Part:BBa_K1658000:Experience

METU_HS_Ankara 2016 Team Characterization

CAD1 (cinnamyl alcohol dehydrogenase) enzyme that we were interested previous year was characterized with Fehling colorimetric assay. This year we wanted to show construct work with pSB1C3 vector on protein gel. After growing and obtaining colonies in Overnight culture, we have got optical density at 1.0 and using several techniques we have extracted protein and loaded on a SDS gel. Without induction, constitutive promoter that we have ligated previous year worked perfectly and give us a lane at 38kDA. The control group was the e. coli dh5a untransformed colonies.

Here you can see image;

First well is ladder. 3rd lane of ladder from bottom stands for 25. 4th lane from bottom stands for 55kDa. First well is loaded with empty dh5a and its lysate is just right next to it 3rd well is our first colony that we have successfully completed ligated into psb1c3 Here you can see 4 lane in-between 25 and 55kDa Extra lane is our CAD1 enzyme denatured protein

To conclude, as you can see, the enzyme was not released in environment and we proved it in wells that we have loaded lysates next to precipitated ones.

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