Team:Paris Bettencourt/Notebook/Drug Screening/Monday 10th June.html

Drug Screening

Day Number^suffix Month

Design of SirA, FdrA, and FdxC genes for synthesis

Mycobacterium tuberculosis
Reference: Pinto et al. 2007. Sulfite Reduction in Mycobacteria doi: 10.1128/JB.00487-07
Primers:
TBsirA F      GGAATTCCATATGTCCGCGAAGGAGAACCCC
TBsirA R      AAGGAAAAAAGCGGCCGCTCATCGCAGGTCGTCCTCCTCGGCCCGGAT We blasted the above primers used to clone sirA from M. tuberculosis into E. coli. We then extracted the sequence from genbank to retrieve the following sequence which includes the restriction sites introduced by Pinto et al.

ggaattccat atgtccgcga aggagaaccc ccaaatgacc actgcacgtc
ccgccaaggc tcgaaatgag ggccagtggg cgctgggaca tcgcgagcca
ctcaacgcca acgaagagct gaagaaggcc ggcaacccgc tcgacgtgcg
ggagcgcatc gaaaacatct acgccaaaca gggtttcgac agcatcgaca
agaccgacct gcgagggcgc tttcgctggt ggggcctgta cacccagcgt
gagcagggct acgacggcac ctggaccggt gacgacaaca tcgacaagct
cgaggccaaa tacttcatga tgcgggtgcg ttgcgacggc ggcgcgctct
cggctgccgc gctgcgcacg ctgggccaga tctcgacgga gttcgcgcgc
gataccgccg atatctccga ccggcagaac gtgcaatacc actggatcga
agtggaaaac gtccctgaaa tctggcgacg gttagacgat gtcggactgc
agaccaccga ggcgtgcggt gactgcccgc gggtagtgct gggctcgccg
ttggccggcg agtcgctcga cgaagtgctc gacccgacct gggcgatcga
ggagatcgtg cgtcgctaca tcggcaagcc cgacttcgcc gacttgccgc
gcaagtacaa gaccgccatc tctggcctgc aggacgtcgc gcacgagatc
aacgacgtcg ccttcatcgg cgtcaaccat cccgagcacg gaccaggcct
ggatctgtgg gtgggcggtg gactgtcgac caacccgatg ctggcccagc
gggtcggcgc ctgggttcca ctgggcgaag tgcccgaggt gtgggcggcg
gtcacctcgg tgtttcgcga ctacggctac cggcgactgc gcgccaaggc
ccggctgaaa tttctgatca aagactgggg catagcgaag ttccgcgaag
tgctcgaaac cgagtacctc aagcgtccgc tgatcgacgg tccggccccc
gaaccggtca agcatccgat cgaccacgtc ggggtgcaac gactcaagaa
cgggctcaac gccgtcggag tcgcccccat cgccgggcgg gtatcgggca
ccatcctcac ggcggtcgcc gacctgatgg cgcgggccgg ttccgaccgg
atccggttca ccccctacca gaagctggtc atcctcgaca ttccggacgc
cttgctcgac gacttgatcg ccggtctgga cgcgctgggg ctgcagtcgc
gcccgtcgca ttggcgccgg aacttgatgg cgtgcagcgg gattgagttc
tgcaagttgt cattcgccga aacccgggtt cgagcacagc atttggtgcc
cgagctggaa cgccggcttg aggacatcaa ctcgcagctc gacgtaccga
tcaccgtcaa catcaacggc tgcccgaact catgtgcgcg aattcaaatc
gccgacatcg gattcaaggg acagatgatc gacgacggac acggcggctc
cgtcgaaggc ttccaggtgc atctgggcgg acacctcggc ctggatgccg
gattcggccg caaactgcgc cagcacaagg tcaccagtga cgaactcggc
gactacatcg accgggtggt gcgcaacttc gtcaaacacc gcagcgaagg
tgaacgcttc gcgcagtggg tcatccgggc cgaggaggac gacctgcgat
gagcggccgc ttttttcctt

