Biomod/2013/Harvard/methods: Difference between revisions
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Creation of the BlaCaM library was executed through [[error-prone PCR | 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 21:42, 11 October 2013
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HARVARD
BIODESIGN
BIOMOD 2013
-
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard"><li>HOME</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/introduction"><li>INTRODUCTION</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/design"><li>DESIGN</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/methods"><li>METHODS</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/protocols"><li>PROTOCOLS</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/team"><li>TEAM</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/lab"><li>LAB NOTEBOOK</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/results"><li>RESULTS</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/references"><li>REFERENCES</li></a></html>
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.
