Team:Technion Israel/Project/Results
Results
Introduction
Expression
After successful overexpression of the 3ɑ-HSD enzyme under pT7 promoter (BBa_K1674002), we conducted a series of experiments based on the 3ɑ-HSD activity measurement protocol, where we measured NADPH fluorescence over time added to E.coli lysates. Every experiment helped us understand a different aspect of the dihydrotestosterone (DHT) reduction reaction using our clones, and characterize the plasmid.
Enzymatic activity as a function of DHT concentration
To get a basic idea of the kinetics of 3ɑ-HSD enzymatic reaction, we first wanted to examine the effect of increase in initial substrate concentration (DHT). Therefore, we sonicated E. coli BL21 cells containing BBa_K1674002 after two hours of induction with IPTG, added 150 µM NADPH to the lysates in a 96-well plate, and inserted into a plate reader pre-heated to 37℃ for 30 minutes to allow for result stabilization. 37℃ mimics human body temperature, which is the environment in which the enzyme will be working in our final product, and is in the optimal temperature range for the enzyme activity.(SOURCE)
In previous experiments we noticed fluctuations in fluorescence during the first 15-30 minutes after the addition of NADPH, even in negative controls, which we assume occurs due to a reaction of NADPH with the phosphate buffer, resulting in a new equilibrium state between NADPH and NADP+.
After 30 minutes we added DHT to each well in various concentrations and measured NADPH fluorescence during 5.5 hours. In a logarithmic time scale we can see linear behavior of NADPH degradation, where the slope represents the degradation rate (Figure 1). When comparing the different E. coli BL21 strains, with and without the 3ɑ-HSD gene, we can see clearly that the graph slope is steeper in presence of 3ɑ-HSD, implying faster NADPH degradation rate due to the specific enzymatic activity.