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Team:ULB-Brussels/Project
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Overall project
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Project Details
Mat & Meth
Team ULB-Brussels, Materials & Methods!
I. E. coli strains
MC1061 : F− araD139, Δ(ara-leu)7696,galE15, galK16, Δ(lac)X74, rpsL (Strr), hsdR2 (rK−mK+), mcrA mcrB1
TOP10 (Invitrogen) : F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 λ-
II. Plasmids
pP70 : plasmid which contains the microcin C7 operon and a resistance to chloramphenicol
pCID909 : plasmid which contains the microcin B17 operon and a resistance to ampicilin
pBAD18: inductible by arabinose vector containing kanamicyn resistance
pBAD33: inductible by arabinose vector containing chloramphenicol resistance
pSB1K3 : standard iGEM plasmid wich has a resistance to Kanamycin
pSB1C3 : standard iGEM plasmid wich has a resistance to Chloramphenicol
pSB1A3 : standard iGEM plasmid wich has a resistance to Ampicilin
III. Primers
The following primers were produced by Sigma-Aldrich and the sequences are shown in a 5’-3’ orientation.
III.I. Primers for first amplification of each gene:
MccBA-FOR
CCTCAGCTACACGTGCACTGATTAAAGAGGAGAAAATGGAATTAAAAGCGAGTGMccBA-REV
GCCGCGAAGCGGCGTCGGCTTGAATGAATTGTTATAACCTCAGATATGTGAACCACTMccBB-FOR
CCTCAGCTACACGTGCACTGATTAAAGAGGAGAAAATGGTGCTCCCTGATATTAAMccBB-REV
GCCGCGAAGCGGCGTCGGCTTGAATGAATTGTTATAACCTTATCTCTCCAGACAGCTMccBC-FOR
CCTCAGCTACACGTGCACTGATTAAAGAGGAGAAAATGTCAAAACACGAACMccBC-REV
GCCGCGAAGCGGCGTCGGCTTGAATGAATTGTTATAACCTTACTGTAGACATTTATCATMccBE-FOR
CCTCAGCTACACGTGCACTGATTAAAGAGGAGAAAATGGTTACATTAAAAATGGCMccBE-REV
GCCGCGAAGCGGCGTCGGCTTGAATGAATTGTTATAACCTCATTGAGAACTCCAGMccBF-FOR
CCTCAGCTACACGTGCACTGATTAAAGAGGAGAAAATGACCATACCTCTMccBF-REV
GCCGCGAAGCGGCGTCGGCTTGAATGAATTGTTATAACCTCAGTCTCCTGTTMccCC-FOR
CCTCAGCTACACGTGCACTGATTAAAGAGGAGAAAATGTTAATTGGTGTCTACMccCC-REV
GCCGCGAAGCGGCGTCGGCTTGAATGAATTGTTATAACCTCATACCATCTCCTTTTTAAMccCF-FOR
CCTCAGCTACACGTGCACTGATTAAAGAGGAGAAAATGATGATACAATCMccCF-REV
GCCGCGAAGCGGCGTCGGCTTGAATGAATTGTTATAACCTTATTTCTCGGTAGIII.II. Primers for second amplification:
Common-FOR
GCAGAATTCGCGGCCGCTTCTAGAGCCTCAGCTACACGTGCACTGCommon-REV
AGCCTGCAGCGGCCGCTACTAGTAGGTTATAACGCTTGAATTAAGCCGCGCCGCGAAGCGGCGTCGGCTTGAAT TTATGGGAATGGTACIII.III. Primers for insertion in immunity pBAD plasmid:
ImMccBG-FOR
CATTCTAGAATTAAAGAGGAGAAAATGGATATAATAGAAAAAAGImMccBG-REV
GCACATGCATCATCCCCCTACImMccCF-FOR
AGCCAAGCTTGCATGTTATTTCTCGGTAGImMccCF-REV
AGCCAAGCTTGCATG TTATGGGAATGGTACImMccCEFOR
ATTCGAGCTCGGTACATGGTGCAGATTATCImMccCEREV
TTTCTCCTCTTTAATTTAACCAATTACTTTTGAATIII.IV. Primers for fixing BE:
MccCB Endo For
TTGCTTTAGGTTCCTTAAGGMccCB Endo Rev
TCAGCTTCTGGTACCTTATGMccCB synthgene For1
AAGGATGATTTCTGGAATTCGCGGCCGMccCB synthgene Rev1
CCTTAAGGAACCTAAAGCAAMccCB synthgene For2
CATAAGGTACCAGAAGCTGAMccCB synthgene Rev2
AGCGGCCGCTACTAGTAGGTTATAACGCTT
