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

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'''Oligo design for XOR Hybrid Promoters
+
==Lux-cI hybrid promoter==
 +
'''Responsible:Xiao Zhu'''
  
−
  Lux-cI hybrid promoter responsible:Xiao Zhu
+
'''Rationale'''
−
“The effect of cI is dominant over LuxR.”(Ron Weiss, part description for R0065)
 
  
−
[http://partsregistry.org/Part:BBa_R0062 Lux/HSL]
+
This hybrid promoter is designed to respond to activation by LuxR when it is bound to AHL and to repression by the phage lambda cI repressor. The repression needs to win over activation. According to Ron Weiss, “the effect of cI is dominant over LuxR”(Ron Weiss, part description for R0065). 
−
 
 
−
[http://partsregistry.org/Part:BBa_R0051 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”).
+
'''References and links'''
  
 
[http://www.pnas.org/cgi/content/full/101/17/6355 Spatiotemporal control of gene expression with pulse-generating networks]
 
[http://www.pnas.org/cgi/content/full/101/17/6355 Spatiotemporal control of gene expression with pulse-generating networks]
Line 14: Line 12:
 
[http://www.pnas.org/cgi/content/full/0307571101/DC1 Its supporting information]
 
[http://www.pnas.org/cgi/content/full/0307571101/DC1 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.
+
[http://partsregistry.org/Part:BBa_R0062 Lux/HSL]
 +
 
 +
[http://partsregistry.org/Part:BBa_R0051 cI]
  
−
'''A.LuxR/HSL + OR1'''
+
'''Design Considerations'''
  
−
'''Entire designed sequence:'''(101bp)
+
We have come up with three possible designs for this promoter.  Designs A and B are based on the idea of replacing the region of the Lux promoter downstream of the -35 either the OR1 or the OR2 cI half-operator.  A) cI OR1, containing the lambda pR -10 region.  B) cI OR2, added LuxR-10 region. 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.
  
−
5’ GCAT GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG TACCTCTGGCGGTGATAA T GGTTGC TACTAGTAGC GGCCGCTGCA GGCAT 3’
+
We hope that doing in this way could lead a stronger regulation for cI OR1/2. 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”).
  
−
'''Forward primer'''(57bp)
+
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.
  
−
5’ GCAT  GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG TACCTC3’
+
'''Design A - LuxR/HSL + OR1'''
  
−
'''Reverse Primer'''(57bp)
+
'''Desired sequence A (101bp)'''
  
−
5’ ATGCCTGCAG CGGCCGCTAC TAGTAGCAAC CATTATCACC GCCAGAGGTA CGTAAAC 3’
+
5’ GCAT  GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG TACCTCTGGCGGTGATAA T GGTTGC TACTAGTAGC GGCCGCTGCA GATGC 3’
  
−
'''BBa_Prefix''' GCAT  GAATTCGCGGCCGCTTCTAGAG
+
'''Annotation of desired sequence A'''
  
−
'''LuxR/HSL Box''' (only includes region upstream of -35 and the first T of -35) ACCTGTAGGA TCGTACAGG T
+
Oligos have 15bp overlap for direct synthesis
  
−
'''LuxR -35''' TTTACG
+
BBa_Prefix: GCAT GAATTCGCGGCCGCTTCTAGAG
  
−
'''cI Half-Operator OR1''' TACCTCTGGCGGTGATAA
+
LuxR/HSL Box (only includes region upstream of -35 and the first T of -35): ACCTGTAGGA TCGTACAGG T
  
−
'''Lambda pR -10'''  GATAAT
+
LuxR -35:  TTTACG
  
−
'''Spacer''' (naturally come after lambda -10): GGTTGC
+
cI Half-Operator OR1: TACCTCTGGCGGTGATAA
  
−
'''Suffix:''' TACTAGTAGCGGCCGCTGCAGGCAT
+
Lambda pR -10: GATAAT
  
 +
Spacer: (naturally occurring just 3' to -10 in cI promoter): GGTTGC
  
−
'''B.LuxR/HSL + OR2'''
+
Suffix: TACTAGTAGCGGCCGCTGCAG ATGC
  
−
'''Entire designed sequence:'''(108bp)
+
'''Forward primer (58bp)'''
  
