Double Plasmids

We tested the affection of β-glucosidase induced by IPTG(1000uM) and Ara(1000uM) in 96-well plates and measuring the A450(pNPG decomposed as substrate to pNP which can be detected at 450nm) every hour. After collected data, we output a graph of A450-time.

We analyzed the pET28a-β-glucosidase plasmid and pSB1C3-pBAD-T7RNAp plasmid in double-transformed E. coli, and using JDesigner we set up the model of the expression toβ-glucosidase . This model displayed the process of pNPG’ decomposition in wells.

Set parameters as：Lac=1000, Ara=1000, pNPG=13 and export the graph to pNPG-time. Modify the parameters(k1, k2_Vmax, k2_Km, k2_Ki, k3_1, k3_2,k4, k5) until the curve fit to the graph output from origin data.

In this result, the parameter (k1, k2_Vmax, k2_Km, k2_Ki, k3_1, k3_2, k4, k5) is 1, 3.4, 2.9, 0.4, 1, 96000, 1, 1.

Enzyme activity

β-glucosidase is used to hydrolyze sugars from saponins of notoginseng. Our Lab has a pET-28a plasmid with β-glucosidase gene. The transcription of β-glucosidase is repressed by LacI protein. But lactose and IPTG can induce the expression of LacI protein. We used a 3L fermentation tank to conduct preliminary experiments, then the enzyme was extracted from bacteria solution using glycine buffer. The result showed us that extracted solution has strong ability to hydrolyze saponins. However, there’s no lactose in notoginseng solid fermentation medium. In order to reduce costs, another plasmid pSB1C3 which contains T7 RNA Polymerase gene under the control of pBAD promoter was transformed into E.coli. This double-plasmid system is expected to be regulated by arabinose, and expresses a large number of T7RNA polymerase to overcome the effect of LacI repression, switch on the expression of β-glucosidase. It’s been reported in scientific literature that the cell wall of notoginseng root cells contains a certain concentration of arabinose. Our ultimate goal is to use notoginseng root to provide nutrients for E.coli in solid state fermentation, where E.coli can hydrolyze saponin of notoginseng as well.