Difference between revisions of "Team:Tokyo-NoKoGen/Project-2"
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To produce 3,3’-diketotorehalose in the cell of <i>E. coli</i>, we constructed following Biobrick ( BBa_K1765000 ). This parts can express the enzyme G3DH from <i>Rhizobium radiobacter</i>, and also two additionally enzymes OtsA, OtsB from <i>E. coli</i>. The two enzymes OtsA and OtsB can convert glucose to trehalose, which can support the function of G3DH in the termite’s gut.<br> | To produce 3,3’-diketotorehalose in the cell of <i>E. coli</i>, we constructed following Biobrick ( BBa_K1765000 ). This parts can express the enzyme G3DH from <i>Rhizobium radiobacter</i>, and also two additionally enzymes OtsA, OtsB from <i>E. coli</i>. The two enzymes OtsA and OtsB can convert glucose to trehalose, which can support the function of G3DH in the termite’s gut.<br> | ||
| − | <center><img src="https://static.igem.org/mediawiki/2015/9/91/NK4.jpeg" width=" | + | <center><img src="https://static.igem.org/mediawiki/2015/9/91/NK4.jpeg" width="900px" height="400px"></center> |
Revision as of 03:15, 19 September 2015
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The present pesticides usually contain neurotoxins, which may affect humans. On the other hand, ExTermite coli can damage termites by producing inhibitory materials for pathway of termite. There are two merits of this insecticidal method.
First, there would be no damage for humans by inhibiting the metabolic pathway that is specific for termite. Therefore, while there is a possibility that neurotoxins affect mammals including human, but this insecticidal method has no effect for humans.
Secondly, this insecticidal method maintains its activity for long term. It can affect not only adult termites, but also larva termites. Therefore, as long as the ExTermite Coli alive in the termite’s gut, it can affect their metabolic pathway regardless of their generation.
Additionally, termites have another metabolic pathway. Termites feed on wood, paper or other cellulose containing materials. They can digest cellulose into glucose in their gut using a characteristic enzyme, cellulase.
ExTermite Coli targets the one of the metabolic pathways in termite to cut their energy source and exterminate termites.
< Details：Higher termite and Lower termite > -
The end product of cellulose digestion is used as an energy sources by termites. However, the method of digestion differs among each species of termites.
Termites can be classified into higher termites and lower termites. In higher termites, cellulose digestion is mediated solely by cellulase secreted by termites themselves. On the other hand, lower termites digest cellulose by cellulase secreted both by termites and by protozore. [2]
We focused on G3DH as an enzyme that can produce inhibitor for the metabolic pathway of termite. (From the specific characteristic of G3DH, it can produce inhibitor of trehalase from trehalose.) The structure of products from trehalose converted by G3DH is similar to trehalose: the substrate of trehalase. Therefore, these products stuck into trehalase resulting in loss of enzymatic activity of G3DH. Therefore, we think that products from G3DH can be used as inhibitor in Extreminator Coli.
< Details：Our strategy to inhibit the trehalase activity> -
3,3’-diketotreharose (3,3’dkT) is a product derived by the enzymatic oxidation of trehalose by G3DH. The third hydroxyl groups of both glucopyraosides of trehalose are oxidized in 3,3’-dkT. 3,3’dkT was revealed to show an inhibitory effect toward the trehalase from pig kidney and Bombyx mori[6]. Therefore, we expect that 3,3’dkT can be used an inhibitor in our system.
To investigate whether 3,3’dkT worked as inhibitor, first, we tried mass production of 3,3’dkT using G3DH.
- < Details：Purification of G3DH>
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To produce 3,3’dkT, we have to purify G3DH. In this system, we used G3DH from Halomonas sp.α-15 strain. To purify G3DH, Halomonas sp.α-15 strain was grown at 30℃ using fermenter. After 27 h, The culture was centrifuged to harvest cells. The cells were disrupted with French press and the lysate was centrifuged to remove cell debris. The cell free extract was ultracentrifuged. Then, G3DH was purified from the supernatant using anion exchange chromatography and hydrophobic interaction chromatography. [4]
< /m2>
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The enzymatic oxidation of trehalose by G3DH was performed in the presence of 100 mM potassium ferricyanide as an electron mediator. The reaction solution was lyophilized and the product was dissolved in methanol to precipitate protein and potassium ferricyanide. The pellet was removed by filtration and methanol was removed by lyophilization. The sample was re-dissolved in distilled water, and the product was purified with ion exchange column chromatography and lyophilization. [6]
The inhibitory effect of G3DH product from trehalose toward trehalase was measured by measuring the amount of glucose generated by enzyme.
