Purification of Enzymes
In order to allow cAMP level to be inversely related to glucose concentration, we need to introduce energy coupling pathways into our system. For the purpose of reducing the complexity of the system, we choose to import glycolysis pathway and lactate dehydrogenase (LDH) to convert glucose to lactate, allowing NAD+ to recycle. Besides, we need to include enzymes that catalyze the conversion of AMP to ATP and AMP to cAMP. Therefore, the first task of our project is to purify these enzymes.
In Vitro Expression
Once the enzymes are successfully purified, the next task is to measure the concentration of each enzymes. For in vitro expression of these enzymes and the functional gene part, we used in vitro expression system provided by ThermoFisher Scientific ©.