Difference between revisions of "Team:Tokyo-NoKoGen/Notebook"

Protocol
Investigation of inhibitory effect of 3,3'-diketotrehalose toward trehalase


1.Grow Halomonas sp. α-15 3 days as M9 Sα plate medium.
(gram-negative, rod-shaped marine bacterium isolated from seawater. This strain is known to produce glucose 3-dehydrogenase (G3DH))
2. Prepare the starter culture from the YM9Slac0.8% medium 3.0 mL. Under the condition at 30℃, 120 rpm for 12 h.

3. Prepare the preculture from the same medium 100mL at 30℃, 120 rpm for 12h.
4. Prepare the main culture from the same medium + FeCl3 (f.c. 200 μM) 2 L. Under the condition at 30℃, 600 rpm for 27 h with a jar fermenter.
5. Harvest the cells and wash with 10,000g. Then centrifuge for 20 min and store them in the deep freezer (-80℃).

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6. Disrupt cells using a French press with 20 mM potassium phosphate buffer (P.P.B.).
7. Obtain the soluble fraction by centrifugation (10,000 g, 5 min) and ultracentrifugation (106,000 g, 60 min), and dialyze it overnight.
8. Purify the dialyzed fraction with Akta FPLC system, anion exchange chromatography and hydrophobic interaction chromatography.
9. Dialyze the purified fraction overnight and store in 20 mM P.P.B. pH 7.0 at 4℃.
10. Check the existence of the protein(G3DH)’s band near the 63 kDa, with SDS-PAGE.


Check the enzyme activity with 20 mM trehalose, G3DH 5.0 U, 20 mM P.P.B(pH7.0) and 100 mM potassium ferricyanide. Mix them and incubate for 10 h at 25℃ under dark conditions.
(※)Enzyme activity was determined by phenazine methosulfate; PMS /2,6-dichloro-N-(4-hydroxyphenyl)-p-benzoquineimine; DCIP assay, substrate: glucose10 mM),


1. After 10 h, lyophilize mixture of 20 mM trehalose, 5.0 U G3DH, 20 mM P.P.B(pH 7.0) and 100 mM potassium ferricyanide.
2. Dissolve the product after lyophilized by 30 mL of methanol and lyophilized again.
3. Re-dissolve the product with distilled water and purified with ion exchange chromatography.
4. Lyophilize the purified fraction.
5. Check the existence of 3,3’-dkt trehalose with TLC.

●Trehalase reaction
1. Mix 10 μL of trehalase solution (0.0035 U) with 40 μL of 3,3’-dkT solution
2. Incubate at 37℃ for 10 min
3. Add 50 μL of trehalose solution
4. Incubate at 37℃ for 10 min
5. Heat at 95℃ for 5 min to stop trehalase reaction


1. Mix 12 μL the solution with 12 μL PMS (0.6 mM), 12 μL DCIP (0.06 mM), 12 μL PQQ-dependent glucose dehydrogenase(PQQGDH), and 72 μL of potassium phosphate buffer (10 mM, pH 7.0)
2. Measure the decrease in absorbance of DCIP (at 600 nm) and calculate PQQGDH activity
3. Draw a standard calibration curve of PQQGDH activity and known various glucose concentration. And from the calibration curve, calculate the glucose concentration in trehalase reaction solution.


●Preparation of enzyme solution
1. Transform the plasmid BBa_K1765000 to E. coli BL21(DE3) strain
2. Pre-culture the E. coli in 3 mL LB medium contain ( 30 μg/mL Cm) at 37 ℃ for 12 h
3. Add 1 mL the pre-culture medium to 100 mM LB medium ( 30 μg/mL Cm) and culture at 20℃ for 48 h
4. Centrifuge the culture medium and wash with 0.85% NaCl twice and get wet cells
5. Crush the cells with ultrasonic
6. Permeate in 10 mM potassium phosphate buffer (pH7.0, containing 1% Triton X-100)
7. Centrifuge the solution and get supernatant as enzyme solution

●G3DH activity assay
1. Mix 20 μL trehalose or cellobiose solution (f.c. 50 mM) with 20 μL PMS (f.c. 0.6 mM), 20 μL DCIP (f.c. 0.06 mM), 20 μL enzyme solution and 120 μL potassium phosphate buffer (10 mM, pH 7.0)
2. Measure the decrease in absorbance of DCIP at 600 nm and calculate G3DH activity

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Chararization of the biobric;BBa_K1765000

●Culture
1. Transform E. coli BL21(DE3) by pSB1C3-G3DH-OtsA-OtsB (BBa_K1765000).
2. Pre-culture transformed E. coli BL21(DE3) in 3 mL LB including 30 μg/mL Chloramphenicol at 37℃ overnight.
3. Culture in 100 mL LB including 30 μg/mL Chloramphenicol, 2.5 % glucose for 48 hr at 20℃.

