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

 
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<m2>Investigation of inhibitory effect of 3,3'-diketotrehalose toward trehalase</m2><br>
 
<m2>Investigation of inhibitory effect of 3,3'-diketotrehalose toward trehalase</m2><br>
 
<br>
 
<br>
−
<m3><Purify the G3DH></m3><br>
+
<m3><u>Purify the G3DH</u></m3><br>
 
<m3>1.Grow <i>Halomonas</i> sp. α-15 3 days as M9 Sα plate medium.<br>
 
<m3>1.Grow <i>Halomonas</i> sp. α-15 3 days as M9 Sα plate medium.<br>
 
　　(gram-negative, rod-shaped marine bacterium isolated from seawater. This strain is known to produce glucose 3-dehydrogenase (G3DH))<br>
 
　　(gram-negative, rod-shaped marine bacterium isolated from seawater. This strain is known to produce glucose 3-dehydrogenase (G3DH))<br>
−
　　2. Prepare the starter culture from the YM9Slac0.8% medium 3.0 mL. Under the condition at 30℃, 120 rpm for 12 h.<br>
+
2. Prepare the starter culture from the YM9Slac0.8% medium 3.0 mL. Under the condition at 30℃, 120 rpm for 12 h.<br>
−
　　3. Prepare the preculture from the same medium 100mL at 30℃, 120 rpm for 12h.<br></m3>
+
3. Prepare the preculture from the same medium 100mL at 30℃, 120 rpm for 12h.<br>
−
<m3>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.<br>
+
<br>
−
　　5. Harvest the cells and wash with 10,000g. Then centrifuge for 20 min and store them in the deep freezer (-80℃).<br></m3>
+
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.<br>
−
<br><center><img src="https://static.igem.org/mediawiki/2015/3/3b/NK7.jpeg"style =position:"rekative";top:"300px";right:"0px"width="700px";height="500px"+></center>><br>
+
5. Harvest the cells and wash with 10,000g. Then centrifuge for 20 min and store them in the deep freezer (-80℃).<br></m3>
 +
<br><center><img src="https://static.igem.org/mediawiki/2015/3/3b/NK7.jpeg"style=position:"relative"; top:"280px"; right:"10px"; width="700px"; height="400px";></center><br>
 +
 
 
<br>
 
<br>
 
<m3>
 
<m3>
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10. Check the existence of the protein(G3DH)’s band near the 63 kDa, with SDS-PAGE. <br></m3>
 
10. Check the existence of the protein(G3DH)’s band near the 63 kDa, with SDS-PAGE. <br></m3>
 
<br>
 
<br>
−
  <m2><Enzyme activity></m2><br>
+
  <m2><u>Enzyme activity</u></m2><br>
−
  <m3>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.<br></m3>
+
  <m3>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.<br>  
−
+
 
  (※)Enzyme activity was determined by phenazine methosulfate; PMS /2,6-dichloro-N-(4-hydroxyphenyl)-p-benzoquineimine; DCIP assay, substrate: glucose10 mM),<br>
 
  (※)Enzyme activity was determined by phenazine methosulfate; PMS /2,6-dichloro-N-(4-hydroxyphenyl)-p-benzoquineimine; DCIP assay, substrate: glucose10 mM),<br>
 
<br></m3>
 
<br></m3>
  
−
<m2><Produce 3,3’-dkt trehalose></m2><br>
+
<m2><u>Produce 3,3’-dkt trehalose</u></m2><br>
 
<m3>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. <br>
 
<m3>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. <br>
 
2. Dissolve the product after lyophilized by 30 mL of methanol and lyophilized again.<br>
 
2. Dissolve the product after lyophilized by 30 mL of methanol and lyophilized again.<br>
 
3. Re-dissolve the product with distilled water and purified with ion exchange chromatography.<br>
 
3. Re-dissolve the product with distilled water and purified with ion exchange chromatography.<br>
 
4. Lyophilize the purified fraction.<br>
 
4. Lyophilize the purified fraction.<br>
−
5. Check the existence of 3,3’-dkt trehalose with TLC.<br></3>
+
5. Check the existence of 3,3’-dkt trehalose with TLC.<br></m3>
−
 
