Part:BBa_K3038006:Experience

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Applications of BBa_K3038006

Manipulations

Enzymatic digestion and ligation in pSB1C3

After amplification of the synthetic gene, sample is purified, the amplicons are digested. TesA with EcorI and SpeI and Mlut_11700 with XbaI and PstI. The pSB1C3 plasmid is digested by EcoRI and PstI.

Design of TesA/Mlut_11700/pSB1C3 with Geneious software.
This map shows the pBAD promoter and its terminator flanking the coding sequence of the TesA and Mlut_11700 protein. A 6 his tag is present on TesA gene, and a c-myc tag is present on Mlut_11700 gene. 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 TesA/Mlut_11700/pSB1C3 ligations.

PCR colony screening

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

User Reviews

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