Oligo design for XOR Hybrid Promoters - Revision history

BoCool: /* Mnt/TetR hybrid promoter */

2008-06-18T16:51:27Z

‎Mnt/TetR hybrid promoter

BoCool: /* Mnt/LacI hybrid promoter */

2008-06-18T16:50:32Z

‎Mnt/LacI hybrid promoter

BoCool: /* Lux-cI hybrid promoter */

2008-06-18T16:49:40Z

‎Lux-cI hybrid promoter

ToEckdahl: /* LsrA/cI hybrid promoter */

2008-06-18T16:37:47Z

‎LsrA/cI hybrid promoter

AnGordon: /* LsrA/cI hybrid promoter */

2008-06-18T16:29:25Z

‎LsrA/cI hybrid promoter

AnGordon: /* LsrA/cI hybrid promoter */

2008-06-18T16:03:13Z

‎LsrA/cI hybrid promoter

BoCool: /* LsrA/cI hybrid promoter */

2008-06-18T15:58:26Z

‎LsrA/cI hybrid promoter

BoCool: /* Lux-cI hybrid promoter */

2008-06-18T15:57:48Z

‎Lux-cI hybrid promoter

BoCool: /* Lux-cI hybrid promoter */

2008-06-18T15:56:41Z

‎Lux-cI hybrid promoter

BoCool: /* Mnt/LacI hybrid promoter */

2008-06-18T15:49:38Z

‎Mnt/LacI hybrid promoter

@@ Line 186: / Line 186: @@
 Biobrick suffix: tactagtagcggccgctgcag TAGC
-'''Forward primer (81 bp)'''
+'''Forward primer pMnt/Tet forward(81 bp)'''
 Gcatgaattcgcggccgcttctagagctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctatagttcc
-'''Reverse primer (81 bp)'''
+'''Reverse primer pMnt/Tet reverse(81 bp)'''
 gcatctgcagcggccgctactagtaatctctatcactgatagggattacagttctctatcactgatagggaactataggag

@@ Line 145: / Line 145: @@
-'''Forward Primer (75bp)'''
+'''Forward Primer pMnt/Lac forward(75bp)'''
 gcatgaattcgcggccgcttctagagctcgaggtgagtgcacagtactaggtccacggtgacctagatctcctat
-'''Reverse Primer (75 bp)'''
+'''Reverse Primer pMnt/Lac reverse(75 bp)'''
 gcatctgcagcggccgctactagtatgtgtgaaattgttatccgctcacaattccacacaactataggagatcta

@@ Line 78: / Line 78: @@
 Suffix: TACTAGTAGCGGCCGCTGCAG ATGC
-'''Forward Primer (63bp)'''
+'''Forward Primer pLuxR/CI forward (63bp)'''
 ’ GCAT  GAATTCGCGGCCGCTTCTAGAG ACCTGTAGGA TCGTACAGG TTTACG TAACACCGTGCG 3’
-'''Reverse Primer (61bp)'''
+'''Reverse Primer pLuxR/CI reverse(61bp)'''
 ’ GCAT CTGCAG CGGCCGCTAC TAGTATTTAT TCGACTATAT CAACACGCAC GGTGTTACGTA 3’

@@ Line 250: / Line 250: @@
 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
+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.

@@ Line 212: / Line 212: @@
 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'''
+'''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.
@@ Line 250: / Line 250: @@
 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
+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.
@@ Line 258: / Line 258: @@
 '''New primer needed - "pLsrA/cI reverse" {82 bp}'''
-GCATCTGCAGCGGCCGCTACTAGTATTATCACCGCCAGAGGTAAAATCTTCAACACGCACGGTGTTAAATGTATTTCTGCTT
+GCAT  CTGCAGCGGCCGCTACTAGTA  TTATCACCGCCAGAGGTAAAATctTCAACACGCACGGTGTTA  AATGTATTTCTGCTT
 Suffix with spacer (reverse complement):  GCAT CTGCAGCGGCCGCTACTAGTA
-x OR2 sites (reverse complement): TCAACACG CACGGTGTTA GATAAA TCAACACG CACGGTGTTA
-nt from 3' end of truncated LsrA promoter (reverse complement): A ATGTATTTCT GCTTTTAATT
+OR1 and OR2 sites with -35 mutations in the last 2 bases:TAACACCGTGCGTGTTGAagATTTTACCTCTGGCGGTGATAA

@@ Line 240: / Line 240: @@
 Oligos have a 15 bp overlap for direct synthesis
-'''Design B - de novo pLsrA/cI promoter'''
+'''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:

@@ Line 56: / Line 56: @@
 ’ GCAT CTGCAG CGGCCGCTAC TAGTAGCAAC CATTATCACC GCCAGAGGTA CGTAAACC 3’
-'''Design B - LuxR/HSL + OR2'''
+'''Design B - LuxR/HSL + OR2''' '''CHOSEN DESIGN'''
 '''Desired sequence B (108bp)'''