Team:UST Beijing/EnzymaticActivity
Enzymatic Activity
We developed a whole cell enzymatic assay using the 96-micro-well plates. PNPG was added to the culture medium, which was degraded into yellow PNP by glycoside hydrolase. Using this assay we analyzed expression pattern of pET28a-β-glucosidase plasmid.
BL21(AI) is originated from BL21 strain which is a E.coli B/r strain. It contains T7 RNApolymerase gene under the control of pBAD promoter. When the concentration of T7 RNApolymerase is higher than LacI protein, T7 RNApolymerase would have priority to bind to pET28a-β-glucosidase plasmid and express β-glucosidase .
Principle： PNPG can be catalyzed by β-glucosidase breaking down to PNP. The solution would change from colorless to yellow. The yellow color of the medium shows the activity of enzyme expressed by BL21(AI).
We added Lac, Ara, PNPG, and a diluted E.coli cell culture in each well of a 96-well plate. After a period of time of culturing, we measured the A620 and A450 using microplate reader.( A450 reflects the concentration of PNP and E.coli cells, A620 reflects only concentration of bacteria cells in this assay set-up.)
We discovered that PNP concentration could be calculated to be proportional to A450real according to this formula： A450real=A450-1.5A620
The catalyst efficiency of enzymes expressed by E.coli is represented by A450real/A620.
After 50 min, the data showed high concentration of IPTG correspond to high efficiency of enzyme, which can prove that IPTG can speed up the process of releasing the inhibition of LacI protein
After adding sample for 327min, those without IPTG in the pores showed more obvious yellow color with the increasing of ARA concentration, which indicated that with the increase of ARA concentration, the expression of the enzyme also increased. This result was the same as our expectations.
In addition, the color after adding IPTG was more obvious than that without IPTG. Because IPTG can combine with lacI, and then remove the inhibition of lacI on the expression of enzyme.
But with the addition of IPTG, yellow was no longer regulated by ARA, the higher the concentration of IPTG, the more obvious this phenomenon was. Because both ARA and IPTG can promote the expression of the enzyme, the effect of ARA concentration on the inhibition of the enzyme was reduced. But IPTG induction had the best concentration, so we found that when the IPTG concentration was about 1000umol/L, the color was less obvious than that of the lower concentration.
