Difference between revisions of "Oligo design for XOR Hybrid Promoters"

Revision as of 01:13, 18 June 2008

Oligo design for XOR Hybrid Promoters

  Lux-cI hybrid promoter responsible:Xiao Zhu

“The effect of cI is dominant over LuxR.”(Ron Weiss, part description for R0065)

Lux/HSL

cI

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”).

Spatiotemporal control of gene expression with pulse-generating networks

Its supporting information

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.

A.LuxR/HSL + OR1

Entire designed sequence:(101bp)

5’ GCAT GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG TACCTCTGGCGGTGATAA T GGTTGC TACTAGTAGC GGCCGCTGCA GGCAT 3’

Forward primer(57bp)

5’ GCAT GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG TACCTC3’

Reverse Primer(57bp)

5’ ATGCCTGCAG CGGCCGCTAC TAGTAGCAAC CATTATCACC GCCAGAGGTA CGTAAAC 3’

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 come after lambda -10): GGTTGC

Suffix: TACTAGTAGCGGCCGCTGCAGGCAT

B.LuxR/HSL + OR2

Entire designed sequence:(108bp)

5’ GCAT GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG TAACACCGTG CGTGTTGA TATAGT CGAATAAA TACTAGTAGCGGCCGCTGCAGGCAT 3’

Forward Primer:(60bp)

5’ GCAT GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG TAACACCGTG 3’

Reverse Primer:(60bp)

5’ ATGCCTGCAG CGGCCGCTAC TAGTATTTAT TCGACTATAT CAACACGCAC GGTGTTACGT 3’

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 use -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 come after LuxR -10) CGAATAAA

Suffix TACTAGTAGCGGCCGCTGCAGGCAT

C.LuxR/HSL + 2xOR2

Entire sequence:(140bp)

5’ GCAT GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG CAAGAAAATG GTTTGTTATAGT TAACACCGTG CGTGTTGA TTTATC TAACACCGTG CGTGTTGA TACTAGTAGCGGCCGCTGCAGGCAT 3’

Forward Primer:(76bp)

5’ GCAT GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG CAAGAAAATG GTTTGTTATAGT TAA3’

Reverse Primer:(76bp)

5’ ATGCCTGCAG CGGCCGCTAC TAGTATCAAC ACGCACGGTG TTAGATAAAT CAACACGCAC GGTGTTAACT ATAACA 3’

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 insert the 6 bp spacer backwards so as to break the cI -35 region) TAACACCGTG CGTGTTGA TTTATC TAACACCGTG CGTGTTGA

Suffix: TACTAGTAGCGGCCGCTGCAGGCAT

  Mnt/LacI hybrid promoter responsible:Robert Cool

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.

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 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.

R0073 and R0010

Biobrick prefix gaattcgcggccgcttctagag

Mnt (everything before and including -10) ctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctatagt

LacI last 35bp (everything after -10) tgtgtggaattgtgagcggataacaatttcacacagagtcgtattaattt

Biobrick suffix tactagtagcggccgctgcag

Entire sequence 138bp

Gcat Gaattcgcggccgcttctagag ctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctatagt tgtgtggaattgtgagcggataacaatttcacaca tactagtagcggccgctgcag atgc

Primers (78bp)

Forward 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.

R0073 and R0040

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