Team:CMUQ/Model
Model
During our work with enzyme kinetics this summer, we found that tests were expensive as reagents were wasted while performing the enzyme kinetic tests. This was because Cas12a was not saturated with its substrate. In other words, the amount of templated DNA needed was not known.
To solve this problem, we mathematically modeled the kinetic activities of Cas12a. Our approach was to perform a small number of enzyme kinetic tests while measuring Cas12a activity in order to determine important kinetic constants pertaining to Cas12a. This includes Km and Vmax which represent the binding affinity and enzymatic activity of Cas12a respectively.
We determined that the optimal amount of template DNA that should be used is 15mM based on experimental data. The Lineweaver Burke plot below indicates how the Km was obtained. The figure was also used to determine the Vmax, this was calculated to be 526.32 umol/min; this is an indication of how fast the reaction can proceed at the enzyme’s maximum rate.
In our experiment, the substrate concentration was varied where the concentrations ranged from 0.125 to 5pmol/ul. 5ul substrate was added to 20ul of DNAse I (0.15kUnitz); where one Kunitz is defined as the amount of DNase I that causes an increase in A260 of 0.001 per minute per milliliter at 25°C, pH 5.0, with highly polymerized DNA as the substrate (Kunitz, M., 1950).
