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In order to study LEC-2 protein, the DNA of the lichen Peltigera membranacea was extracted. It was used to synthetize by PCR lec-2 gene using specific primers designed with NcoI and EcoRI recognition sites. The lec-2 PCR product was then purified using column before to be double digested using NcoI and EcoRI-HF restriction enzymes. A gel extraction was then used.
Plasmids pET28a was isolated using spin column method and double digested using NcoI and EcoRI restriction enzymes.
Plasmids en PCR product was then ligated together using T4 DNA ligase. A first chemical transformation was effected to insert the plasmid into TOP10 competent cells.
The plasmids were then removed from TOP10 competent cell to BL21(C43) competent cells by Sheeba S. Manoharan. IPTG was then added to induce the expression of lec-2.
Samples were taken every hour to identify the production peak of lec-2 for a potential important production.
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