Biomod/2013/Harvard/methods

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Methods

Input

Creation of the BlaCaM library was executed through 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.