+
<br>
−
<m2><Measurement of inhibitory effect></m2><br>
+
<m2><u>Measurement of inhibitory effect</u></m2><br>
 
<m3>●Trehalase reaction<br>
 
<m3>●Trehalase reaction<br>
 
1. Mix 10 μL of trehalase solution (0.0035 U) with 40 μL of 3,3’-dkT solution<br>
 
1. Mix 10 μL of trehalase solution (0.0035 U) with 40 μL of 3,3’-dkT solution<br>
Line 184: Line 185:
 
3. Add 50 μL of trehalose solution<br>
 
3. Add 50 μL of trehalose solution<br>
 
4. Incubate at 37℃ for 10 min<br>
 
4. Incubate at 37℃ for 10 min<br>
−
5. Heat at 95℃ for 5 min to stop trehalase reaction<br>
+
5. Heat at 95℃ for 5 min to stop trehalase reaction</m3>
−
<br>
+
<br><br>
−
<m2><Measurement of glucose concentration></m2><br>
+
<m2><u>Measurement of glucose concentration</u></m2><br>
 
<m3>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)<br>
 
<m3>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)<br>
 
2. Measure the decrease in absorbance of DCIP (at 600 nm) and calculate PQQGDH activity<br>
 
2. Measure the decrease in absorbance of DCIP (at 600 nm) and calculate PQQGDH activity<br>
Line 192: Line 193:
 
And from the calibration curve, calculate the glucose concentration in trehalase reaction solution.<br></m3>
 
And from the calibration curve, calculate the glucose concentration in trehalase reaction solution.<br></m3>
 
<br>
 
<br>
−
<m2><G3DH activity assay></m2><br>
+
<m2><u>G3DH activity assay</u></m2><br>
 
<m3>●Preparation of enzyme solution<br>
 
<m3>●Preparation of enzyme solution<br>
 
1. Transform the plasmid BBa_K1765000 to <i>E. coli</i> BL21(DE3) strain<br>  
 
1. Transform the plasmid BBa_K1765000 to <i>E. coli</i> BL21(DE3) strain<br>  
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<br></m3>
 
<br></m3>
 
<m3>----------------------------------------------------------------------------------------------------<br></m3>
 
<m3>----------------------------------------------------------------------------------------------------<br></m3>
−
<br><m2></m2>
+
<br>
−
<m1>Chararization of the biobric;BBa_K1765000<m1><br>
+
<br>
−
<m2><Preparation of enzyme solution</m2><br>
+
<m1>Chaprarization of the biobric;BBa_K1765000</m1><br>
 +
<m2><u>Preparation of enzyme solution</u></m2><br>
 
<m3>●Culture<br>
 
<m3>●Culture<br>
 
1. Transform <i>E. coli</i> BL21(DE3) by pSB1C3-<i>G3DH-OtsA-OtsB</i> (BBa_K1765000).<br>
 
1. Transform <i>E. coli</i> BL21(DE3) by pSB1C3-<i>G3DH-OtsA-OtsB</i> (BBa_K1765000).<br>
 
2. Pre-culture transformed <i>E. coli</i> BL21(DE3) in 3 mL LB including 30 μg/mL Chloramphenicol at 37℃ overnight.<br>
 
2. Pre-culture transformed <i>E. coli</i> BL21(DE3) in 3 mL LB including 30 μg/mL Chloramphenicol at 37℃ overnight.<br>
−
3. Culture in 100 mL LB including 30 μg/mL Chloramphenicol, 2.5 % glucose for 48 hr at 20℃.<br></m3>
+
3. Culture in 100 mL LB including 30 μg/mL Chloramphenicol, 2.5 % glucose for 48 hr at 20℃.<br>
 