This sequence was then inserted into Geneious. The PstI and EcoRI sites were manually removed and replaced with silent mutations. A NcoI site was inserted at the start codon and an extra glycine was added after the start codon for the NcoI site to function properly. We chose glycine as it is small an unlikely to interfere with either polar or non-polar domains within the protein. We used GGC as the codon as it is the highest used glycine codon in e. coli. We also added an AvrII site on the 3’ end of the site. We then added biobrick restriction sites on the ends of the sequence (EcoRI, XbaI, SpeI, PstI). This will allow us to clone into our expression vector pACYCDuet-1 with NcoI and AvrII enzymes and into a biobrick registry with EcoRI and PstI. The insertion into pACYCDuet-1 removes both protein tags and cuts out the second MCS resulting in vector pACYCDuet-TBsirA. The resulting sequence for TB SirA follows:

catggaattc tagaccatgg gctccgcgaa ggagaacccc caaatgacca ctgcacgtcc cgccaaggct cgaaatgagg gccagtgggc gctgggacat cgcgagccac tcaacgccaa cgaagagctg aagaaggccg gcaacccgct cgacgtgcgg gagcgcatcg aaaacatcta cgccaaacag ggtttcgaca gcatcgacaa gaccgacctg cgagggcgct ttcgctggtg gggcctgtac acccagcgtg agcagggcta cgacggcacc tggaccggtg acgacaacat cgacaagctc gaggccaaat acttcatgat gcgggtgcgt tgcgacggcg gcgcgctctc ggctgccgcg ctgcgcacgc tgggccagat ctcgacggag ttcgcgcgcg ataccgccga tatctccgac cggcagaacg tgcaatacca ctggatcgaa gtggaaaacg tccctgaaat ctggcgacgg ttagacgatg tcggacttca gaccaccgag gcgtgcggtg actgcccgcg ggtagtgctg ggctcgccgt tggccggcga gtcgctcgac gaagtgctcg acccgacctg ggcgatcgag gagatcgtgc gtcgctacat cggcaagccc gacttcgccg acttgccgcg caagtacaag accgccatct ctggccttca ggacgtcgcg cacgagatca acgacgtcgc cttcatcggc gtcaaccatc ccgagcacgg accaggcctg gatctgtggg tgggcggtgg actgtcgacc aacccgatgc tggcccagcg ggtcggcgcc tgggttccac tgggcgaagt gcccgaggtg tgggcggcgg tcacctcggt gtttcgcgac tacggctacc ggcgactgcg cgccaaggcc cggctgaaat ttctgatcaa agactggggc atagcgaagt tccgcgaagt gctcgaaacc gagtacctca agcgtccgct gatcgacggt ccggcccccg aaccggtcaa gcatccgatc gaccacgtcg gggtgcaacg actcaagaac gggctcaacg ccgtcggagt cgcccccatc gccgggcggg tatcgggcac catcctcacg gcggtcgccg acctgatggc gcgggccggt tccgaccgga tccggttcac cccctaccag aagctggtca tcctcgacat tccggacgcc ttgctcgacg acttgatcgc cggtctggac gcgctggggc ttcagtcgcg cccgtcgcat tggcgccgga acttgatggc gtgcagcggg attgagttct gcaagttgtc attcgccgaa acccgggttc gagcacagca tttggtgccc gagctggaac gccggcttga ggacatcaac tcgcagctcg acgtaccgat caccgtcaac atcaacggct gcccgaactc atgtgcgcga gttcaaatcg ccgacatcgg attcaaggga cagatgatcg acgacggaca cggcggctcc gtcgaaggct tccaggtgca tctgggcgga cacctcggcc tggatgccgg attcggccgc aaactgcgcc agcacaaggt caccagtgac gaactcggcg actacatcga ccgggtggtg cgcaacttcg tcaaacaccg cagcgaaggt gaacgcttcg cgcagtgggt catccgggcc gaggaggacg acctgcgatg agcggccgcc taggactact agtctgcagt ttttcctt

Mycobacterium smegmatis
This reference also contains the cloning of sirA from T. smegmatis. We used the primers below taken from Pinto et al, blasted against the T. smegmatis genome and extracted the sirA sequence from genbank as above.
Primers:
smegsirA F      TACAGCTGATGCTCGAAGACGAGTACTTCAT
smegsirA R      CCCAAGCTTTCACGTTGCCTACCTCAAATCCGCTTCGTC

