Step I.
Firstly, we wanted to design a BioBrick consisting of our methanol – dependent promoter. It is the core of our project, so we decided to mainly focus on it. In theory the plan that we prepared was clear and easy – but, of course, nothing is predictable in lab J The part itself fortunately didn’t include any restriction sites that we would have to get rid of, so the only thing that we had to do was isolating genomic DNA from M.organophilum, PCR with primers matching our promoter sequence and ligation of the PCR product with plasmid Backbone.
Step II.
The second step that we planned to do was to check if our BioBrick is properly working. The bacterium on which we wanted to test it was E.coli TOP10 F’ strain. We decided that the reporter protein which we will use is GFP and catechol oxidase.
Step III.
After checking, if the BioBrick which we constructed is working, we wanted to ligate our part with pKT230 plasmid. The aim of this procedure was to create a plasmid that would be maintained by Zymomonas mobilis. After a thorough research we decided, that Zymomonas mobilis, although it’s hard to transform, would be a best host to our plasmid due to its high resistance to ethanol.
Step IV.
The last step was to measure the efficiency of reporter protein production by Zymomonas mobilis in different concentrations of methanol.
Results.
Unfortunately, due to lack of time, we couldn’t execute our whole project plan. What we achieved, was creation of the methanol-dependent promoter BioBrick. We didn’t have time to measure how well it’s working. We also constructed pKT230 plasmid with an insert consisting of methanol-dependent promoter and GFP. We hope to continue or work after the Jamboree. Next step ahead of us: Z.mobilis transformation!
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