As a team, we finally decided on inputs and outputs for our project. After a long discussion, we chose Eucalyptol as our output because we wanted a more rare or hard to obtain substance. For our input, we chose to use the spent grains from beer production here in Milwaukee. This provided us with direction to further our research on the pathways and enzymes involved.
In small groups we researched the different pathways and enzymes involved in the output of Eucalyptol and found the pathways incorporated in its production. The glucose from the spent grains will travel into the citric acid cycle, then through the mevalonate pathway, and then a final enzyme will synthesize the Eucalyptol from Geranyl-PP. We found the different enzymes incorporated in the different pathways that do not already exist in E.coli and found their sequences in online databases.
To continue our research, we looked at different promoters, terminators, and plasmids in the parts registry that are available for our use. We also looked at past iGEM projects to investigate which ones they used and their reasoning behind that to help us make our decision. We decided on using the T7 promoter to control the expression of our genes and the standard double terminator.
In groups, we researched the possible locations of where to incorporate terminators and promoters in the biobrick parts, and we decided to use one promoter and one terminator for each biobrick part or gene to promote stable gene expression. We also decided to use two genes or two biobrick parts per plasmid to optimize expression and ensure that the plasmids do not reach capacity. Our plan is to group our output genes as follows:
*Plasmid 1: HMG-CoA synthase and HMG-CoA reductase
*Plasmid 2: Mevalonate kinase and Phosphomevalonate kinase
*Plasmid 3: Mevalonate-5-pyrophosphate decarboxylase and 1,8-cineole synthase
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