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User:Allancrossman/Gibson
From 2011.igem.org
(Difference between revisions)
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'''Primer strategy:''' to create homology between part A and part B, use >= 20 bases from the end of A and the start of B; gene A's reverse primer is the reverse complement of gene B's forward primer. It is possible to add bases in the junction. If a stop codon is needed, I only use one stop codon in case we need to use a template that completely lacks stops; in that case one stop codon will be added by the PCR. | '''Primer strategy:''' to create homology between part A and part B, use >= 20 bases from the end of A and the start of B; gene A's reverse primer is the reverse complement of gene B's forward primer. It is possible to add bases in the junction. If a stop codon is needed, I only use one stop codon in case we need to use a template that completely lacks stops; in that case one stop codon will be added by the PCR. | ||
| - | '''Note that (before any PCR) the INP part has to be created by attaching linker2fs and linker2rt to BBa_K523008.''' | + | '''Note that (before any PCR) the INP part has to be created by attaching linker2fs and linker2rt to BBa_K523008. Note that we must use the new, serine-rich linker to avoid certain issues.''' |
==Plac-RBS-LacZ-RBS-INP[spacer]-malS== | ==Plac-RBS-LacZ-RBS-INP[spacer]-malS== | ||
Revision as of 11:49, 8 August 2011
In the event that a straight-up Gibson assembly becomes desirable...
Primer strategy: to create homology between part A and part B, use >= 20 bases from the end of A and the start of B; gene A's reverse primer is the reverse complement of gene B's forward primer. It is possible to add bases in the junction. If a stop codon is needed, I only use one stop codon in case we need to use a template that completely lacks stops; in that case one stop codon will be added by the PCR.
Note that (before any PCR) the INP part has to be created by attaching linker2fs and linker2rt to BBa_K523008. Note that we must use the new, serine-rich linker to avoid certain issues.
Plac-RBS-LacZ-RBS-INP[spacer]-malS
- Templates:
- Vector = pSB1C3
- Plac-RBS-LacZ-RBS = <partinfo>BBa_K523005</partinfo>
- INP[spacer] - make by cutting <partinfo>BBa_K523008</partinfo> with SpeI and ligating linker2fs and linker2rt
- malS = <partinfo>BBa_K523001</partinfo>
Primers
Vector
- Forward: gtctgggcagggcaacagtaa tactagtagcggccgctgcag
- Reverse: gttaacggcgggatataacatgag ctctagaagcggccgcgaattc
Plac-RBS-LacZ-RBS
- Forward: gaattcgcggccgcttctagag ctcatgttatatcccgccgttaac
- Reverse: gctttatccagggtcatTAT TTAcctccttgagctctagtatc
INP[spacer]
- Forward: gatactagagctcaaggaggTAA ATAatgaccctggataaagc
- Reverse: cccggagaagtccagctggc ccgccacgctgctcgggctg
malS
- Forward: cagcccgagcagcgtggcgg gccagctggacttctccggg
- Reverse: ctgcagcggccgctactagta ttactgttgccctgcccagac
Plac-RBS-LacZ-RBS-INP[spacer]-Endoglucanase
- Templates:
- Vector = pSB1C3
- Plac-RBS-LacZ-RBS = <partinfo>BBa_K523005</partinfo>
- INP[spacer] - make by cutting <partinfo>BBa_K523008</partinfo> with SpeI and ligating linker2fs and linker2rt
- Endoglucanase - use <partinfo>BBa_K118023</partinfo>. Don't use anything else; in particular, our previous primers to PCR this altered some codons and the product doesn't match the new primers below. So use K118023.
