Oligo design for XOR Hybrid Promoters
Contents
Lux-cI hybrid promoter responsible:Xiao Zhu
Rationale
“The effect of cI is dominant over LuxR.”(Ron Weiss, part description for R0065)
The first 2 designs are based on the same idea to replace the downstream of Lux/HSL -35 region with A). cI OR1 (containing -10 region); B). cI OR2, added LuxR-10 region. We hope that doing in this way could lead a stronger regulation for cI OR1/2. Based on what we have found up to now, promoters which are constructed just by inserting OR1/2 downstream of Lux are all very leaky. There’s a journal talking about a research that used Lux-cI hybrid promoter in their system. What they did is to insert cI OR1 upstream of Lux +1(they had a mutant for OR1 sequence), and it’s leaky (“In this case, even without AHL, leaky CI expression completely represses luxPRcI-OR1”).
References
Spatiotemporal control of gene expression with pulse-generating networks
Design Considerations
Design C, to use OR2 twice, is because “it requires the binding of two cI repressor dimers for maximal repression”, but as BBa_R0065 has shown, if we put both OR2 and OR1 downstream of Lux, the promoter won’t work very well, it’s very leaky at high copy number. And we found that ETHZ iGEM’07 team were building these hybrid promoters as well. They used OR2 twice. We don’t know whether OR2 has any advantages over OR1 for functioning.
Design A - LuxR/HSL + OR1
Desired sequence (101bp)
5’ GCAT GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG TACCTCTGGCGGTGATAA T GGTTGC TACTAGTAGC GGCCGCTGCA GGCAT 3’
Annotation of desired sequence
BBa_Prefix: GCAT GAATTCGCGGCCGCTTCTAGAG
LuxR/HSL Box (only includes region upstream of -35 and the first T of -35): ACCTGTAGGA TCGTACAGG T
LuxR -35: TTTACG
cI Half-Operator OR1: TACCTCTGGCGGTGATAA
Lambda pR -10: GATAAT
Spacer: (naturally occurring just 3' to -10 in cI promoter): GGTTGC
Suffix: TACTAGTAGCGGCCGCTGCAG ATGC
Forward primer (57bp)
5’ GCAT GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG TACCTC3’
Reverse Primer (57bp)
5’ GCAT CTGCAG CGGCCGCTAC TAGTAGCAAC CATTATCACC GCCAGAGGTA CGTAAAC 3’
Design B - LuxR/HSL + OR2
Desired sequence (108bp)
5’ GCAT GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG TAACACCGTG CGTGTTGA TATAGT CGAATAAA TACTAGTAGCGGCCGCTGCAGGCAT 3’
Annotated desired sequence
BBa_Prefix: GCAT GAATTCGCGGCCGCTTCTAGAG
LuxR/HSL Box (only includes region upstream of -35 and the first T of -35): ACCTGTAGGA TCGTACAGG T
LuxR -35: TTTACG
Lambda Half-Operator OR2: TAACACCGTG CGTGTTGA
LuxR -10: TATAGT (we used the -10 region from LuxR in order to reduce the risk that LuxR -35 region won’t work well with -10 region from other sources.)
Spacer (naturally occurs after LuxR -10): CGAATAAA
Suffix: TACTAGTAGCGGCCGCTGCAG ATCG
Forward Primer (60bp)
5’ GCAT GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG TAACACCGTG 3’
Reverse Primer (60bp)
5’ GCAT CTGCAG CGGCCGCTAC TAGTATTTAT TCGACTATAT CAACACGCAC GGTGTTACGT 3’
Design C - LuxR/HSL + 2xOR2
Desired sequence: (140bp)
5’ GCAT GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG CAAGAAAATG GTTTGTTATAGT TAACACCGTG CGTGTTGA TTTATC TAACACCGTG CGTGTTGA TACTAGTAGCGGCCGCTGCAGGCAT 3’
Annotated desired sequence
BBa_Prefix: GCAT GAATTCGCGGCCGCTTCTAGAG
Lux/HSL (the whole sequence, including -35 and -10): ACCTGTAGGA TCGTACAGG TTTACG CAAGAAAATG GTTTGTTATAGT
Lux -35: TTTACG
Lux -10: TATAGT
2xOR2 with 6 bp that naturally exist in Lambda promoter between OR2 and OR1 (we inserted the 6 bp spacer backwards so as to break the cI -35 region): TAACACCGTG CGTGTTGA TTTATC TAACACCGTG CGTGTTGA
Suffix: TACTAGTAGCGGCCGCTGCAG ATGC
Forward Primer: (76bp)
5’ GCAT GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG CAAGAAAATG GTTTGTTATAGT TAA3’
Reverse Primer: (76bp)
5’ GCAT CTGCAG CGGCCGCTAC TAGTATCAAC ACGCACGGTG TTAGATAAAT CAACACGCAC GGTGTTAACT ATAACA 3’
Mnt/LacI hybrid promoter responsible:Robert Cool
Rationale
This promoter is a modified version of the Mnt promoter that is also responsive to LacI. The promoter should be repressed by Mnt repressor. It should also be repressed by LacI, and in the absence of Mnt repressor, should be induced by IPTG.
References and Links
Design considerations Mnt repressor binds as a tetramer to two half-operator sites (http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=11226234 ). Introduction of a LacI binding site between the Mnt promoter -10 sequence and the start site for transcription should allow for repression by LacI. Accordingly, the hybrid promoter was designed by 1) Remove all bases of mnt promoter 3’ to -10: gagtcgtattaattt will be replaced by tgtgtggaattgtga, and 2) Position lacI binding site (composed of an inverted repeat) from the lacI regulated promoter (R0010) immediately 3’ to truncated mnt promoter.
