Team:Edinburgh/crtI

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crtI

AMINO ACID SEQUENCE

MKPTTVIGAGFGGLALAIRLQAAGIPVLLLEQRDKPGGRAYVYE
DQGFTFDAGPTVITDPSAIEELFALAGKQLKEYVELLPVTPFYRLCWESGKVFNYDND
QTRLEAQIQQFNPRDVEGYRQFLDYSRAVFKEGYLKLGTVPFLSFRDMLRAAPQLAKL
QAWRSVYSKVASYIEDEHLRQAFSFHSLLVGGNPFATSSIYTLIHALEREWGVWFPRG
GTGALVQGMIKLFQDLGGEVVLNARVSHMETTGNKIEAVHLEDGRRFLTQAVASNADV
VHTYRDLLSQHPAAVKQSNKLQTKRMSNSLFVLYFGLNHHHDQLAHHTVCFGPRYREL
IDEIFNHDGLAEDFSLYLHAPCVTDSSLAPEGCGSYYVLAPVPHLGTANLDWTVEGPK
LRDRIFAYLEQHYMPGLRSQLVTHRMFTPFDFRDQLNAYHGSAFSVEPVLTQSAWFRP
HNRDKTITNLYLVGAGTHPGAGIPGVIGSAKATAGLMLEDLI*

DNA SEQUENCE

3541 ttgcaagcca ttatgacgac tcatcgttaa agagcgacta catgaaacca actacggtaa
3601 ttggtgcagg cttcggtggc ctggcactgg caattcgtct acaagctgcg gggatccccg
3661 tcttactgct tgaacaacgt gataaacccg gcggtcgggc ttatgtctac gaggatcagg
3721 ggtttacctt tgatgcaggc ccgacggtta tcaccgatcc cagtgccatt gaagaactgt
3781 ttgcactggc aggaaaacag ttaaaagagt atgtcgaact gctgccggtt acgccgtttt
3841 accgcctgtg ttgggagtca gggaaggtct ttaattacga taacgatcaa acccggctcg
3901 aagcgcagat tcagcagttt aatccccgcg atgtcgaagg ttatcgtcag tttctggact
3961 attcacgcgc ggtgtttaaa gaaggctatc taaagctcgg tactgtccct tttttatcgt
4021 tcagagacat gcttcgcgcc gcacctcaac tggcgaaa ct gcag gcatgg agaagcgttt
4081 acagtaaggt tgccagttac atcgaagatg aacatctgcg ccaggcgttt tctttccact
4141 cgctgttggt gggcggcaat cccttcgcca cctcatccat ttatacgttg atacacgcgc
4201 tggagcgtga gtggggcgtc tggtttccgc gtggcggcac cggcgcatta gttcagggga
4261 tgataaagct gtttcaggat ctgggtggcg aagtcgtgtt aaacgccaga gtcagccata
4321 tggaaacgac aggaaacaag attgaagccg tgcatttaga ggacggtcgc aggttcctga
4381 cgcaagccgt cgcgtcaaat gcagatgtgg ttcataccta tcgcgacctg ttaagccagc
4441 accctgccgc ggttaagcag tccaacaaa c tgcag actaa gcgcatgagt aactctctgt
4501 ttgtgctcta ttttggtttg aatcaccatc atgatcagct cgcgcatcac acggtttgtt
4561 tcggcccgcg ttaccgcgag ctgattgacg aaatttttaa tcatgatggc ctcgcagagg
4621 acttctcact ttatctgcac gcgccctgtg tcacggattc gtcactggcg cctgaaggtt
4681 gcggcagtta ctatgtgttg gcgccggtgc cgcatttagg caccgcgaac ctcgactgga
4741 cggttgaggg gccaaaacta cgcgaccgta tttttgcgta ccttgagcag cattacatgc
4801 ctggcttacg gagtcagctg gtcacgcacc ggatgtttac gccgtttgat tttcgcgacc
4861 agcttaatgc ctatcatggc tcagcctttt ctgtggagcc cgttcttacc cagagcgcct
4921 ggtttcggcc gcataaccgc gataaaacca ttactaatct ctacctggtc ggcgcaggca
4981 cgcatcccgg cgcaggcatt cctggcgtca tcggctcggc aaaagcgaca gcaggtttga
5041 tgctggagga tctgatatga ataatccgtc gttactcaat catgcggtcg aaacgatggc

BIOBRICKTM PRIMERS

Forward: gat gaattc gcggccgc t tctag atg aaa cca act acg gta att g
Reverse: gct actagt a tta tt a tat cag atc ctc cag cat c

MUTAGENIC PRIMERS

The native crtI sequence contains two internal PstI sites. These were removed by MABEL site directed mutagenesis by Douglas Leslie (Honours project, 2008). Initially mutagenesis by megaprimer PCR was attempted (see iGEM 2007 lab book page 57, also file 'mutagenicPrimersForcrtI20aug07.doc'), using primers crtImut1 and crtImut2, but this was not successful, so primers crtImutf1 and crtImutf2 were ordered so that the MABEL protocol (invented for this purpose) could be used instead.The mutagenic primers were as follows:

   * crtImut1: gct tct cca tgc ttg cag ttt cgc cag
   * crtImut2: cat gcg ctt agt ttg cag ttt gtt gg
   * crtImutf1: gtt tac agt aag gtt gcc : none, no, 51.81 C.
   * crtImutf2: agt aac tct ctg ttt gtg c : very weak, no, 51.46 C

In both cases, the PstI site CTGCAG was converted to CTGCAA, a silent mutation.

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