Primers
Vector
- Forward: cgcgcaacgccaggtggtaa tactagtagcggccgctgcag
- Reverse: [as for malS]
Plac-RBS-LacZ-RBS
- Forward: [as for malS]
- Reverse: [as for malS]
INP[spacer]
- Forward: [as for malS]
- Reverse: gtcgacgcggcagccgggagc ccgccacgctgctcgggctg
Endoglucanase
- Forward: cagcccgagcagcgtggcgg gctcccggctgccgcgtcgac
- Reverse: ctgcagcggccgctactagta ttaccacctggcgttgcgcg
Plac-RBS-LacZ-RBS-INP[spacer]-Exoglucanase
- Templates:
- Vector = pSB1C3
- Plac-RBS-LacZ-RBS = <partinfo>BBa_K523005</partinfo>
- INP[spacer] - make by cutting <partinfo>BBa_K523008</partinfo> with SpeI and ligating linker2fs and linker2rt
- Exoglucanase = <partinfo>BBa_K118022</partinfo>
Primers
Vector
- Forward: cgccctgcacggtcggctaa tactagtagcggccgctgcag
- Reverse: [as for malS]
Plac-RBS-LacZ-RBS
- Forward: [as for malS]
- Reverse: [as for malS]
INP[spacer]
- Forward: [as for malS]
- Reverse: gcctccttgagcgtggtcgc ccgccacgctgctcgggctg
Exoglucanase
- Forward: cagcccgagcagcgtggcgg gcgaccacgctcaaggaggc
- Reverse: ctgcagcggccgctactagta ttagccgaccgtgcagggcg
Plac-RBS-LacZ-RBS-INP[spacer]-B.Glucosidase
- Templates:
- Vector = pSB1C3
- Plac-RBS-LacZ-RBS = <partinfo>BBa_K523005</partinfo>
- INP[spacer] - make by cutting <partinfo>BBa_K523008</partinfo> with SpeI and ligating linker2fs and linker2rt
- B-glucosidase = <partinfo>BBa_K392008</partinfo>
Primers
Vector
- Forward: ccgcgacctaccagccctga tactagtagcggccgctgcag
- Reverse: [as for malS]
Plac-RBS-LacZ-RBS
- Forward: [as for malS]
- Reverse: [as for malS]
INP[spacer]
- Forward: [as for malS]
- Reverse: gcctgctggaaccggtcgcc ccgccacgctgctcgggctg
B-Glucosidase
- Forward: cagcccgagcagcgtggcgg ggcgaccggttccagcaggc
- Reverse: ctgcagcggccgctactagta tcagggctggtaggtcgcgg
Appendix: Sequences
Only the relevant parts of the sequence are shown; any flanking regions have not been shown, since they are irrelevant to the planning of primers. Since Gibson is scarless, the concatenation of the sequences involved is the final sequence.
Vector pSB1C3
tactagtagcggccgctgcagtccggcaaaaaagggcaaggtgtcaccaccctgcccttt ttctttaaaaccgaaaagattacttcgcgttatgcaggcttcctcgctcactgactcgct gcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggtt atccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggc caggaaccgtaaaaaggccgcgttgctggcgtttttccacaggctccgcccccctgacga gcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagata ccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttac cggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctg taggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccc cgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaag acacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgt aggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagt atttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttg atccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattac gcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctca gtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcac ctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaac ttggtctgacagctcgaggcttggattctcaccaataaaaaacgcccggcggcaaccgag