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 [[Biomod/2013/Harvard/protocols#Agilent_GeneMorph_II_Error-Prone_PCR |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 [[Biomod/2013/Harvard/protocols#Gibson_Assembly|Gibson assembly]]. Chemically competent E. coli was [[Biomod/2013/Harvard/protocols#Chemically_Competent_Cells_Transformation|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, [[Biomod/2013/Harvard/protocols#Glycerol_Stock|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 [[Biomod/2013/Harvard/protocols#BlaCaM_Library_Assay|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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Need protocols for:
 +
Ni-resin 96 well plate purification
 +
BlaCaM library expression
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Revision as of 23:46, 11 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 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 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.

Need protocols for:

Ni-resin 96 well plate purification
BlaCaM library expression
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