Part:BBa_K3038004:Experience

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Manipulations

PCR amplification

Following the design of the synthetic gene, it is amplified by PCR thanks to the design of primers upstream and downstream of the sequence.

Enzymatic digestion and ligation in pSB1C3

After amplification of the synthetic gene, sample is purified, the amplicons are digested with restriction enzymes EcoRI and PstI. Similarly for the cloning vector pSB1C3. The insert (Mlut_11700) is then ligated into the plasmid.

Design of Mlut_11700/pSB1C3 with Geneious software.
This map shows the terminator corresponding to the pBAD, flanking the coding sequence of the Mlut_11700 protein. A tag is also present in N-ter. Finally, in the plasmid is present and chloramphenicol resistance cassette.

Cloning into thermocompetent cells JM109

The thermocompetent E. coli JM109 bacteria are then transformed and clones are obtained.

Clones on a selective LB medium (+ chloramphenicol 25 µg/mL) following the transformation of E. coli thermocompetent cells with the Mlut_11700/pSB1C3 ligations.

PCR colony screening

After bacterial transformation, colony PCR is performed with the forward and reverse primer hybridizing into the plasmid. The PCR products are loaded on 0.8% agarose gel.

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