[[Davidson_Missouri_W/colony_PCR | Colony PCR]]

[[Standard PCR]]

[[Resuspending Oligos]]

[[Direct Synthesis with Overlapping Oligos]]

[[Nystatin Plates]]

Oligo design for XOR Hybrid Promoters

2008-06-18T16:29:25Z

AnGordon: /* LsrA/cI hybrid promoter */

==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'''

[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/0307571101/DC1 Its supporting information]

[http://partsregistry.org/Part:BBa_R0062 Lux/HSL]

[http://partsregistry.org/Part:BBa_R0051 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 (63bp)'''

5’ GCAT GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG TAACACCGTGCG 3’

'''Reverse Primer (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'''

[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.

'''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 (75bp)'''

gcatgaattcgcggccgcttctagagctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctat

'''Reverse Primer (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'''

[http://partsregistry.org/wiki/index.php?title=Part:BBa_R0073 R0073] [http://partsregistry.org/Part:BBa_R0040 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 (81 bp)'''

Gcatgaattcgcggccgcttctagagctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctatagttcc

'''Reverse primer (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'''

[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]

'''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 [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:

AACCGTGA AAATCAAAAT AGCATAAAT TGTGATCTATT CGTCGGAAAT ATGTGCAATG TCCACCTAAG GTTATGAACA AATTAAAAGC AGAAA'''TACAT T'''TGTTCA'''A'''AA 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: 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.

'''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

Oligo design for XOR Hybrid Promoters

2008-06-18T16:03:13Z

AnGordon: /* LsrA/cI hybrid promoter */

==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'''

[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/0307571101/DC1 Its supporting information]

[http://partsregistry.org/Part:BBa_R0062 Lux/HSL]

[http://partsregistry.org/Part:BBa_R0051 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 (63bp)'''

5’ GCAT GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG TAACACCGTGCG 3’

'''Reverse Primer (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'''

[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.

'''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 (75bp)'''

gcatgaattcgcggccgcttctagagctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctat

'''Reverse Primer (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'''

[http://partsregistry.org/wiki/index.php?title=Part:BBa_R0073 R0073] [http://partsregistry.org/Part:BBa_R0040 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 (81 bp)'''

Gcatgaattcgcggccgcttctagagctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctatagttcc

'''Reverse primer (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'''

[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]

'''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 - 2xOR2 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 [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:

AACCGTGA AAATCAAAAT AGCATAAAT TGTGATCTATT CGTCGGAAAT ATGTGCAATG TCCACCTAAG GTTATGAACA AATTAAAAGC AGAAA'''TACAT T'''TGTTCA'''A'''AA 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.

'''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}'''

GCATCTGCAGCGGCCGCTACTAGTATTATCACCGCCAGAGGTAAAATCTTCAACACGCACGGTGTTAAATGTATTTCTGCTT

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