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Team:Washington/Celiacs/Methods
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==Mutagenizing Kumamolisin== | ==Mutagenizing Kumamolisin== | ||
| + | ===Producing ssDNA=== | ||
| + | Once we had obtained feasible mutants from the Foldit program, we ordered these mutations in oligonucleotide primers to be used in Kunkel mutagenesis. To perform the mutagenesis, we first transformed the plaasmid containing wild-type kumamolisin into chemically competent cells of ''Escherichia coli'' CJ236. These cells were then grown and harvested for their single stranded DNA (ssDNA). | ||
| + | ===Kunkel Mutagenesis=== | ||
| + | The ssDNA produced then seved as a template srand to which the oligonucleotide primers could bind. | ||
| + | |||
==Using a Whole Cell Lysate Assay to Test Feasability of Mutant== | ==Using a Whole Cell Lysate Assay to Test Feasability of Mutant== | ||
==Purifying Mutants to Accurately Assess Activity== | ==Purifying Mutants to Accurately Assess Activity== | ||
INSERT INFO HERE... LOOK AT OTHER PAGES FOR EXAMPLES OF FORMATING AND INSERTING PICTURES. CLICK THE EDIT BUTTON ON THE UPPER LEFT SIDE OF THE PAGE AFTER YOU HAVE SIGNED IN. | INSERT INFO HERE... LOOK AT OTHER PAGES FOR EXAMPLES OF FORMATING AND INSERTING PICTURES. CLICK THE EDIT BUTTON ON THE UPPER LEFT SIDE OF THE PAGE AFTER YOU HAVE SIGNED IN. | ||
Revision as of 22:27, 9 September 2011
Contents |
Redesigning Kumamolisin to Have Higer Activity at Low pH
Using Foldit to Produce Mutations
In order to create mutations from wild-type kumamolisin, we first loaded the enzyme structure into the protein editing game Foldit. Foldit allows the user to modify the way in which a protein folds by changing the amino acid sequence. Using Foldit, we modified the amino acid residues around the active site of kumamolisin which contained within it a Proline-Glutamine-Lysine-Proline (PQLP) motif found throughout the gliadin peptide of gluten. With the PQLP locked in place along with the key active site amino acid residues, mutations were made which appeared to stabilize PQLP within the active site. Using this method, we produced several mutants at a time which could potentially increase the enzyme's proteolytic activity towards gliadin.
Mutagenizing Kumamolisin
Producing ssDNA
Once we had obtained feasible mutants from the Foldit program, we ordered these mutations in oligonucleotide primers to be used in Kunkel mutagenesis. To perform the mutagenesis, we first transformed the plaasmid containing wild-type kumamolisin into chemically competent cells of Escherichia coli CJ236. These cells were then grown and harvested for their single stranded DNA (ssDNA).
Kunkel Mutagenesis
The ssDNA produced then seved as a template srand to which the oligonucleotide primers could bind.
Using a Whole Cell Lysate Assay to Test Feasability of Mutant
Purifying Mutants to Accurately Assess Activity
INSERT INFO HERE... LOOK AT OTHER PAGES FOR EXAMPLES OF FORMATING AND INSERTING PICTURES. CLICK THE EDIT BUTTON ON THE UPPER LEFT SIDE OF THE PAGE AFTER YOU HAVE SIGNED IN.
