Our composite component, BBa_K4289000, is an enzyme that could be used to develop the screen glucokinase agonists platform.
As early as 2014, the iGEM14_Saarland team was committed to transferring glucose to glucose-6-phosphate and producing HA (BBa_K1469003). Based on their work, by reading literature and consulting experts in related fields, we found that glucokinase (hGK2) also could to screen glucokinase agonists in vitro. Therefore, we further optimized the glucokinase agonists screening platform and constructed a new composite part BBa_K4516019 to screen more kinds of glucokinase agonists that can be used to decrease blood sugar and promote islet β-cell survival.
n order to prove the function of our new composite part hGK2, we transferred the recombinant plasmid into E. coli M13 cells to express the hGK2 protein. We purified the protein and established an in vitro glucokinase agonists screening platform, and found compound 13926, which has an effect on decreasing blood sugar. Then, by culturing INS-832/13 cells with compound 13926, we detected the effect of compound 13926 in promoting islet β-cell survival. As a result, it was further confirmed that compound 13926 has the effect of decreasing blood sugar and promoting islet β-cell survival.

GK is a kind of isozyme of hexokinase that expresses in islet β-cell. GK takes part in the first step of glycolysis and transfers Glucose into Glucose 6-phosphate(G6P). The appetency of GK, as the receptor of glucose in the cells, is mainly responsible for promoting insulin release and biosynthesis under glucose stimulation, as well as regulating pancreatic islets β cell survival and proliferation.

We design the plasmid: The DNA fragment of the hGK2 was inserted into the BamHI and SalI sites of the pQE-30 vector (Figure 1). We transformed the recombinant plasmid into E. coli M15 competent cells and sent the constructed recombinant plasmid to a sequencing company for sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region, and the plasmid was successfully constructed.

We established a screening system for glucokinase agonists. The total volume of the reaction system is 120μL.

Finally, we used Flexstation 3 Multifunction microplate reader workstation to add 12 μL ATP to initiate the reaction and immediately detect the change in absorbance at 340 nm. The Maximum reaction rate is reflected by the slope of the curve.

As we can learn from the result (Figure 2), compound 13926 was finally discovered (maximum agitation rate, 1.21; EC50, 87 nM), which with higher activation activity than PF-04937319 (EC50, 930 nM).

Incubate 2×105 of INS-832/13 cells in a 24-well cell culture plate with 500μL culture medium overnight. The next day, replace the medium with 500μL of KRB buffer (115 mM NaCl, 5 mM KCl, 24 mM NaHCO3, 10 mM HEPES, 2.5 mM CaCl2, 1 mM MgCl2, 0.1% BSA, pH = 7.3) with 2.8mM glucose and cultured for 2h. Then replace the medium with KRB buffer containing 5.5mM glucose and 20μM compound 13926. Incubate the cells for 2h. The supernatant of the medium was transferred to a 1.5 mL centrifuge tube and centrifuged at 600 rpm, 4℃ for 5 min. Dilute the supernatant and test it with an insulin assay kit. After the cells were lysed at 100μL RIPA, the protein concentration was determined as an internal reference using the BCA protein concentration determination kit. hGK2 enzyme activity detection platform was established and a positive compound PF-04937319 was used to prove the activity of hGK2 (Figure 3A).

Ins-832/13 cells were incubated with a density of 5 × 105 for each well. Incubate the cells in a 96-well plate. Culture them overnight in a 5% CO2, 37°C cell incubator. On the second day, add STZ with a final concentration of 0.4 mM and 13926 with a final concentration of 20 μM. Incubate them for 24 h. On the third day, discarded the medium, add the medium containing 0.5 mg / mL MTT, and continue to incubate for 4 h. After that, aspirate the medium. Add 100 μL DMSO to each hole, and shake the plate at 400 rpm for 10 min. Detected the absorbance at 490 nm with a spectrum max M5 multi-functional microplate reader (Figure 3B).
The results indicated that 13926 effectively promoted insulin secretion and protected it from STZ damage in Ins-832/13 cells.