−
5’ GCAT  GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG TAACACCGTG CGTGTTGA TATAGT CGAATAAA TACTAGTAGCGGCCGCTGCAGGCAT 3’
+
5’ GCAT  GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG TACCTCT3’
  
−
'''Forward Primer:'''(60bp)
+
'''Reverse Primer (58bp)'''
  
−
5’ GCAT GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG TAACACCGTG 3’
+
5’ GCAT CTGCAG CGGCCGCTAC TAGTAGCAAC CATTATCACC GCCAGAGGTA CGTAAACC 3’
  
−
'''Reverse Primer:'''(60bp)
+
'''Design B - LuxR/HSL + OR2''' '''CHOSEN DESIGN'''
  
−
5’ ATGCCTGCAG CGGCCGCTAC TAGTATTTAT TCGACTATAT CAACACGCAC GGTGTTACGT 3’
+
'''Desired sequence B (108bp)'''
  
−
'''BBa_Prefix''' GCAT  GAATTCGCGGCCGCTTCTAGAG
+
5’ GCAT  GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG TAACACCGTG CGTGTTGA TATAGT CGAATAAA TACTAGTAGCGGCCGCTGCAGATGC 3’
  
−
'''LuxR/HSL Box''' (only includes region upstream of -35 and the first T of -35) ACCTGTAGGA TCGTACAGG T
+
'''Annotated desired sequence B'''
  
−
'''LuxR -35''' TTTACG
+
BBa_Prefix: GCAT  GAATTCGCGGCCGCTTCTAGAG
  
−
'''Lambda Half-Operator OR2''' TAACACCGTG CGTGTTGA
+
LuxR/HSL Box (only includes region upstream of -35 and the first T of -35): ACCTGTAGGA TCGTACAGG T
  
−
'''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.)
+
LuxR -35: TTTACG
  
−
'''Spacer: '''(naturally come after LuxR -10) CGAATAAA
+
Lambda Half-Operator OR2: TAACACCGTG CGTGTTGA
  
−
'''Suffix''' TACTAGTAGCGGCCGCTGCAGGCAT
+
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
  
−
'''C.LuxR/HSL + 2xOR2'''
+
Suffix: TACTAGTAGCGGCCGCTGCAG ATGC
  
−
'''Entire sequence:'''(140bp)
+
'''Forward Primer pLuxR/CI forward (63bp)'''
  
−
5’ GCAT  GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG CAAGAAAATG GTTTGTTATAGT  TAACACCGTG CGTGTTGA  TTTATC TAACACCGTG CGTGTTGA TACTAGTAGCGGCCGCTGCAGGCAT 3’
+
5’ GCAT  GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG TAACACCGTGCG 3’
  
−
'''Forward Primer:'''(76bp)
+
'''Reverse Primer pLuxR/CI reverse(61bp)'''
  
−
5’ GCAT GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG CAAGAAAATG GTTTGTTATAGT  TAA3’
+
5’ GCAT CTGCAG CGGCCGCTAC TAGTATTTAT TCGACTATAT CAACACGCAC GGTGTTACGTA 3’
  
−
'''Reverse Primer:'''(76bp)
 
  
−
5’ ATGCCTGCAG CGGCCGCTAC TAGTATCAAC ACGCACGGTG TTAGATAAAT CAACACGCAC GGTGTTAACT ATAACA 3’
+
'''Design C - LuxR/HSL + 2xOR2'''
  
−
'''BBa_Prefix''' GCAT  GAATTCGCGGCCGCTTCTAGAG
+
'''Desired sequence C (141bp)'''
  
−
'''Lux/HSL'''(the whole sequence, including -35 and -10) ACCTGTAGGA TCGTACAGG TTTACG CAAGAAAATG GTTTGTTATAGT
+
5’ GCAT  GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG CAAGAAAATG GTTTGTTATAGT C TAACACCGTG CGTGTTGA  TTTATC TAACACCGTG CGTGTTGA TACTAGTAGCGGCCGCTGCAGATGC 3’
  