<br>
 
<br>
−
<m3>
+
●Harvest<br>
−
　●Harvest<br>
+
 
1. Centrifuge 100 mL of culture for 10 minutes at 4,000×g and 4 °C, and discard supernatant.br>
 
1. Centrifuge 100 mL of culture for 10 minutes at 4,000×g and 4 °C, and discard supernatant.br>
 
2. Add 0.85 % NaCl and resuspend.<br>
 
2. Add 0.85 % NaCl and resuspend.<br>
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5. Add 30 mL of 0.85% NaCl and resuspend.<br>
 
5. Add 30 mL of 0.85% NaCl and resuspend.<br>
 
6. Take suspension into new 50 mL conical tube.<br>
 
6. Take suspension into new 50 mL conical tube.<br>
−
7. Centrifuge for 10 minutes at 5,000×g and 4 °C, and discard supernatant.<br></m3>
+
7. Centrifuge for 10 minutes at 5,000×g and 4 °C, and discard supernatant.<br>
−
 
+
<br>
−
　●Crush and solubilize Membrane protein<br>
+
●Crush and solubilize Membrane protein<br>
−
</m3>1. Resuspend pellet (engineered E.coli) with 10 mM potassium phosphate buffer (pH 7) in pellet 1g with buffer 3mL.<br>
+
1. Resuspend pellet (engineered E.coli) with 10 mM potassium phosphate buffer (pH 7) in pellet 1g with buffer 3mL.<br>
 
2. Crush the engineered <i>E. coli</i> by ultrasonic cell crushing apparatus for 30s (repeat 30times).<br>
 
2. Crush the engineered <i>E. coli</i> by ultrasonic cell crushing apparatus for 30s (repeat 30times).<br>
 
3. Add 10 mM potassium phosphate buffer including 2% Triton-X 100(pH 7) of equivalent as step2 P.P.B buffer.<br>
 
3. Add 10 mM potassium phosphate buffer including 2% Triton-X 100(pH 7) of equivalent as step2 P.P.B buffer.<br>
Line 232: Line 233:
 
5. Incubate it for 1 h with shaker.<br>
 
5. Incubate it for 1 h with shaker.<br>
 
6. Centrifuge it for 15 minutes at 9,000×g, 4 °C.<br>
 
6. Centrifuge it for 15 minutes at 9,000×g, 4 °C.<br>
−
7. Put 800 μL of supernatant into new 1.5 mL tube.<br></m3>
+
7. Put 800 μL of supernatant into new 1.5 mL tube.</m3>
 
<br>
 
<br>
−
 
+
<br>
−
 
+
<m2><u>SDS-PAGE analysis</u></m2><br>
−
<m2><SDS analysis></m2><br>
+
 
<m3>1. Mix gently equivalent amount of protein solution sample and sodium dodecyl sulfate (SDS) buffer.<br>
 
<m3>1. Mix gently equivalent amount of protein solution sample and sodium dodecyl sulfate (SDS) buffer.<br>
 
2. Set polyacrylamide gel(10%- 20%) in electrophoretic tank and Pour the running buffer (25mM Tris-HCl, 200mM Glycine, 0.1% SDS)<br>
 
2. Set polyacrylamide gel(10%- 20%) in electrophoretic tank and Pour the running buffer (25mM Tris-HCl, 200mM Glycine, 0.1% SDS)<br>
Line 243: Line 243:
 
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.<br></m3>
 
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.<br></m3>
 
<br>
 
<br>
−
<m2><TLC analysis></m2><br>
+
<m2><u>TLC analysis</u></m2><br>
 
<m3>1.Drip glucose solution, trehalose solution as standard and enzyme solution from <i>E. coli</i> which transformed either by pSB1C3-<i>G3DH-OtsA-OtsB</i> or empty pSB1C3 on thin-layer chromatography (TLC) plate.<br>
 
<m3>1.Drip glucose solution, trehalose solution as standard and enzyme solution from <i>E. coli</i> which transformed either by pSB1C3-<i>G3DH-OtsA-OtsB</i> or empty pSB1C3 on thin-layer chromatography (TLC) plate.<br>
 
