Difference between revisions of "Part:BBa K4289008"

Revision as of 08:04, 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.

Sequence and Features