We began by transforming our plasmid into E. coli., then did miniprep to isolate the plasmid so that we had enough to work with.
We obtained the bicistronic CYP2e1/NPR plasmid in the pCW vector for further testing.
Confirmation of expression of this bicistronic plasmid through successful transformation (this means that the plasmid can be expressed in conjunction with the aldB gene already in bacteria).
Step 2: Isolating the Gene
We next isolated our gene by cutting it with restriction enzymes.
Step 3: Putting our Gene into iGEM Backbone
We then ligated our gene into the iGEM backbone. However, the restriction sites of each didn't fit together, so we created our own gene fragment to connect the two.
Isolation of the human NADPH P450 reductase gene for further implantation with other CYP P450 enzymes (need to be expressed with one another to work) and modified the gene fragment to fit the Biobrick RFC requirements.
Step 4: Cloning
Finally, we transformed the new plasmid into E. coli and then did miniprep to isolate the plasmid.