2.Run the prepared plate with acetnitril:H<sub>2</sub>O=7:3<br>
 
2.Run the prepared plate with acetnitril:H<sub>2</sub>O=7:3<br>
Line 250: Line 250:
 
<br></m3>
 
<br></m3>
  
−
<m2><G3DH activity assay></m2><br>
+
<m2><u>G3DH activity assay</u></m2><br>
 
<m3>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)<br>
 
<m3>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)<br>
 
2. Measure the decrease in absorbance of DCIP at 600 nm and calculate G3DH activity<br>
 
2. Measure the decrease in absorbance of DCIP at 600 nm and calculate G3DH activity<br>
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<m3>---------------------------------------------------------------------------------------<br>
 
<m3>---------------------------------------------------------------------------------------<br>
  
−
<m1>electrode experiments</m1><br>
+
<m1><b>electrode experiments</b></m1><br>
−
<m2><Extract gut solution></m2><br>
+
<m2><u>Extract gut solution</u></m2><br>
 
1.Pick up five termites<br>
 
1.Pick up five termites<br>
 
2.Freeze them and separate the head and body<br>
 
2.Freeze them and separate the head and body<br>
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7.Get supernatant and take heat block at 50,95 ℃<br>
 
7.Get supernatant and take heat block at 50,95 ℃<br>
 
<br>
 
<br>
−
<m2>Experiment</m2><br>
+
<m2><u>Electrical experiment</u></m2><br>
 
<m3>
 
<m3>
 
1.Prepare the screen printed carbon electrode(SPCE)<br>
 
1.Prepare the screen printed carbon electrode(SPCE)<br>
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---------------------------------------------------------------------------------------<br>
 
---------------------------------------------------------------------------------------<br>
 
<br>
 
<br>
−
<m1>Pseudo Egg</m1><br>
+
<m1><b>Pseudo Egg</b></m1><br>
−
<m2><Administration 3,3’-dkt to termite></m2><br>
+
<m2><u>Administration 3,3’-dkt to termite</u></m2><br>
 
1.Add 300 μL 3,3’-dkt(or 100 mM torehalose)(f.c. 100mM) solution to kimtowel (2 cm2) on petri dish<br>
 
1.Add 300 μL 3,3’-dkt(or 100 mM torehalose)(f.c. 100mM) solution to kimtowel (2 cm2) on petri dish<br>
 
2.Release 8 worker termites (C. formosanus) to the petri dish<br>
 
2.Release 8 worker termites (C. formosanus) to the petri dish<br>
 
3.Keep observing them for days <br>
 
3.Keep observing them for days <br>
 
<br>
 
<br>
−
<m2>Immobilization evaluation of E.coli on artificial egg</m2><br>
+
<m2><u>Immobilization evaluation of E.coli on artificial egg</u></m2><br>
 
1.Make artificial eggs by mixing of sodium alginate (10% w/w) and LB medium and drop into the mixture solution of CaCl2.<br>
 
1.Make artificial eggs by mixing of sodium alginate (10% w/w) and LB medium and drop into the mixture solution of CaCl2.<br>
 
2.Add the 10 μL LB medium containing RFP expressing <i>E.coli</i>
 
2.Add the 10 μL LB medium containing RFP expressing <i>E.coli</i>
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text-align:center;
 
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position:relative;
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margin-left:auto;
 
margin-left:auto;
 
margin-right:auto;
 
margin-right:auto;
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     border-width: thick;
 
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border-color: #ADFF2F;
 
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Latest revision as of 10:23, 21 November 2015

　

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

Purify the G3DH
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℃).


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.

Enzyme activity
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),

 Produce 3,3’-dkt trehalose
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.
Measurement of inhibitory effect
●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

Measurement of glucose concentration
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.
G3DH activity assay
●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

 ----------------------------------------------------------------------------------------------------

Chaprarization of the biobric;BBa_K1765000
Preparation of enzyme solution
●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.br> 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-PAGE 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

 ---------------------------------------------------------------------------------------
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 ℃

Electrical 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

---------------------------------------------------------------------------------------

Pseudo Egg
Administration 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