cgttctgaacaaatccagatggagttctgaggtcattactggatctatcaacaggagtcc aagcgagctcgatatcaaattacgccccgccctgccactcatcgcagtactgttgtaatt cattaagcattctgccgacatggaagccatcacaaacggcatgatgaacctgaatcgcca gcggcatcagcaccttgtcgccttgcgtataatatttgcccatggtgaaaacgggggcga agaagttgtccatattggccacgtttaaatcaaaactggtgaaactcacccagggattgg ctgagacgaaaaacatattctcaataaaccctttagggaaataggccaggttttcaccgt aacacgccacatcttgcgaatatatgtgtagaaactgccggaaatcgtcgtggtattcac tccagagcgatgaaaacgtttcagtttgctcatggaaaacggtgtaacaagggtgaacac tatcccatatcaccagctcaccgtctttcattgccatacgaaattccggatgagcattca tcaggcgggcaagaatgtgaataaaggccggataaaacttgtgcttatttttctttacgg tctttaaaaaggccgtaatatccagctgaacggtctggttataggtacattgagcaactg actgaaatgcctcaaaatgttctttacgatgccattgggatatatcaacggtggtatatc cagtgatttttttctccattttagcttccttagctcctgaaaatctcgataactcaaaaa atacgcccggtagtgatcttatttcattatggtgaaagttggaacctcttacgtgcccga tcaactcgagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaa aataggcgtatcacgaggcagaatttcagataaaaaaaatccttagctttcgctaaggat gatttctggaattcgcggccgcttctagag
Plac-RBS-LacZ-RBS
(Three bases added at end to give RBS room to work)
ctcatgttatatcccgccgttaaccaccatcaaacaggattttcgcctgctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcg gtgaagggcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcat taatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttac actttatgcttccggctcgtatgttgtgtgaaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacggattcactggccgtcg ttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccg caccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctttgcctggtttccggcaccagaagcggtgccggaaagctggctggagtga tactagagctcaaggaggTAA
INP[with serine-rich linker]
(Three bases added at start to give RBS room to work)
ATAatgaccctggataaagcgctggtgctgcgcacttgtgccaataatatggccgaccattgtggcctgatctggcctgcttctggtacagttgaaagc cgctattggcagtctacacgtcgtcacgaaaacgggctggttgggctgctgtggggtgccggaacatccgcctttctgagcgttcatgccgatgctcgtt ggattgtttgtgaggttgctgttgccgacatcattagcctggaagaaccgggtatggtgaaatttcctcgtgctgaagtggtccatgttggtgatcgtat tagcgcctcccatttcatttcagctcgtcaggctgatccagcaagcacatcaacaagcacctctaccagtacactgacaccaatgccaaccgctatccct actcctatgccggctgtagcctctgttactctgcctgtggccgaacaagctcgccacgaagtctttgacgttgcctctgtgtctgctgctgctgctcctg ttaacactctgcctgttaccacgcctcaaaatctgcaaacggcgacctatggctcaacactgagtggtgataaccattctcgtctgattgccgggtatgg ctctaacgaaaccgccggaaaccattctgatctgatcggtggtcacgattgtacgctgatggcaggtgatcaatctcgtctgaccgctggcaaaaattcc gttctgaccgctggggcacgttctaaactgatcgggagcgaaggctctactctgtccgccggagaagattctaccctgatctttcgtctgtgggatggta aacgttatcgtcagctggtcgctcgtacaggtgaaaatggtgtggaagccgacattccgtattatgtgaatgaggacgatgatattgtggacaaaccgga cgaagatgatgattggatcgaagtgaaaggtactagcagcagcattgcgagcagcagcccgagcagcgtggcgg
malS [mature peptide only, aa 18 onwards]
