Biomod/2013/Harvard/methods

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Methods

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Creation of the BlaCaM library was executed through error-prone PCR of the calmodulin region of the encoding gene. Mutant CaM fragments were then assembled into the rest of the BlaCaM gene and its containing vector via . Chemically competent E. coli was with the plasmid and grown on LB-agar plates. Individual colonies were picked off the plates and grown in 96-well deep-well plates. From those plates, of the library were made and of the BlaCaM protein was induced. 96-well plates containing expressed BlaCaM were with individual Ni resin columns. Purified library members were then with CENTA substrate for baseline β-lactamase activity, and activity with the peptides M13 and S. aureus δ-toxin. Members displaying increased activity with δ-toxin were assayed again in triplicate to confirm. A second round of mutagenesis was then carried on any remaining members to continue the evolution.

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