Team:ULB-Brussels/Materiel&Methods
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Material & Methods
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1. 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-
2.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: arabinose-inducible vector containing kanamicyn resistance
pBAD33: arabinose-inducible vector containing chloramphenicol resistance
pSB1K3 : standard iGEM plasmid which has a resistance to Kanamycin
pSB1C3 : standard iGEM plasmid which has a resistance to Chloramphenicol
pSB1A3 : standard iGEM plasmid which has a resistance to Ampicilin
3. Primers
The following primers were produced by Sigma-Aldrich and the sequences are shown in a 5’-3’ orientation.
3.1. Primers for first amplification of each gene
MccBA-FOR
5'-CCTCAGCTACACGTGCACTGATTAAAGAGGAGAAAATGGAATTAAAAGCGAGTG-3'MccBA-REV
5'-GCCGCGAAGCGGCGTCGGCTTGAATGAATTGTTATAACCTCAGATATGTGAACCACT-3'MccBB-FOR
5'-CCTCAGCTACACGTGCACTGATTAAAGAGGAGAAAATGGTGCTCCCTGATATTAA-3'MccBB-REV
5'-GCCGCGAAGCGGCGTCGGCTTGAATGAATTGTTATAACCTTATCTCTCCAGACAGCT-3'MccBC-FOR
5'-CCTCAGCTACACGTGCACTGATTAAAGAGGAGAAAATGTCAAAACACGAAC-3'MccBC-REV
5'-GCCGCGAAGCGGCGTCGGCTTGAATGAATTGTTATAACCTTACTGTAGACATTTATCAT-3'MccBE-FOR
5'-CCTCAGCTACACGTGCACTGATTAAAGAGGAGAAAATGGTTACATTAAAAATGGC-3'MccBE-REV
5'-GCCGCGAAGCGGCGTCGGCTTGAATGAATTGTTATAACCTCATTGAGAACTCCAG-3'MccBF-FOR
5'-CCTCAGCTACACGTGCACTGATTAAAGAGGAGAAAATGACCATACCTCT-3'MccBF-REV
5'-GCCGCGAAGCGGCGTCGGCTTGAATGAATTGTTATAACCTCAGTCTCCTGTT-3'MccCC-FOR
5'-CCTCAGCTACACGTGCACTGATTAAAGAGGAGAAAATGTTAATTGGTGTCTAC-3'MccCC-REV
5'-GCCGCGAAGCGGCGTCGGCTTGAATGAATTGTTATAACCTCATACCATCTCCTTTTTAA-3'MccCF-FOR
5'-CCTCAGCTACACGTGCACTGATTAAAGAGGAGAAAATGATGATACAATC-3'MccCF-REV
5'-GCCGCGAAGCGGCGTCGGCTTGAATGAATTGTTATAACCTTATTTCTCGGTAG-3'3.2. Primers for second amplification
Common-FOR
5'-GCAGAATTCGCGGCCGCTTCTAGAGCCTCAGCTACACGTGCACTG-3'Common-REV
5'-AGCCTGCAGCGGCCGCTACTAGTAGGTTATAACGCTTGAATTAAGCCGCGCCGCGAAGCGGCGTCGGCTTGAAT TTATGGGAATGGTAC-3'3.3. Primers for insertion in immunity pBAD plasmid
ImMccBG-FOR
5'-CATTCTAGAATTAAAGAGGAGAAAATGGATATAATAGAAAAAAG-3'ImMccBG-REV
5'-GCACATGCATCATCCCCCTAC-3'ImMccCF-FOR
5'-AGCCAAGCTTGCATGTTATTTCTCGGTAG-3'ImMccCF-REV
5'-AGCCAAGCTTGCATG TTATGGGAATGGTAC-3'ImMccCEFOR
5'-ATTCGAGCTCGGTACATGGTGCAGATTATC-3'ImMccCEREV
5'-TTTCTCCTCTTTAATTTAACCAATTACTTTTGAAT-3'3.4. Primers for fixing BE
MccCB Endo For
5'-TTGCTTTAGGTTCCTTAAGG-3'MccCB Endo Rev
5'-TCAGCTTCTGGTACCTTATG-3'MccCB synthgene For1
5'-AAGGATGATTTCTGGAATTCGCGGCCG-3'MccCB synthgene Rev1
5'-CCTTAAGGAACCTAAAGCAA-3'MccCB synthgene For2
5'-CATAAGGTACCAGAAGCTGA-3'MccCB synthgene Rev2
5'-AGCGGCCGCTACTAGTAGGTTATAACGCTT-3'4. Kits
Gel purification kit: Sigma-Aldrich, GenEluteTm PCR Clean-up (ref:NA1020)
Mini prep kit: Zymo Research, ZyppyTM Plasmid Miniprep Kit (ref: D4036)
5. Restriction
• Run a PCR to amplify the genes of interest with floating restriction sites.
• Digest the PCR product and the vector by the appropriate restriction enzymes.
Restriction mix for the PCR products (insert):
*Xµl of PCR products (X depending on the concentration of insert of the PCR products).
*1µl of each restriction enzyme.
*2µl appropriate buffer 10X.
*Add Yµl bi-distilled water to reach a final volume of 20µl.
Restriction mix for the vector:
*200ng of vector.
*1µl of each restriction enzyme.
*2µl appropriate buffer 10X.
*Add Xµl bi-distilled water to reach a final volume of 20µl.
• Incubate for one hour at 37°C
6. Ligation
• The ligation reaction proceeds with 100ng of restricted vector. The quantity of the PCR products is calculated according to the following formula: (100ng vector x PCR product size x 3)/(vector size) = PCR product quantity in ng.
Ligation mix:
*1µl T4 DNA ligase.
*2µl buffer (10x).
*Add the two restrictions reactions in a final volume of 20µl.
*Add bi-distilled water for a final volume of 20µl.
*ADN
• Incubate the ligation reaction overnight at room temperature.
• Electroporate the ligation on bacteria.
7. Preparation of electrocompetent cells
• Dilute 100x an overnight culture in LB medium and incubate at 30°C with shaking until the OD600nm reaches 0.5.
• Centrifuge in a cold rotor at 4500 rpm 4°C for 30 minutes.
• Remove supernatant, resuspend in 20ml cold H20, then centrifuge as above. Repeat this step twice.
• Remove supernatant, resuspend in 10ml cold glycerol, then centrifuge as above.
• Remove final supernatant carefully, resuspend in 1ml cold glycerol.
• Store it at -80°C.
8. Electroporation
DNA dialysis:
• Fill a petri dish with dH2O.
• Make float a round 0.025µm Millipore dialysis filter on dH2O.
• Place the DNA on the Filter for 20 min, then take it back.
Electroporation:
• Add dialysed DNA in 50µl of electrocompetent cells.
• Put mixture in an electroporation cuvette.
• Electroporate it at 2,500 volt, 200Ω and 25µFD.
• Add quickly 1ml of LB.
• Put the bacteria for 1 hours at 37°C.
• Plate the volume you need on dish (with appropriate antibiotics).