Biomod/2013/Harvard/methods

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=Methods=
=Methods=
==Input==
==Input==
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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 [[Gibson assembly | ]]. Chemically competent E. coli was [[transformed | ]] 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, [[glycerol stocks | ]] of the library were made and [[expression | ]] of the BlaCaM protein was induced. 96-well plates containing expressed BlaCaM were [[purified using plates | ]] with individual Ni resin columns. Purified library members were then [[assayed | ]] with CENTA substrate for baseline β-lactamase activity, and activity with the peptides M13 and <i>S aureus</i> 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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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 [[Gibson assembly | ]]. Chemically competent E. coli was [[transformed | ]] 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, [[glycerol stocks | ]] of the library were made and [[expression | ]] of the BlaCaM protein was induced. 96-well plates containing expressed BlaCaM were [[purified using plates | ]] with individual Ni resin columns. Purified library members were then [[assayed | ]] with CENTA substrate for baseline β-lactamase activity, and activity with the peptides M13 and <i>S. aureus</i> 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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Revision as of 00:42, 12 October 2013

Methods

Input

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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