You and your colleagues are working on engineering a fast-growing, heat-tolerant (can grow well at 45°C) strain of Saccharomyces cerevisiae to have additional characteristics.
The goal of your project is to produce a strain that can use amylose (starch) as a substrate and can reverse the final step of the ethanol fermentation process efficiently. Your culture collection contains some heat-tolerant microbial isolates that are amylolytic and others that are efficient at ethanol production. Because you are an expert biochemist, your part of the project is to decide which amylase gene and which alcohol dehydrogenase gene to engineer into your fastgrowing strain.
Your lab technician has already prepared extracts of amylase and alcohol dehydrogenase from the relevant cultures. They have also prepared genomic DNA extracts from these cultures.
Describe your approach to the remainder of your task using only techniques that you have learned about during this lab course.
Here is a list of the things you will need to do:
1. Ensure that your enzyme extracts are pure protein, and that they contain ONLY the protein of choice.
2. Make enzyme extract solutions that are 1 mg / mL. Remember these are cell extracts, so you will have to determine their concentration and then dilute to make them 1 mg / mL. 3. Confirm that the chosen amylase has the strongest β-amylolytic activity (conversion of amylose to maltose)
4. Confirm that the chosen alcohol dehydrogenase is most efficient at the conversion of ethanol to acetaldehyde compared to the wild type (you already have the wild type data from one of your previous activities)
5. Determine the nucleotide sequences of the chosen amylase and alcohol dehydrogenase genes
So techniques learned are Henderson-hasselbalch, chromophores, the beer-lambert law, pcr, agarose gel electrophoresis and sequence analysis, SDS, qualitative analysis of carbs using benedicts, seliwanoffs, barfoeds and Iodine tests. Enzume kinetics using Michaelis-Menten curve and a lineweaver-burk plot, using spectrophotometric to identify unknown and protein substances with Biuret, UV, and CBB assays.