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Team:Tokyo-NoKoGen/g3dh
From 2014.igem.org
About G3DH
The glucose-3-dehydrogenase (G3DH) operon from Rhizobium tumefaciens EHA101 encodes the heterotrimer G3DH, which converts glucose to 3-ketoglucose. The deduced primary structure and secondary structure of G3DH are similar to sorbitol dehydrogenase from Gluconobacter oxidans and 2-keto-D-gluconate dehydrogenases from Erwinia herbicola. These enzymes are composed of three subuints; catalytic subunit, cytochrome c subunit, and small subunit. Catalytic domain has flavin adenine dinucleotide (FAD) as a cofactor. And cytochrome c subunit bounds to cytoplasmic membrane. The function of small subunit is unknown.
This G3DH we use also has cytochrome c subunit. Cytochrome c subunit needs to be matured by cytochrome c maturation enzymes (CCM). Therefore, we use the plasmid, pEC86 which has the gene of CCM.
And this G3DH is a homolog of G3DH from a marine bacterium Halomonas sp. α-15 which shows wide substrate specificity. It is already reported that G3DH from Halomonas sp. α-15 can also convert trehalose (a disaccharide made up of two glucose moieties) into 3,3'-diketotrehalose, which is trehalase inhibitor. Therefore, the G3DH we use can also convert trehalose to 3,3'-diketotrehalose.
Reference
(1) Cloning and Expression of Glucose 3-Dehydrogenase from Halomonas sp. α-15 in Escherichia coli.,(2001),K Kojima et al.,
(2) Overproduction of the Bradyrhizobium japonicum c-Type Cytochrome Subunits of the cbb3 Oxidase in Escherichia coli.,(1998),E Arslan et al.,
1.Construction of Biobrick
G3DH gene were cloned from pTrc99a-G3DH which originated from Agrobacterium tumefaciens. PCR products were inserted pSB1C3. Original G3DH gene has two illegal restriction sites. In order to remove these restriction sites, the G3DH gene were amplified by overlap extension PCR. We designed two primer sets for overlap extension PCR.
Fig.1. Remove illegal restriction sites
G3DH gene fragments were amplified and three PCR products were connected.
G3DH (removed illegal restriction sites) were ligated with four promoters and double terminator (BBa_B0010 and BBa_B0012).
One of those promoters is arabinose inducible, and the others are constitutive promoter.
Fig.2 Constructed plasmid
2. Evaluation
This is the method of culturing and extraction of production (Fig. 1).
We cultured E. coli TOP10 transformed with two plasmids; G3DH expression vector, and cytochrome c maturation enzymes expression vector (pEC86) in LB medium containing 20 mM Trehalose. For expression of G3DH, we used four different promoters.
When OD660 achieves 0.6, 0.2 % arabinose was added to the medium for induction in the case we used pBAD as a promoter.
After culturing for 20 hours at 37 ℃, we extracted products by boiling and centrifugation.
0.007U trehalase and 20 mM trehalose mixed sample and incubated 30h.
First, we investigated expression of G3DH by SDS-PAGE analysis. Second, we measured the glucose dehydrogenase activity of G3DH. Finally we tried to detect 3,3'-dkT by thin-layer chromatography (TLC).
Fig. 1 evaluation of G3DH
By SDS-PAGE analysis, there was the band which showed about 68 kDa on G3DH under the constitutive promoter P106 and P100, pBAD. Therefore, we confirmed the expression of G3DH (Fig. 2).
Fig. 2 SDS-PAGE analysis
3. GDH activity assay
Trehalase inhibition measurement (Fig. 3)
Fig. 3 Trehalose inhibition activity assay
This is the result of trehalase inhibition activity assay. Each value of activity was normalized at the value of activity of empty vector which were not induced by arabinose. Samples of G3DH which induced by P100 and P106 activity was lower than that of empty vector.
We concluded that G3DH which induced by P100 and P106 expressed G3DH. And G3DH converted trehalose to 3,3'-diketotorehalose .
