Difference between revisions of "Part:BBa K4289008"

Revision as of 08:11, 26 September 2022

pQE-30-GK

A recombinant plasmid for expressing and purifying GK protein.

BBa_K4289008 pQE-30-GK

Contribution

Type 2 diabetes accounts for 90% of all kinds of diabetes caused by a declining response to insulin, the victims suffer from huge side effects. And the number of cases is increasing rapidly. Moreover, it is gradually spreading among younger crowds. Therefore, we find it helpful to many people to design a drug used to prevent symptoms of type 2 diabetes.

Glucokinase is a monomeric enzyme and serves as a “glucose-sensor” or “glucose receptor” in pancreatic cells and the liver, eliciting glucose-stimulated insulin secretion, and as glucose “gate-keeper” in hepatocytes, promoting hepatic glucose uptake and glycogen synthesis and storage. Due to its essential role in glucose homeostasis, it is important to develop a screening platform for GK activators. In this project, we chose pQE30 as a protein expression vector and purified the hGK2 protein in E. coli M15, and then the protein could be used in the screening platform.

Engineering

How we design our plasmid

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).

Figure 1. The plasmid map in this project.

How we build our plasmid

In order to obtain the target fragments, we selected the appropriate endonuclease and digested both the DNA fragments and plasmid carrier simultaneously. We digested the DNA fragment hGK2 and plasmid pQE-30 with BamHI and SalI. Then we obtained the target DNA fragments (Figure 2) and ligated the fragments with T4 DNA ligase. Afterward, we transformed the recombinant plasmid into E. coli M15 competent cells and coated on the LB culture medium plate.

Figure 2. Gel electrophoresis results of target gene fragments. A. double enzymes digested hGK2 DNA fragments, B. double enzymes digested pQE-30 plasmid.

We inoculated 3 single colonies and extracted the plasmids. To verify the plasmids, we digested these plasmids with BamHI and SalI (Figure 3A). We send 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 (Figure 3B), and the plasmid was successfully constructed. So far, we have successfully constructed the pQE30-hGK2 vector.

Figure 3 Agarose gel electrophoresis diagram of the clone. (A) Verify the colony in lanes 1-3 (B) Sequenced results mapped to the plasmid.

In Figure 3A, we can see that there are obvious bands of hGK2 and pQE30, proving that our recombinant cloning products were constructed successfully. In Figure 3B, we can see that there is no difference in the result of the template and construction, which represents the success of construction. This meant that we can carry out subsequent cell transfection and characterization qualitative detection.

Protein expression and verification

In order to purify the protein, we cultured the M13 transformants in LB (Ampicillin/Chloramphenicol) and add IPTG to induce the protein expression when the OD600 reached 0.6-0.8. After overnight induction and culture, we collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins. Next, we used Ni-NTA column purification to purify the hGK2 protein. As shown in Figure 4, there are several clear bands which means the hGK2 protein was successfully expressed in the strain.

Figure 4. SDS-PAGE detection of hGK2.

improvement

Our composite component, BBa_K4289008, 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_K4289008 to screen more kinds of glucokinase agonists that can be used to decrease blood sugar and promote islet β-cell survival.

In 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.

2. Results of Compounds Screening Using this Platform

a) To Establish the screen system for glucokinase agonists

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

Sequence and Features