Difference between revisions of "Biomod/2013/Harvard/methods"

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=Methods=
 
=Methods=
 
==Input==
 
==Input==
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 (0.4mM IPTG, 18h, 18°C). 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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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 (0.4mM IPTG, 18h, 18°C). 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.
  
 
==Output==  
 
==Output==  
 
Variations of GlucCaM gene was created by splitting the Gluc gene and assembling the split halves around the calmodulin gene along with its containing vector via [[Biomod/2013/Harvard/protocols#Gibson_Assembly|Gibson assembly]]. Individual Gluc halves and mutated GlucCam genes were created with similar methods. Chemically competent E. coli cells were [[Biomod/2013/Harvard/protocols#Chemically_Competent_Cells_Transformation|transformed]] with the plasmid and grown on LB-agar plates. Individual colonies were [[Biomod/2013/Harvard/protocols#Bacterial_Cell_Cloning_/_Inoculation_of_Cells|picked and inoculated]] overnight. Overnight cultures are then [[Biomod/2013/Harvard/protocols#Qiagen_Miniprep|miniprepped]] and sent in for [[Biomod/2013/Harvard/protocols#GeneWiz_Sequencing_Prep|sequencing]], and proteins are then [[Biomod/2013/Harvard/protocols#GLucCaM_Expression|expressed]] from desirable colonies. The resulting cell culture is spun down, resuspended, and sonicated. [[Biomod/2013/Harvard/protocols#GLucCaM/Gluc_Assay|GLuc or GLucCaM assay]] is performed on the sonicated protein solution to determine strength and sensitivity of output signal to varying conditions.  
 
Variations of GlucCaM gene was created by splitting the Gluc gene and assembling the split halves around the calmodulin gene along with its containing vector via [[Biomod/2013/Harvard/protocols#Gibson_Assembly|Gibson assembly]]. Individual Gluc halves and mutated GlucCam genes were created with similar methods. Chemically competent E. coli cells were [[Biomod/2013/Harvard/protocols#Chemically_Competent_Cells_Transformation|transformed]] with the plasmid and grown on LB-agar plates. Individual colonies were [[Biomod/2013/Harvard/protocols#Bacterial_Cell_Cloning_/_Inoculation_of_Cells|picked and inoculated]] overnight. Overnight cultures are then [[Biomod/2013/Harvard/protocols#Qiagen_Miniprep|miniprepped]] and sent in for [[Biomod/2013/Harvard/protocols#GeneWiz_Sequencing_Prep|sequencing]], and proteins are then [[Biomod/2013/Harvard/protocols#GLucCaM_Expression|expressed]] from desirable colonies. The resulting cell culture is spun down, resuspended, and sonicated. [[Biomod/2013/Harvard/protocols#GLucCaM/Gluc_Assay|GLuc or GLucCaM assay]] is performed on the sonicated protein solution to determine strength and sensitivity of output signal to varying conditions.  
 
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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 (0.4mM IPTG, 18h, 18°C). 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.

Output

Variations of GlucCaM gene was created by splitting the Gluc gene and assembling the split halves around the calmodulin gene along with its containing vector via Gibson assembly. Individual Gluc halves and mutated GlucCam genes were created with similar methods. Chemically competent E. coli cells were transformed with the plasmid and grown on LB-agar plates. Individual colonies were picked and inoculated overnight. Overnight cultures are then miniprepped and sent in for sequencing, and proteins are then expressed from desirable colonies. The resulting cell culture is spun down, resuspended, and sonicated. GLuc or GLucCaM assay is performed on the sonicated protein solution to determine strength and sensitivity of output signal to varying conditions.