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Team:Washington/Celiacs/Results
From 2011.igem.org
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In order to determine whether our proposed mutations to the wild-type Kumamolisin improved the ability of the enzyme to break down PQLP, we tested each mutant with a whole cell lysate fluorescence assay. Cells harboring the expressed mutants were lysed at pH 4, mimicking the gastric environment. The released enzymes, after being roughly separated from cell material, were added to a fluorescent PQLP that had been conjugated to a quencher. Thus, no fluorescence was achieved until the peptide had been cleaved and the fluorophore had been released from the quencher. This allowed a relative assessment of rate of enzyme activity by measuring increase in fluorescence of the system. | In order to determine whether our proposed mutations to the wild-type Kumamolisin improved the ability of the enzyme to break down PQLP, we tested each mutant with a whole cell lysate fluorescence assay. Cells harboring the expressed mutants were lysed at pH 4, mimicking the gastric environment. The released enzymes, after being roughly separated from cell material, were added to a fluorescent PQLP that had been conjugated to a quencher. Thus, no fluorescence was achieved until the peptide had been cleaved and the fluorophore had been released from the quencher. This allowed a relative assessment of rate of enzyme activity by measuring increase in fluorescence of the system. | ||
Revision as of 02:02, 16 September 2011
Testing our designed mutants for activity on PQLP
Using a whole cell lysate assay to screen a large number of mutants for good activity
In order to determine whether our proposed mutations to the wild-type Kumamolisin improved the ability of the enzyme to break down PQLP, we tested each mutant with a whole cell lysate fluorescence assay. Cells harboring the expressed mutants were lysed at pH 4, mimicking the gastric environment. The released enzymes, after being roughly separated from cell material, were added to a fluorescent PQLP that had been conjugated to a quencher. Thus, no fluorescence was achieved until the peptide had been cleaved and the fluorophore had been released from the quencher. This allowed a relative assessment of rate of enzyme activity by measuring increase in fluorescence of the system.
As one might expect, our first screen of mutants showed some mutants with a decrease in activity from the wild-type, some showed no change, and some actually showed great increase in activity. One single point mutant showed close to a 1000% increase, or about an 11-fold rise in activity from wild-type Kumamolisin.