−
'''Lux -35:''' TTTACG
+
'''Annotated desired sequence C'''
  
−
'''Lux -10:''' TATAGT
+
BBa_Prefix: GCAT  GAATTCGCGGCCGCTTCTAGAG
  
−
'''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
+
Lux/HSL (the whole sequence, including -35 and -10): ACCTGTAGGA TCGTACAGG TTTACG CAAGAAAATG GTTTGTTATAGT C
  
−
'''Suffix:''' TACTAGTAGCGGCCGCTGCAGGCAT
+
Lux -35: TTTACG
  
 +
Lux -10: TATAGT
  
−
  Mnt/LacI hybrid promoter responsible:Robert Cool
+
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
  
−
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.
+
Suffix: TACTAGTAGCGGCCGCTGCAG ATGC
  
−
'''Design features: '''
+
'''Forward Primer (78bp)'''
−
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.
 
  
 +
5’ GCAT  GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG CAAGAAAATG GTTTGTTATAGT C TAAC 3’
  
 +
'''Reverse Primer (78bp)'''
  
 +
5’ GCAT CTGCAGCGGCCGCTACTAGTA TCAACACGCA CGGTGTTAGA TAAATCAACA CGCACGGTGT TAGACTATA ACAA 3’
  
−
'''[http://partsregistry.org/wiki/index.php?title=Part:BBa_R0073 R0073] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010 R0010]'''
+
==Mnt/LacI hybrid promoter==
  
−
'''Biobrick prefix'''       gaattcgcggccgcttctagag 
+
'''Responsible:Robert Cool'''
  
 +
'''Rationale'''
  
−
'''Mnt (everything before and including -10)'''      ctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctatagt
+
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'''
  
−
'''LacI last 35bp (everything after -10)'''      tgtgtggaattgtgagcggataacaatttcacacagagtcgtattaattt
+
[http://partsregistry.org/wiki/index.php?title=Part:BBa_R0073 R0073][http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010 R0010]
  
 +
'''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.
  
−
'''Biobrick suffix'''       tactagtagcggccgctgcag
+
'''Entire desired sequence (138 bp)'''
  
 +
Gcat Gaattcgcggccgcttctagag ctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctatagt  tgtgtggaattgtgagcggataacaatttcacaca  tactagtagcggccgctgcag atgc
  
 +
'''Annotated desired sequence'''
  
−
'''Entire sequence 138bp'''
+
Biobrick prefix: GCAT gaattcgcggccgcttctagag 
  
−
Gcat Gaattcgcggccgcttctagag ctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctatagt   tgtgtggaattgtgagcggataacaatttcacaca  tactagtagcggccgctgcag atgc
+
Mnt promoter (everything before and including -10): ctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctatagt
  
 +
LacI binding site (last 35bp, everything after -10):  tgtgtggaattgtgagcggataacaatttcacacagagtcgtattaattt
  
 +
Biobrick suffix: tactagtagcggccgctgcag ATCG
  
−
'''Primers (78bp)'''
 
  
 +
'''Forward Primer pMnt/Lac forward(75bp)'''
  
−
'''Forward'''      gcatgaattcgcggccgcttctagagctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctatagt
+
gcatgaattcgcggccgcttctagagctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctat
  
 +
'''Reverse Primer pMnt/Lac reverse(75 bp)'''
  
−
'''Reverse (reverse complement)''' gcatctgcagcggccgctactagtatgtgtgaaattgttatccgctcacaattccacacaactataggagatctaggt
+
gcatctgcagcggccgctactagtatgtgtgaaattgttatccgctcacaattccacacaactataggagatcta
  
 +
==Mnt/TetR hybrid promoter==
  
 +
'''Responsible:Robert Cool'''
  
 +
'''Background'''
  
 +
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.
  