tacagctgat gctcgaagac gagtacttca tgctgcgcgt gcgctgcgat ggtggcgcgctgaccact gcagcgctgc gcacgctcgg cggcatctcg accgagttcg cgcgcgacac cgccgacatc tccgaccgcg agaacgtcca gtaccactgg atccaggtcg agaacatgcc cgagatctgg aagcgcctcg acgccgtcgg cctgcagacc accgaggcgt gcggcgactg cccgcgtgtg gtcctcggct cgccgctggc cggtgagtcc ctcgacgagg tgatcgacgg gacccccgcg atcgacgaga tcgtgcgccg ctacatcggc aagcccgagt actcgaacct gccgcgcaag ttcaagaccg cgatctcggg gcttcaggac gtggtccacg aggtcaacga cgtcgcgttc atcggcgtca accaccccga gcacggcccg ggcttcgacc tgtgggtcgg tggcggcctg tcgaccaacc cgatgctggc ccagcgcgtc ggggtgtggg tgccgctcga cgaggtgccc gacgtctggg agggcgtcgt cagcatcttc cgcgactacg gttaccggcg tctgcggtcg aaggcgcggc tgaagttcct gatcaaggac tggggcgtcg aaaagttcag ggaagtgctg gaaaccgagt acctcaagcg ccccctgatc gacggcccgg cacccgaacc ggtgacccgc cccatcgacc acgtcggtgt gcagaagctc aagaacggcc tcaacgccgt gggcgtcgcc ccgatcgcgg gtcgcgtctc gggcacgatc ctgaccaagg tggccgatct cgccgaggcc gccgggtccg accggatccg cttcacgccg taccagaagc tgatcatcct cgacgtgccc gacgacaaga tcgacgaact gcgcgctggc ctcgacgcgc tcggactgcc gtcgacgccg tcgcactggc gccgcaacct catggcgtgc acgggtatcg agttctgcaa gctgagcttc gccgagaccc gcaagcgtgc ccaggtgctg gttcccgagc tggagaaacg gctcgacgac atcaacgccc agctcgacgt gcccatcacg gtcaacatca acggctgccc caactcgtgc gcccgtatcc aggtcgccga catcgggttc aagggccaga tggtcgacga tggcaacggc cccgaggagg gtttccaggt gcatctgggc ggcagcctgg gcctggacag cgggttcggc cgcaagctgc gccagcacaa ggtgctctcg tccgagctcg gcgactacat cgagcgcgtc gtgcgcaact tcgtgaaaca acgcgaggac ggcgagcgtt tcgcccagtg ggccgtgcgg gccgacgaag cggatttgag gtaggcaacg tgaaagcttg gg

As above, the sequence was imported into geneious, the biobrick restriction sites were manually removed from the sequence and restriction sites were added to the 5’ and 3’ ends in the same fashion as TB sirA. This resulted in the following sequence for synthesis:

Tacagaattc tagaccatgg gcctcgaaga cgagtacttc atgctgcgcg tgcgctgcga tggtggcgcg ctgaccacta cagcgctgcg cacgctcggc ggcatctcga ccgagttcgc gcgcgacacc gccgacatct ccgaccgcga gaacgtccag taccactgga tccaggtcga gaacatgccc gagatctgga agcgcctcga cgccgtcggc ctacagacca ccgaggcgtg cggcgactgc ccgcgtgtgg tcctcggctc gccgctggcc ggtgagtccc tcgacgaggt gatcgacggg acccccgcga tcgacgagat cgtgcgccgc tacatcggca agcccgagta ctcgaacctg ccgcgcaagt tcaagaccgc gatctcgggg cttcaggacg tggtccacga ggtcaacgac gtcgcgttca tcggcgtcaa ccaccccgag cacggcccgg gcttcgacct gtgggtcggt ggcggcctgt cgaccaaccc gatgctggcc cagcgcgtcg gggtgtgggt gccgctcgac gaggtgcccg acgtctggga gggcgtcgtc agcatcttcc gcgactacgg ttaccggcgt ctgcggtcga aggcgcggct gaagttcctg atcaaggact ggggcgtcga aaagttcagg gaagtgctgg aaaccgagta cctcaagcgc cccctgatcg acggcccggc acccgaaccg gtgacccgcc ccatcgacca cgtcggtgtg cagaagctca agaacggcct caacgccgtg ggcgtcgccc cgatcgcggg tcgcgtctcg ggcacgatcc tgaccaaggt ggccgatctc gccgaggccg ccgggtccga ccggatccgc ttcacgccgt accagaagct gatcatcctc gacgtgcccg acgacaagat cgacgaactg cgcgctggcc tcgacgcgct cggactgccg tcgacgccgt cgcactggcg ccgcaacctc atggcgtgca cgggtatcga gttctgcaag ctgagcttcg ccgagacccg caagcgtgcc caggtgctgg ttcccgagct ggagaaacgg ctcgacgaca tcaacgccca gctcgacgtg cccatcacgg tcaacatcaa cggctgcccc aactcgtgcg cccgtatcca ggtcgccgac atcgggttca agggccagat ggtcgacgat ggcaacggcc ccgaggaggg tttccaggtg catctgggcg gcagcctggg cctggacagc gggttcggcc gcaagctgcg ccagcacaag gtgctctcgt ccgagctcgg cgactacatc gagcgcgtcg tgcgcaactt cgtgaaacaa cgcgaggacg gcgagcgttt cgcccagtgg gccgtgcggg ccgacgaagc ggatttgagg taggcaacgt gaaagcctag gtactagtct gcagtggg