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Team:UC Davis/Potentiostat Design
From 2014.igem.org
Inspiration and Iteration
Our biosensor required a potentiostat. A potentiostat is an instrument capable of maintaining a voltage bias between electrodes. The bias encourages diffusion but more importantly the transfer of electrons which are recorded, and ultimately related to the species present in solution.
Build
Flowchart!!! Kunkel!!! DNA!!!
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.
Click on “Build” to learn more about how we turned DNA sequences into enzymes
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.
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