 +
'''References and links'''
  
 +
[http://partsregistry.org/wiki/index.php?title=Part:BBa_R0073 R0073] [http://partsregistry.org/Part:BBa_R0040 R0040]
  
−
  Mnt/TetR hybrid promoter responsible:Robert Cool
+
'''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 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.
  
−
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.
+
'''Entire desired sequence (149 bp)'''
  
−
'''Design features:'''
+
Gcat gaattcgcggccgcttctagag ctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctatagt tccctatcagtgatagaga actgta atccctatcagtgatagagat tactagtagcggccgctgcag atgc
−
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.
 
  
 +
'''Annotated desired sequence'''
  
 +
Biobrick prefix: GCAT gaattcgcggccgcttctagag
  
−
'''[http://partsregistry.org/wiki/index.php?title=Part:BBa_R0073 R0073] and [http://partsregistry.org/Part:BBa_R0040 R0040]'''
+
Mnt promoter (everything before and including -10):   ctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctatagt
  
 +
TetR binding site, tetR1: tccctatcagtgatagaga
  
−
'''Biobrick prefix'''      gaattcgcggccgcttctagag
+
Spacer: actgta
  
 +
TetR binding site, tetR2: atccctatcagtgatagagat
  
−
'''Mnt (everything before and including -10)'''    ctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctatagt
+
Biobrick suffix: tactagtagcggccgctgcag TAGC
  
 +
'''Forward primer pMnt/Tet forward(81 bp)'''     
  
−
'''tetR1'''      tccctatcagtgatagaga
+
Gcatgaattcgcggccgcttctagagctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctatagttcc
  
−
 
−
'''Spacer'''      actgta
 
  
 +
'''Reverse primer pMnt/Tet reverse(81 bp)'''
  
−
'''TetR 2 binding site'''        atccctatcagtgatagagat
+
gcatctgcagcggccgctactagtaatctctatcactgatagggattacagttctctatcactgatagggaactataggag
  
 +
==LsrA/cI hybrid promoter==
  
−
'''Biobrick suffix'''         tactagtagcggccgctgcag
+
'''Responsible: Andrew Gordon'''
  
 +
'''Rationale'''
  
−
'''Entire sequence 143bp'''
+
This hybrid promoter uses the LsrA promoter that is capable of being repressed by LsrR - induction can occur with phospho-AI-2.  Binding sites for cI repressor also occur. As a result the promoter is off in the absence of AI-2 and on in the presence of AI-2, but always off in the presence of cI. 
  
−
Gcat gaattcgcggccgcttctagag ctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctatagt tccctatcagtgatagaga actgta atccctatcagtgatagagat tactagtagcggccgctgcag atgc
+
'''References and links'''
  
−
'''Primers (81bp)'''
+
[http://jb.asm.org/cgi/content/full/187/6/2066?view=long&pmid=15743955#F7 Wang et al]
 +
[http://fruitfly.org:9005/seq_tools/promoter.html Promoter predictor]
  
−
'''Forward'''       gcatctgcagcggccgctactagtaatctctatcactgatagggattacagttctctatcactgatagggaactataggag
+
'''Design considerations'''
  
 +
The promoter region for LsrA should remain unaltered while cI binding sites (OR2) are introduced downstream.  The OR2 sites with cI bound should not allow transcription. The cI binding sites have a natural 6 bp spacer between them. It would be desirable to position the cI binding sites immediately downstream of the -10 region of the LsrA promoter.  However, experimental evidence for the txn start (+1) appears to be lacking. 
 +
 +
'''Design A - OR1 cI Cassette'''
  
−
'''Reverse (reverse complement)''' gcatctgcagcggccgctactagtaatctctatcactgatagggattctctatcactgatagggaactataggagatct
+
This will involve direct synthesis from two oligos of a biobricked part that contains two copies of the cI binding sites.  These can then be assembled behind the existing pLsrA promoter.
  
