Difference between revisions of "Part:BBa K4289007"
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==Engineering==
 
==Engineering==
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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.  
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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 1) 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.  
 
[[File:T-Nanjing-HS-BBa-K4289007-figure1.jpg|500px|thumb|center|Figure 1. Gel electrophoresis results of target gene fragments. A. double enzymes digested hGK2 DNA fragments.]]
 
[[File:T-Nanjing-HS-BBa-K4289007-figure1.jpg|500px|thumb|center|Figure 1. Gel electrophoresis results of target gene fragments. A. double enzymes digested hGK2 DNA fragments.]]
  

Latest revision as of 07:35, 6 October 2022

GK


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Contribution

GK, as a glucose receptor in pancreatic islets β cells, is mainly responsible for promoting insulin release and biosynthesis under glucose stimulation. The glucokinase agonists have a significant hypoglycemic effect and can significantly improve pancreatic islets’ β Cell function. So it is promising to screen glucokinase agonists for clinical treatment with GK protein.

In the past iGEM teams, there is no very detailed and complete screen platform of glucokinase agonists. For this reason, our team provides some valuable information for future iGEM teams by developing an in vitro screen platform.

Engineering

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 1) 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 1. Gel electrophoresis results of target gene fragments. A. double enzymes digested hGK2 DNA fragments.

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 2. SDS-PAGE detection of hGK2.