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Team:UC Davis/Protein Engineering
From 2014.igem.org
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Once we had identified the aldehyde dehydrogenases we wanted to screen for specificities, we needed order DNA so we could express the enzymes and assay them in the lab. For enzymes identified in the literature, sequences were pulled from UniProt Knowledge Base. We ordered DNA from Life Technologies, cloned the genes into the pET29b-(+) plasmid vector using the Gibson assembly method, and transformed the assembled plasmids into BLR strain E. coli for expression. For our engineered mutants, Kunkel mutagenesis was used to introduce desired mutations into the plasmid DNA. Expressed aldehyde dehydrogenase enzymes were purified using affinity chromatography.<br><br> | Once we had identified the aldehyde dehydrogenases we wanted to screen for specificities, we needed order DNA so we could express the enzymes and assay them in the lab. For enzymes identified in the literature, sequences were pulled from UniProt Knowledge Base. We ordered DNA from Life Technologies, cloned the genes into the pET29b-(+) plasmid vector using the Gibson assembly method, and transformed the assembled plasmids into BLR strain E. coli for expression. For our engineered mutants, Kunkel mutagenesis was used to introduce desired mutations into the plasmid DNA. Expressed aldehyde dehydrogenase enzymes were purified using affinity chromatography.<br><br> | ||
Revision as of 23:27, 15 October 2014
Design
Enzyme Picture!!!
Our research on the chemical composition of olive oil revealed that rancid olive oil contains a different composition of medium saturated, long saturated, and unsaturated aldehydes as compared to fresh olive oil. To differentiate between medium saturated, long saturated, and unsaturated aldehydes in our electrochemical biosensor, we needed enzymes which would selectively use these compounds as substrates and produce a product which may be easily measured using both spectrophotometric (for enzyme characterization and engineering) and electrochemical techniques. We found that the aldehyde dehydrogenase family of enzymes would serve as the perfect catalyst for the needs of our sensor. We used two approaches to identify enzymes with the desired specificities we would use in our biosensor: bioprospecting and rational design.
Click on “Design” to learn more about how we found our enzymes
Our research on the chemical composition of olive oil revealed that rancid olive oil contains a different composition of medium saturated, long saturated, and unsaturated aldehydes as compared to fresh olive oil. To differentiate between medium saturated, long saturated, and unsaturated aldehydes in our electrochemical biosensor, we needed enzymes which would selectively use these compounds as substrates and produce a product which may be easily measured using both spectrophotometric (for enzyme characterization and engineering) and electrochemical techniques. We found that the aldehyde dehydrogenase family of enzymes would serve as the perfect catalyst for the needs of our sensor. We used two approaches to identify enzymes with the desired specificities we would use in our biosensor: bioprospecting and rational design.
Click on “Design” to learn more about how we found our enzymes
Build
Click on “Build” to learn more about how we turned DNA sequences into enzymes
Test
PLATE READER!!! CURVES!!!
To determine the specificity profiles of our aldehyde dehydrogenases, we needed to develop a simple, high-throughput assay which we could ultimately use to determine the aldehyde composition of a sample of olive oil. We developed a simple spectrophotometric plate assay which measures the concentration of NADH in a solution. Using this assay, we screened all 26 aldehyde dehydrogenases against 16 aldehyde substrates and identified four enzymes with unique specificities. We created full Michaelis-Menten curves and calculated the kinetic constants for the four enyzmes we identified on all sixteen aldehyde substrates. We also developed a protocol for extracting aldehydes from olive oil which could be used with our plate assay.
Click on “Test” to learn more about the results of our assays
To determine the specificity profiles of our aldehyde dehydrogenases, we needed to develop a simple, high-throughput assay which we could ultimately use to determine the aldehyde composition of a sample of olive oil. We developed a simple spectrophotometric plate assay which measures the concentration of NADH in a solution. Using this assay, we screened all 26 aldehyde dehydrogenases against 16 aldehyde substrates and identified four enzymes with unique specificities. We created full Michaelis-Menten curves and calculated the kinetic constants for the four enyzmes we identified on all sixteen aldehyde substrates. We also developed a protocol for extracting aldehydes from olive oil which could be used with our plate assay.
Click on “Test” to learn more about the results of our assays