gccagctggacttctc cggggtttcccgcctttagcgaacaggggacaggaacatttgtcagccacgcgcagttgcccaaaggtacgcgtccactaacgctaaattttgaccaaca gtgctggcagcctgcggatgcgataaaactcaatcagatgctttccctgcaaccttgtagcaacacgccgcctcaatggcgattgttcagggacggcgaa tatacgctgcaaatagacacccgctccggtacgccaacattgatgatttccatccagaacgccgccgaaccggtagcaagcctggtccgtgaatgcccga aatgggatggattaccgctcacagtggatgtcagcgccactttcccggaaggagccgccgtacgggattattacagccagcaaattgcgatagtgaagaa cggtcaaataatgttacaacccgctgccaccagcaacggtttactcctgctggaacgggcagaaactgacacatccgcccctttcgactggcataacgcc acggtttactttgtgctgacagatcgtttcgaaaacggcgatcccagtaatgaccagagttacggacgtcataaagacggtatggcggaaattggcactt ttcacggcggcgatttacgcggcctgaccaacaaactggattacctccagcagttgggcgttaatgctttatggataagcgccccatttgagcaaattca cggctgggtcggcggcggtacaaaaggcgatttcccgcattatgcctaccacggttattacacacaggactggacgaatcttgatgccaatatgggcaac gaagccgatctacggacgctggttgatagcgcacatcagcgcggtattcgtattctctttgatgtcgtgatgaaccacaccggctatgccacgctggcgg atatgcaggagtatcagtttggcgcgttatatctttctggtgacgaagtgaaaaaatcgctgggtgaacgctggagcgactggaaacctgccgccgggca aacctggcatagctttaacgattacattaatttcagcgacaaaacaggctgggataaatggtggggaaaaaactggatcagaacggatatcggcgattac gacaatcctggattcgacgatctcactatgtcgctagcctttttgccggatatcaaaaccgaatcaactaccgcttctggtctgccggtgttctataaaa acaaaatggatacccacgccaaagccattgacggctatacgccgcgcgattacttaacccactggttaagtcagtgggtccgcgactatgggattgatgg ttttcgggtcgataccgccaaacatgttgagttgcccgcctggcagcaactgaaaaccgaagccagcgccgcgcttcgcgaatggaaaaaagctaacccc gacaaagcattagatgacaaacctttctggatgaccggtgaagcctggggccacggcgtgatgcaaagtgactactatcgccacggcttcgatgcgatga tcaatttcgattatcaggagcaggcggcgaaagcagtcgactgtctggcgcagatggatacgacctggcagcaaatggcggagaaattgcagggtttcaa cgtgttgagctacctctcgtcgcatgatacccgcctgttccgtgaagggggcgacaaagcagcagagttattactattagcgccaggcgcggtacaaatc ttttatggtgatgaatcctcgcgtccgttcggtcctacaggttctgatccgctgcaaggtacacgttcggatatgaactggcaggatgttagcggtaaat ctgccgccagcgtcgcgcactggcagaaaatcagccagttccgcgcccgccatcccgcaattggcgcgggcaaacaaacgacacttttgctgaagcaggg ctacggctttgttcgtgagcatggcgacgataaagtgctggtcgtctgggcagggcaacagtaa
Endoglucanase BBa_K118023 [mature peptide only, aa 32 onwards]
gctcccg gctgccgcgtcgactacgccgtcaccaaccagtggcccggcggcttcggcgccaacgtcacgatcaccaacctcggcgaccccgtctcgtcgtggaagct cgactggacctacaccgcaggccagcggatccagcagctgtggaacggcaccgcgtcgaccaacggcggccaggtctccgtcaccagcctgccctggaac ggcagcatcccgaccggcggcacggcgtcgttcgggttcaacggctcgtgggccgggtccaacccgacgccggcgtcgttctcgctcaacggcaccacct gcacgggcaccgtgccgacgaccagccccacgccgaccccgacgccgacgacccccacgccgacgccgaccccgacccccacccccacgccgacggtcac gccgcagccgaccagcggcttctacgtcgacccgacgacgcagggctaccgcgcgtggcaggccgcgtccggcacggacaaggcgctgctcgagaagatc gcgctcaccccgcaggcgtactgggtcggcaactgggccgacgcgtcgcacgcgcaggccgaggtcgccgactacaccggccgcgccgtcgcggccggga agacgccgatgctcgtcgtctacgcgatcccgggccgcgactgcggctcgcactccggcggtggtgtgtccgagtccgagtacgcgcgctgggtcgacac cgtcgcgcagggcatcaagggcaacccgatcgtgatcctcgagcccgacgcgctcgcgcagctcggcgactgctccggccagggtgaccgcgtcggcttc ctcaagtacgccgccaagtcgctcaccctcaagggcgcgcgcgtctacatcgacgcgggccacgcgaagtggctgtcggtcgacacgccggtgaaccgcc tcaaccaggtcggcttcgagtacgcggtgggcttcgcgctcaacacgtcgaactaccagacgacggcggacagcaaggcgtacggccagcagatctcgca gcggctgggcggcaagaagttcgtcatcgacacctcgcgcaacggcaacggctcgaacggcgagtggtgcaacccgcgcggccgcgcgctcggcgaacgc ccggtcgcggtgaacgacggctccggcctggacgcgctcctgtgggtcaagctgcccggcgagtccgacggcgcgtgcaacggcggcccggccgccggcc agtggtggcaggagatcgccctggagatggcgcgcaacgccaggtggtaa
Exoglucanase BBa_K118022 [mature peptide only, aa 43 onwards]
