Team:Paris Bettencourt/Notebook/Drug Screening/Wednesday 12th June.html

Drug Screening

Wednesday 12^th June

Design of FdxC and FdxA genes for synthesis

Mycobacterium tuberculosis
Reference: Cole, S.T. et al. 1998. Deciphering the biology of Mycobacterium tuber- culosis from the complete genome sequence. doi:10.1038/31159.
We couldn’t find a reference of FdxC being cloned into E. coli and so we took the sequence data from genbank which is as follows:

gtgacgtaca cgatcgccga accctgtgtc gacatcaagg acaaggcatg
cattgaggag tgcccggtcg attgcatcta cgagggcgcc cggatgctgt
atatccaccc cgacgaatgc gtcgactgtg gggcttgcga gccggtctgc
cccgttgaag ctatcttcta cgaagacgat gtgcccgaac agtggagcca
ttacacccag atcaacgccg atttcttcgc cgagctggga tcgccgggcg
gtgcggccaa ggttggcatg accgagaacg acccgcaagc ggtcaaggat
ctggcgccgc agagcgagga cgcctga

The Gtg was replaced with the NcoI cut site and a glycine codon (ccatgggc) and a HindIII site was added to the end of the sequence. The biobrick restriction sites were then added to the ends of the sequence resulting in the following sequence:

cttagaattc tagaccatgg gcacgtacac gatcgccgaa ccctgtgtcg
acatcaagga caaggcatgc attgaggagt gcccggtcga ttgcatctac
gagggcgccc ggatgctgta tatccacccc gacgaatgcg tcgactgtgg
ggcttgcgag ccggtctgcc ccgttgaagc tatcttctac gaagacgatg
tgcccgaaca gtggagccat tacacccaga tcaacgccga tttcttcgcc
gagctgggat cgccgggcgg tgcggccaag gttggcatga ccgagaacga
cccgcaagcg gtcaaggatc tggcgccgca gagcgaggac gcctgaagct
tgactagtct gcagaaatg

Mycobacterium smegmatis
Reference: Ricagno S. et al. 2008. The crystal structure of FdxA, a 7Fe ferredoxin from Mycobacterium smegmatis. http://dx.doi.org/10.1016/j.bbrc.2007.06.013 http://www.metacyc.org/MSME246196/NEW-IMAGE?type=GENE-IN-MAP&object=GJ4Y-1124
Sequence:

atgacatacg tgatcggccg gccgtgcgtg gacgtcaaag accgcgcatg
cgtggatgag tgcccggtcg actgcatcta cgagggcgcg cggatgctct
acatccaccc cgacgaatgt gtcgactgtg gcgcgtgtga acccgtgtgc
ccggtggagg cgatctacta cgaggacgac ctgcccgagg atctgcagcc
gtaccaggag gagaacgcga agttcttcac cgatgtcctg cccgggcgtg
cccaaccgct cggatcaccg ggcggtgccg cgaaactcgg tgtcgtggac
gccgatacgc ccatggttgc ggagctaccg ccgcaggggg attga

The NcoI cut site and a glycine codon (ccatgggc) replaced the atg in green and a HindIII site was added to the end of the sequence. The biobrick restriction sites were then added to the ends of the sequence resulting in the following sequence:

cttagaattc tagaccatgg gcacatacgt gatcggccgg ccgtgcgtgg
acgtcaaaga ccgcgcatgc gtggatgagt gcccggtcga ctgcatctac
gagggcgcgc ggatgctcta catccacccc gacgaatgtg tcgactgtgg
cgcgtgtgaa cccgtgtgcc cggtggaggc gatctactac gaggacgacc
tgcccgagga tcttcagccg taccaggagg agaacgcgaa gttcttcacc
gatgtcctgc ccgggcgtgc ccaaccgctc ggatcaccgg gcggtgccgc
gaaactcggt gtcgtggacg ccgatacgcc catggttgcg gagctaccgc
cgcaggggga ttgagcctag gtactagtct gcagtggg