−
  LsrA/CI hybrid promoter responsible: Andrew Gordon
+
'''Entire desired sequence'''
  
−
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. 
+
TAACACCGTGCGTGTTGATTTATCTAACACCGTGCGTGTTGA
  
−
'''2xOR2 Repressor Primer Design'''
+
Prefix: gcat gaattcgcggccgcttctagag
  
−
'''2xOR2 Sequence'''
+
5' half of part: TAACACCGTGCGTGTTGATTTATCTAAC
  
−
TAACACCGTGCGTGTTGATTTATCTAACACCGTGCGTGTTGA
+
'''Forward oligo, pLsr/cI forward'''
  
−
'''Forward Sequence'''
+
gcat  gaattcgcggccgcttctagag TAACACCGTGCGTGTTGATTTATCTAAC
  
−
TAACACCGTGCGTGTTGATTTATCTAAC
+
3' half of part: TTGATTTATCTAACACCGTGCGTGTTGA
  
−
'''Prefix'''
+
Reverse complement of 3' half of part: TAACACCGTGCGTGTTGATTTATCTAAC
  
−
gcat gaattcgcggccgcttctagag
+
Suffix: gcat ctgcagcggccgctactagta
  
−
'''Forward Primer LsrAp'''
+
'''Reverse oligo pLsrA/cI reverse'''
  
−
gcat  gaattcgcggccgcttctagag TAACACCGTGCGTGTTGATTTATCTAAC
+
gcat  ctgcagcggccgctactagta  TCAACACGCTCGGTGTTAGATAAATCAA
  
−
'''Forward of Second Squence'''
+
Oligos have a 15 bp overlap for direct synthesis
  
−
TTGATTTATCTAACACCGTGCGTGTTGA
+
'''Design B - de novo pLsrA/cI promoter''' '''CHOSEN DESIGN'''
  
−
'''Reverse Complement of Second Squence'''
+
According to [http://jb.asm.org/cgi/content/full/187/6/2066?view=long&pmid=15743955#F7 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:
  
−
TAACACCGTGCGTGTTGATTTATCTAAC
+
AACCGTGA AAATCAAAAT AGCATAAAT TGTGATCTATT CGTCGGAAAT ATGTGCAATG TCCACCTAAG GTTATGAACA AATTAAAAGC AGAAA'''TACAT T'''TGTTCA'''A'''AA CTCACCTGCA AAACTGAA
  
−
'''Suffix'''
+
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.
  
−
tactagtagcggccgctgcag
+
Truncated LsrA promoter: AACCGTGA AAATCAAAAT AGCATAAAT TGTGATCTATT CGTCGGAAAT ATGTGCAATG TCCACCTAAG GTTATGAACA AATTAAAAGC AGAAATACAT T
  
−
'''Suffix reverse complement'''
+
cI binding site, OR1 and OR2 with 6 bp that naturally exists in Lambda promoter between OR2 and OR1, a 6 bp spacer has been inserted between the two OR sites so as to mutate the cI -35 region: TAACACCGTGCGTGTTGAagATTTTACCTCTGGCGGTGATAA
  
−
gcat  ctgcagcggccgctactagta
+
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.
  
−
'''Reverse Primer LsrAp'''
+
'''Entire Desired Sequence''' AACCGTGA AAATCAAAAT AGCATAAAT TGTGATCTATT CGTCGGAAAT ATGTGCAATG TCCACCTAAG GTTATGAACA AATTAAAAGC AGAAATACAT T  TAACACCGTG CGTGTTGA TTTATC TAACACCGTG CGTGTTGA
  
−
gcat  ctgcagcggccgctactagta  TCAACACGCTCGGTGTTAGATAAATCAA
+
'''New primer needed - "pLsrA/cI reverse" {82 bp}'''
  
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Primers have a 15bp overlap for direct synthesis
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GCAT  CTGCAGCGGCCGCTACTAGTA  TTATCACCGCCAGAGGTAAAATctTCAACACGCACGGTGTTA  AATGTATTTCTGCTT
  
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'''Alternative design for LsrR/CI promoter'''
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Suffix with spacer (reverse complement):  GCAT CTGCAGCGGCCGCTACTAGTA 
  