Entire desired sequence (138 bp)
Gcat Gaattcgcggccgcttctagag ctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctatagt tgtgtggaattgtgagcggataacaatttcacaca tactagtagcggccgctgcag atgc
Annotated desired sequence
Biobrick prefix: GCAT gaattcgcggccgcttctagag
Mnt promoter (everything before and including -10): ctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctatagt
LacI binding site (last 35bp, everything after -10): tgtgtggaattgtgagcggataacaatttcacacagagtcgtattaattt
Biobrick suffix: tactagtagcggccgctgcag ATCG
Forward Primer (78bp)
gcatgaattcgcggccgcttctagagctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctatagt
Reverse (reverse complement) gcatctgcagcggccgctactagtatgtgtgaaattgttatccgctcacaattccacacaactataggagatctaggt
Mnt/TetR hybrid promoter responsible:Robert Cool
This promoter is a modified version of the Mnt promoter that is also responsive to TetR. The promoter should be repressed by Mnt repressor. It should also be repressed by TetR, and in the absence of Mnt repressor, should be induced by aTc.
Design features: Mnt repressor binds as a tetramer to two half-operator sites (http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=11226234 ). Introduction of TetR binding sites between the Mnt promoter -10 sequence and the start site for transcription should allow for repression by TetR. Accordingly, the hybrid promoter was designed by 1) Remove all bases of mnt promoter 3’ to -10: gagtcgtattaattt will be replaced by tccctatcagtgata, and 2) truncate the second half of ptet -10 and mutate -35 so that it function to bind tetR (replace ttgaca with actgta), but is not a promoter, and 3) place the tetR1 and tetR2 binding sites 3’ to truncated mnt -10 sequence.
Biobrick prefix gaattcgcggccgcttctagag
Mnt (everything before and including -10) ctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctatagt
tetR1 tccctatcagtgatagaga
Spacer actgta
TetR 2 binding site atccctatcagtgatagagat
Biobrick suffix tactagtagcggccgctgcag
Entire sequence 143bp
Gcat gaattcgcggccgcttctagag ctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctatagt tccctatcagtgatagaga actgta atccctatcagtgatagagat tactagtagcggccgctgcag atgc
Primers (81bp)
Forward gcatctgcagcggccgctactagtaatctctatcactgatagggattacagttctctatcactgatagggaactataggag
Reverse (reverse complement) gcatctgcagcggccgctactagtaatctctatcactgatagggattctctatcactgatagggaactataggagatct
LsrA/CI hybrid promoter responsible: Andrew Gordon
This hybrid promoter uses the CI promoter as a repressor. The promoter region for LsrA is unaffected awhile CI is allowed to bind to the OR2 sites. The OR2 sites with CI bound do not allow transcription. The CI binding sites have a natural 6bp spacer.
2xOR2 Repressor Primer Design
2xOR2 Sequence
TAACACCGTGCGTGTTGATTTATCTAACACCGTGCGTGTTGA
Forward Sequence
TAACACCGTGCGTGTTGATTTATCTAAC
Prefix
gcat gaattcgcggccgcttctagag
Forward Primer LsrAp
gcat gaattcgcggccgcttctagag TAACACCGTGCGTGTTGATTTATCTAAC
Forward of Second Squence
TTGATTTATCTAACACCGTGCGTGTTGA
Reverse Complement of Second Squence
TAACACCGTGCGTGTTGATTTATCTAAC
Suffix
tactagtagcggccgctgcag
Suffix reverse complement
gcat ctgcagcggccgctactagta
Reverse Primer LsrAp
gcat ctgcagcggccgctactagta TCAACACGCTCGGTGTTAGATAAATCAA
Primers have a 15bp overlap for direct synthesis
Alternative design for LsrR/CI promoter
According to Wang et al, the start site for transcription predicted by the BDGP software (we could not replicate this!) is as indicated below in bold on the sequence of the pLsrA promoter that we built:
AACCGTGA AAATCAAAAT AGCATAAAT TGTGATCTATT CGTCGGAAAT ATGTGCAATG TCCACCTAAG GTTATGAACA AATTAAAAGC AGAAATACAT TTGTTCAAAA CTCACCTGCA AAACTGAA
So, a possible design for a hybrid LsrR/CI promoter would be to place two binding sites for cI immediately downstream of the -10 sequence of the LsrA promoter.
Truncated LsrA promoter: AACCGTGA AAATCAAAAT AGCATAAAT TGTGATCTATT CGTCGGAAAT ATGTGCAATG TCCACCTAAG GTTATGAACA AATTAAAAGC AGAAATACAT T
cI binding site, 2x OR2 with 6 bp that naturally exists in Lambda promoter between OR2 and OR1, a 6 bp spacer has been inserted between the two OR2 sites so as to mutate the cI -35 region: TAACACCGTG CGTGTTGA TTTATC TAACACCGTG CGTGTTGA
Synthesis could be accomplished with the already purchased primer "pLsrA forward" and a new primer that binds to the last portion of the truncated LsrA promoter.
New primer needed - "pLsrA/cI reverse" - 82 bp
Suffix with spacer (reverse complement): GCAT CTGCAGCGGCCGCTACTAGTA 2x OR2 sites (reverse complement): TCAACACG CACGGTGTTA GATAAA TCAACACG CACGGTGTTA 15 nt from 3' end of truncated LsrA promoter (reverse complement): A ATGTATTTCT GCTTTTAATT
GCAT CTGCAGCGGCCGCTACTAGTA TCAACACG CACGGTGTTA GATAAA TCAACACG CACGGTGTTA A ATGTATTTCT GCTT
