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Team:Copenhagen/Results
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We mutated the CYP79A2 to remove a restrictionsite. Afterwards we inserted the CYP in the shipping plasmid. We confirmed both the mutation and the insertion by sequencing. We tried to insert the CYP79A2 in the ekspressionvector BBa_J04500 and although it looked like the gene was inserted when we got it sequenced it wasn't a perfect fit. We picked the best fitting and expressed them in BL21 cells. A SDS gel didn't show any increase in protein amount for the band-size corresponding to the CYP. A membrane preperation induced with CYPs substrate phenylalanine showed no production of oxime. Our original A2 template have been reported to work (Ref to Kenneth article) and we therefore suspect the mutations and/or the prefix suffix to have influenced the activity. To explore this, we plan to analyse the device BBa_ K527001 - the cypa2 inserted in an expressionvector by the DTU_Denmark2 user assembly method which doesn't contain prefix/suffix but the mutation |
We mutated the CYPB1 to remove an illigal restrictionsite and confirmed this by sequencing. We tried to insert the CYP in the expression vector as well as the shipping plasmid. But we was unable to get good results of a colony PCR reaction because our primers didn't bind. (An ekstra set of primers didn't either). Due to time pressure we turned to another assembly methods. Our good friends at DTU (The powerpuff team) have designed an assembly method using the User enzyme and they got our mutated CYPB1 and made user-friendly assembly tales on it, and on the promoter, RBS and terminator which we had used from the kit. We thereafter made a very easy, quick and succesful assembly. Afterwards we expressed the CYP and confirmed that a membrane preperation of BL21 cells were able to confirm tyrosine to oxime with a TLC TLC
CO-measurement A specific characteristic of p450's, is their absorbance of light at 450 nm in their active state. As a part of our characterization, we have performed CO-measurements on the purified p450 membrane fractions, which supposedly should contain a enriched amount of p450 enzyme. CO (carbon monoxide) is added to a membrane sample, by blowing bubbles at approximately 1 bubble/sec in 60 seconds. The reason for doing this, is that the CO binds the heme group of cytochrome p450, which holds the p450 in its active conformation. |
We have prevented fungus growth with oximes. |
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