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According to [http://jb.asm.org/cgi/content/full/187/6/2066?view=long&pmid=15743955#F7 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:
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OR1 and OR2 sites with -35 mutations in the last 2 bases:TAACACCGTGCGTGTTGAagATTTTACCTCTGGCGGTGATAA
  
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AACCGTGA AAATCAAAAT AGCATAAAT TGTGATCTATT CGTCGGAAAT ATGTGCAATG TCCACCTAAG GTTATGAACA AATTAAAAGC AGAAATACAT TTGTTCA'''A'''AA CTCACCTGCA AAACTGAA
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OR1 and OR2 sites with -35 mutations in the last 2 bases(reverse complement): TTATCACCGCCAGAGGTAAAATctTCAACACGCACGGTGTTA
  
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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.
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15 nt from 3' end of truncated LsrA promoter (reverse complement): A ATGTATTTCT GCTT

Latest revision as of 16:51, 18 June 2008

Lux-cI hybrid promoter

Responsible:Xiao Zhu

Rationale

This hybrid promoter is designed to respond to activation by LuxR when it is bound to AHL and to repression by the phage lambda cI repressor. The repression needs to win over activation. According to Ron Weiss, “the effect of cI is dominant over LuxR”(Ron Weiss, part description for R0065).

References and links

Spatiotemporal control of gene expression with pulse-generating networks

Its supporting information

Lux/HSL

cI

Design Considerations

We have come up with three possible designs for this promoter. Designs A and B are based on the idea of replacing the region of the Lux promoter downstream of the -35 either the OR1 or the OR2 cI half-operator. A) cI OR1, containing the lambda pR -10 region. B) cI OR2, added LuxR-10 region. 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.

We hope that doing in this way could lead a stronger regulation for cI OR1/2. 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”).

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 A (101bp)

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

Annotation of desired sequence A

Oligos have 15bp overlap for direct synthesis

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 (58bp)

5’ GCAT GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG TACCTCT3’

Reverse Primer (58bp)

5’ GCAT CTGCAG CGGCCGCTAC TAGTAGCAAC CATTATCACC GCCAGAGGTA CGTAAACC 3’

Design B - LuxR/HSL + OR2 CHOSEN DESIGN

Desired sequence B (108bp)

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

Annotated desired sequence B

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 ATGC

Forward Primer pLuxR/CI forward (63bp)

5’ GCAT GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG TAACACCGTGCG 3’

Reverse Primer pLuxR/CI reverse(61bp)

5’ GCAT CTGCAG CGGCCGCTAC TAGTATTTAT TCGACTATAT CAACACGCAC GGTGTTACGTA 3’


Design C - LuxR/HSL + 2xOR2

Desired sequence C (141bp)

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

Annotated desired sequence C

BBa_Prefix: GCAT GAATTCGCGGCCGCTTCTAGAG

Lux/HSL (the whole sequence, including -35 and -10): ACCTGTAGGA TCGTACAGG TTTACG CAAGAAAATG GTTTGTTATAGT C

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 (78bp)

5’ GCAT GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG CAAGAAAATG GTTTGTTATAGT C TAAC 3’

Reverse Primer (78bp)

5’ GCAT CTGCAGCGGCCGCTACTAGTA TCAACACGCA CGGTGTTAGA TAAATCAACA CGCACGGTGT TAGACTATA ACAA 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

R0073R0010

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 pMnt/Lac forward(75bp)

gcatgaattcgcggccgcttctagagctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctat

Reverse Primer pMnt/Lac reverse(75 bp)

gcatctgcagcggccgctactagtatgtgtgaaattgttatccgctcacaattccacacaactataggagatcta

Mnt/TetR hybrid promoter

Responsible:Robert Cool

Background

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.

References and links

R0073 R0040

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 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.