gcgaccacgctcaaggaggccgccgacggcgccggccgggacttcggcttcgcgctcgaccccaaccggctctc ggaggcgcagtacaaggcgatcgccgacagcgagttcaacctcgtcgtcgccgagaacgcgatgaagtgggacgccaccgagccctcgcagaacagcttc tccttcggcgcgggcgaccgcgtcgcgagctacgccgccgacaccggcaaggagctgtacggccacacgctcgtatggcactcgcagctgcccgactggg cgaagaacctcaacggctccgcgttcgagagcgcgatggtcaaccacgtgacgaaggtcgccgaccacttcgagggcaaggtcgcgtcgtgggacgtcgt caacgaggcgttcgccgacggcggcggccgccggcaggactcggcgttccagcagaagctcggcaacggctacatcgagaccgcgttccgggcggcacgt gcggcggacccgaccgccaagctgtgcatcaacgactacaacgtcgagggcatcaacgcgaagagcaactcgctctacgacctcgtcaaggacttcaagg cgcgcggcgtcccgctcgactgcgtcgggttccagtcgcacctcatcgtcggccaggtgccgggcgacttccggcagaacctgcaacggttcgcggacct gggcgtggacgtgcgcatcaccgagctcgacatccgcatgcggacgccctccgacgcgaccaagctcgcgacccaggcggccgactacaagaaggtcgtg caggcctgcatgcaggtgacccgctgccagggcgtgaccgtctggggcatcaccgacaagtactcgtgggtgccggacgtcttcccgggcgagggggccg cgctggtgtgggacgcgagctacgccaagaagccggcctacgccgccgtgatggaggccttcggcgcgagcccgacgccgacgcccaccacgccgacccc gacgcccacgacgccgacgccgaccccgacgtccggtccggccgggtgccaggtgctgtggggcgtcaaccagtggaacaccggcttcaccgcgaacgtc accgtgaagaacacgtcctccgctccggtcgacggctggacgctcacgttcagcttcccgtccggccagcaggtcacccaggcgtggagctcgacggtca cgcagtccggctcggccgtgacggtccgcaacgccccgtggaacggctcgatcccggcgggcggcaccgcgcagttcggcttcaacggctcgcacacggg caccaacgccgcgccgacggcgttctcgctcaacggcacgccctgcacggtcggctaa
B-glucosidase BBa_K392008 (or whatever we have)
ggcgaccggttccagcaggccggtcgcccacgccgccgcggcccggcgagggccgttaaccgtaccggtcaagaagacgcgtcgacggggtcgaggg agcggtcccacgcgtgtatcgtatcgtttcgacaccgccacccggccaccgggcacgcaccggggacgcagcagtccccgccccggccaccccctgtcac cgaaacccgaaggaccctcatgaccaccacgcgcccctcgggccgccagttctccgacgacttcctgtggggctcggccacggcctcctaccagatcgag ggcgcgcacgacgagggcggtcgcggcccgtcgatctgggacacgttctcccgcaccccgggcaaggtcctcaacggcgacaccggcgacgtcgcggtcg accactaccaccgcgtccccgaggacgtcgagatcatgaagagcctcggcctccaggcctaccggttctcgatcgcctggccccgcatccagccgaccgg ctcgggcgagttcaaccaggccgggctcgacttctactccgacctcgtcgaccggctcatcgccgccggcatcaagcccgtcgcgacgctgtaccactgg gacctcccgcagcccctggaggacgagggcggctgggcgaaccgcgcgacggcgtaccggttcgtcgagtacgcgcgcaagctcgccgaggtcctcggca agcgcgtcgacctgtggaccacgctcaacgagccgtggtgctccgcgttcctcggctacgcctcaggcgtgcacgccccggcgtcaccgacccggtcaag gccctgcgcgccgtccaccacctcaacctcgcgcacggcctcgcgggccgcgcgatccgcgaggtcctcggcgagaacgcgcccgtctcgatcacgtgaa cctgcacgtgacgcgcgccgccgacgactccgccgactccgtcgaggccaagcgccgcatcgacaccatcgcgaacgaggtcttcctcggcccgctgctc gacggcgagtaccccaaggaggtcttcgcggacaccgcgcaccacaccgactggtccttcgtcgagccgggcgacctcgagctcatccgcatcccgctca ccgtgctcggcgtcaactactactcgaccggccgcgtgaagaagggcagcgcgcgggcgccgacccgggcaagccgggccccgacggccaccgcgcctcc gagcactcctcgtgggtcggcgccgacgaggtcgagtggctcccgcagcccggcccgcacaccgcgatgggctggaacatcgagcccgacggcctcgtcg acctgctgctcgagctgcgcgaccgttaccccagccagcccctggcgatcacggagaacggtgccgcgttctacgacacggtcagcgaggacggccgcgt gcacgaccccgagcgcgtcggctacctgcacgaccacgtcgacgccgtcggcgaggccatcgacaagggcgccgacgtgcgcgcctacttcgtgtggtcg ctgctcgacaacttcgagtggcgctacggctacgaccgccggttcggcatcgtgcgcgtcgactacgacacgcacgagcgcatcgtgaaggactcgggcc tctggtaccgcgagctcgtgcgcacccgcaccatcgctccggccgaggacgccgcgacctaccagccctga
