow">cccacgcgcgtggG ATACT<font style="background-color: yellow">cccacgcgcgtggg

These promoters are too weak under my testing conditions. (Some were not clonable).

Homodimers: -35                   -10        +1                            ______                 ______       _ BBa_R2108  tttatcaaaaagagtgTTGACAtttttaagt<font style="background-color: yellow">cccacgcgTGGG ATtagattcataaatttgagagaggagtt BBa_R2110 tttatcaaaaagagtgTTGACAtttttaagct<font style="background-color: yellow">cccacgcGTGGG Ttagattcataaatttgagagaggagtt BBa_R2112 tttatcaaaaagagtgTTGACAtttttaat<font style="background-color: yellow">cccacgcgtGGG AATtagattcataaatttgagagaggagtt BBa_R2109 tttatcaaaaagagtgTTGAT<font style="background-color: yellow">Cccacgcgtggg atatagGATACTtagattcataaatttgagagaggagtt BBa_R2111 tttatcaaaaagagtgTTGACT<font style="background-color: yellow">cccacgcgtggg aatagGATACTtagattcataaatttgagagaggagtt BBa_R2113 tttatcaaaaagagtgTTGT<font style="background-color: yellow">CCcacgcgtggg actatagGATACTtagattcataaatttgagagaggagtt BBa_R2114 tttatcaaaaagagtgTTGACT<font style="background-color: yellow">cccacgcgtggg aatagGATATTtagattcataaatttgagagaggagtt

Promoter design

 * 1) Lutz-NAR-1997 pmid=9092630
 * 2) Lanzer-PNAS-1988 pmid=3057497
 * 3) Besse-EMBO-1986 pmid=3015603
 * 4) Gralla-Cell-1991 pmid=1868543
 * 5) Collado-Vides-MicrobioRev-1991 pmid=1943993
 * 6) Burr-NAR-2000 pmid=10756184
 * 7) Voskuil-MolMicrobiol-1995 pmid=7494476
 * 8) Ellinger-JMB-1994 pmid=8006961
 * 9) Kammerer-EMBO-1986 pmid=3539590

Structures

 * 1) Wolfe-Structure-2000 pmid=10903945
 * 2) Murakami-Science-2002a pmid=12016306
 * 3) Murakami-Science-2002b pmid=12016307

Promoter libraries

 * 1) Alper-PNAS-2005 pmid=16502313
 * 2) Hammer-TrendsBiotechnol-2006 pmid=16406119
 * 3) Fischer-TrendsBiotechmol-2006 pmid=16380177