Entire desired sequence (149 bp)

Gcat gaattcgcggccgcttctagag ctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctatagt tccctatcagtgatagaga actgta atccctatcagtgatagagat tactagtagcggccgctgcag atgc

Annotated desired sequence

Biobrick prefix: GCAT gaattcgcggccgcttctagag

Mnt promoter (everything before and including -10): ctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctatagt

TetR binding site, tetR1: tccctatcagtgatagaga

Spacer: actgta

TetR binding site, tetR2: atccctatcagtgatagagat

Biobrick suffix: tactagtagcggccgctgcag TAGC

Forward primer pMnt/Tet forward(81 bp)

Gcatgaattcgcggccgcttctagagctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctatagttcc


Reverse primer pMnt/Tet reverse(81 bp)

gcatctgcagcggccgctactagtaatctctatcactgatagggattacagttctctatcactgatagggaactataggag

LsrA/cI hybrid promoter

Responsible: Andrew Gordon

Rationale

This hybrid promoter uses the LsrA promoter that is capable of being repressed by LsrR - induction can occur with phospho-AI-2. Binding sites for cI repressor also occur. As a result the promoter is off in the absence of AI-2 and on in the presence of AI-2, but always off in the presence of cI.

References and links

Wang et al Promoter predictor

Design considerations

The promoter region for LsrA should remain unaltered while cI binding sites (OR2) are introduced downstream. The OR2 sites with cI bound should not allow transcription. The cI binding sites have a natural 6 bp spacer between them. It would be desirable to position the cI binding sites immediately downstream of the -10 region of the LsrA promoter. However, experimental evidence for the txn start (+1) appears to be lacking.

Design A - OR1 cI Cassette

This will involve direct synthesis from two oligos of a biobricked part that contains two copies of the cI binding sites. These can then be assembled behind the existing pLsrA promoter.

Entire desired sequence

TAACACCGTGCGTGTTGATTTATCTAACACCGTGCGTGTTGA

Prefix: gcat gaattcgcggccgcttctagag

5' half of part: TAACACCGTGCGTGTTGATTTATCTAAC

Forward oligo, pLsr/cI forward

gcat gaattcgcggccgcttctagag TAACACCGTGCGTGTTGATTTATCTAAC

3' half of part: TTGATTTATCTAACACCGTGCGTGTTGA

Reverse complement of 3' half of part: TAACACCGTGCGTGTTGATTTATCTAAC

Suffix: gcat ctgcagcggccgctactagta

Reverse oligo pLsrA/cI reverse

gcat ctgcagcggccgctactagta TCAACACGCTCGGTGTTAGATAAATCAA

Oligos have a 15 bp overlap for direct synthesis

Design B - de novo pLsrA/cI promoter CHOSEN DESIGN

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, OR1 and OR2 with 6 bp that naturally exists in Lambda promoter between OR2 and OR1, a 6 bp spacer has been inserted between the two OR sites so as to mutate the cI -35 region: TAACACCGTGCGTGTTGAagATTTTACCTCTGGCGGTGATAA

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.

Entire Desired Sequence AACCGTGA AAATCAAAAT AGCATAAAT TGTGATCTATT CGTCGGAAAT ATGTGCAATG TCCACCTAAG GTTATGAACA AATTAAAAGC AGAAATACAT T TAACACCGTG CGTGTTGA TTTATC TAACACCGTG CGTGTTGA

New primer needed - "pLsrA/cI reverse" {82 bp}

GCAT CTGCAGCGGCCGCTACTAGTA TTATCACCGCCAGAGGTAAAATctTCAACACGCACGGTGTTA AATGTATTTCTGCTT

Suffix with spacer (reverse complement): GCAT CTGCAGCGGCCGCTACTAGTA

OR1 and OR2 sites with -35 mutations in the last 2 bases:TAACACCGTGCGTGTTGAagATTTTACCTCTGGCGGTGATAA

OR1 and OR2 sites with -35 mutations in the last 2 bases(reverse complement): TTATCACCGCCAGAGGTAAAATctTCAACACGCACGGTGTTA

15 nt from 3' end of truncated LsrA promoter (reverse complement): A ATGTATTTCT GCTT

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