　●Harvest
1. Centrifuge 100 mL of culture for 10 minutes at 4,000×g and 4 °C, and discard supernatant.
2. Add 0.85 % NaCl and resuspend.
3. Centrifuge for 10 minutes at 4,000×g and 4 °C, and discard supernatant.
4. Repreat wash (steps 2-3)
5. Add 30 mL of 0.85% NaCl and resuspend.
6. Take suspension into new 50 mL conical tube.
7. Centrifuge for 10 minutes at 5,000×g and 4 °C, and discard supernatant.

　●Crush and solubilize Membrane protein
1. Resuspend pellet (engineered E.coli) with 10 mM potassium phosphate buffer (pH 7) in pellet 1g with buffer 3mL.
2. Crush the engineered E. coli by ultrasonic cell crushing apparatus for 30s (repeat 30times).
3. Add 10 mM potassium phosphate buffer including 2% Triton-X 100(pH 7) of equivalent as step2 P.P.B buffer.
4. Take 1 mL of the disrupted cell suspension into new 1.5 mL tube.
5. Incubate it for 1 h with shaker.
6. Centrifuge it for 15 minutes at 9,000×g, 4 °C.
7. Put 800 μL of supernatant into new 1.5 mL tube.


●SDS analysis
1. Mix gently equivalent amount of protein solution sample and sodium dodecyl sulfate (SDS) buffer.
2. Set polyacrylamide gel(10%- 20%) in electrophoretic tank and Pour the running buffer (25mM Tris-HCl, 200mM Glycine, 0.1% SDS)
3. Apply 2μL of protein marker into the end lane and 15 μL of mixture of step 1 into other wells respectively.
4. Start running the electrophoresis at 20 mA for 90 minutes.
5. Dye the gel with Coomassie brilliant blue staining solution for 1 h and wash the gel with distilled water to decolor with shaking overnight.


●TLC analysis
1.Drip glucose solution, trehalose solution as standard and enzyme solution from E. coli which transformed either by pSB1C3-G3DH-OtsA-OtsB or empty pSB1C3 on thin-layer chromatography (TLC) plate.
2.Run the prepared plate with acetnitril:H₂O=7:3
3.Dip into 5% H₂SO₄ in EtOH
4.Heat TLC plate


●G3DH activity assay
1. Mix 20 μL trehalose or cellobiose solution (f.c. 50 mM) with 20 μL PMS (f.c. 0.6 mM), 20 μL DCIP (f.c. 0.06 mM), 20 μL enzyme solution and 120 μL potassium phosphate buffer (10 mM, pH 7.0)
2. Measure the decrease in absorbance of DCIP at 600 nm and calculate G3DH activity

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electrode experiments
●Extract gut solution
1.Pick up five termites
2.Freeze them and separate the head and body
3.Push the middle of the termite bodies
4.Extract the guts and mix to the 50 μL distillated water
5.Vortex them well
6.Ultracentrifuge the solution 2 times (13,000g, 10 min)
7.Get supernatant and take heat block at 50,95 ℃

●Experiment
1.Prepare the screen printed carbon electrode(SPCE)
2.Mix 2 μL gut solution and 1 μL Trehalose (100 mM) and 1 μL G3DH
3.Wait 1 minute
4.Incubate 1.5 μL of mixture to the SPCE
5.Apply 0.6 V vs. Ag/AgCl and measure response current

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Pseudo Egg
Administration of 3,3’-dkt to termite
1.Add 300 μL 3,3’-dkt(or 100 mM torehalose)(f.c. 100mM) solution to kimtowel (2 cm2) on petri dish
2.Release 8 worker termites (C. formosanus) to the petri dish
3.Keep observing them for days

●Immobilization evaluation of E.coli on artificial egg
1.Make artificial eggs by mixing of sodium alginate (10% w/w) and LB medium and drop into the mixture solution of CaCl2.
2.Add the 10 μL LB medium containing RFP expressing E.coli 3.Wash by 10 μL MQ or 10mg/ml β-Glucosidase or 10 mg/mL Lysozyme
4.0, 1, 3 days after making artificial eggs, and put these artificial eggs to LB plate