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Measurement of trehalase activity was performed based on the enzymatic method. The amount of glucose produced by each enzyme was measured by enzymatically.
Trehalase or solution was incubated in the absence or presence of inhibitor (product of G3DH reaction). Then, substrate (trehalose or cellobiose) was added to trehalase or cellulose solution and the enzymatic conversion to glucose was allowed by incubating at 30 ℃ for 30 min. After that, the reaction was terminated by heating at 95℃ for 10 min. [6] Glucose produced by the trehalase or cellulase reaction was measured using PQQ-dependent glucose dehydrogenase, phenazine methosulfate (PMS), and 2,6-dichlorophenolindophenol (DCIP). The decrease in absorbance of DCIP at 600 nm was compared with the standard calibration curve for glucose, which was prepared using the same reagents with known glucose concentrations. [6]
From these results, we could clarify the inhibitory effect of each G3DH products from trehalose toward trehalase.
To produce 3,3’-diketotorehalose in the cell of E. coli, we constructed following Biobrick ( BBa_K1765000 ). This parts can express the enzyme G3DH from Rhizobium radiobacter, and also two additionally enzymes OtsA, OtsB from E. coli. The two enzymes OtsA and OtsB can convert glucose to trehalose, which can support the function of G3DH in the termite’s gut.
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Termite can digest cellulose into glucose by cellulose, and also they can convert glucose into trehalose. In our strategy, we are targeting inhibition of metabolic pathway by converting trehalose into trehalase inhibitor by G3DH. However, there is no proof for that termites stock their energy source in their gut. If there is no trehalose in the termite’s gut, G3DH can’t produce the inhibitor for the metabolic pathway of termite. To avoid this, we planned to produce trehalose by OtsA and OtsB. By coding these two enzymes in the same biobrick, G3DH can produce metabolic pathway inihibitor by converting trehalose produced by OtsA, OtsB.
After preparation of enzyme solution, we confirmed the function of OtsA, OtsB to check whether each of the enzymes can catalyze the conversion of glucose to trehalose. Using the purified OtsA, OtsB, we converted glucose into trehalose and checked the products by thin layer chromatography (TLC).
To check the activity of G3DH toward trehalose, we measured G3DH activity. We mixed G3DH solution to reaction reagent containing trehalose, PMS and DCIP. Then we measured the decrease of absorbance of DCIP at 600 nm to calculate the activity of G3DH.
From the results, we showed that the product derived from trehalose by G3DH has the inhibitory effect to trehalase.
Additionally, we built up one hypothesis about another possibility of G3DH. We focused on the wide substrate specificity of G3DH, and thought that it is possible for G3DH to catalyze of oxidation of cellobiose. Also, the product derived from cellobiose may work as the inhibitor of cellulase.
According to our hypothesis, we checked the activity of G3DH towards cellobiose. The method activity assay of G3DH toward cellobiose was same to toward trehalase. Figure (5) shows the new fact that G3DH has activity toward cellobiose. As we couldn’t check whether the product has the inhibitory activity towards cellulose, we are going to investigate whether the product can work as inhibitor.
Through above experiments, we confirmed that our constructed biobrick BBa_K1765000 can work as we expected. Also, we showed the inhibitory effect of G3DH products from trehalose toward trehalase. Additionally, we found that G3DH has the activity toward cellobiose. There is great possibility that cellulase derivative derived from the enzymatic oxidation of cellulase by G3DH has inhibitory effect to cellulase, and it can work as the metabolic pathway inhibitor.
These results show that enzymes produced by ExTermite Coli can work as inhibitor of metabolic pathway of termites.
[2] R.W.0’ Brien and Slaytor (1982) Role of Microorganisms in the Metabolism of Termites, Australian Journal of Biological Science, 35(3), 239-262 [3] Tsugawa, W. et al. (1998) Fluorescent measurement of 1,5-anhydro-D-glucitol based on a novel marine bacterial glucose dehydrogenase, Enzyme and microbial Technology, 22, 269-274 [4] Kojima, K. et al. (2001) Cloning and Expression of Glucose 3-Dehydrogenase from Halomonas sp. α-15 in Escherichia coli, Biochemical and Biophysical Research Communication, 282, 21-27 [5]Hayano, K. et al. (1967) Purification and properties of 3-ketosucrose-forming enzyme from the cells of Agrobacterium tumefaciens, The Journal of Biological Chemistry, 242, 3665-3672 [6] Sode, K. et al. (2001) Enzymatic synthesis of novel trehalose derivative, 3,3’-diketotrehalose, and its potential application as the trehalose enzyme inhibitor, FEBS Letters, 489, 42-45
