Difference between revisions of "Team:Tokyo Tech/AmiE Assay"

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<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-3- </span>title</p></div>
 
<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-3- </span>title</p></div>
 
<p class="normal_text"><br>
 
<p class="normal_text"><br>
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                                         <!--英訳がやべえ、なえさんよろしく-->
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                                         <!--英訳がやべえ、適当に直したけどチェックよろしくお願いします-->
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               1. Inoculate A into 3 mL LB medium containing ampicillin (50 microg / mL), and incubate at 37°C, 180 rpm for 12 h, so that the final concentration of glucose becomes 0.2%. <br><br>
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               1. Inoculate A into 3 mL LB medium containing ampicillin (50 microg / mL), and add glucose so that the final concentration becomes 0.02%.<br>Inoculate B and C into 3 mL LB culture containing ampicillin (50 microg / mL).<br><br>
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2. Inoculate B and C into 3 mL LB culture containing ampicillin (50 microg / mL), and incubate at 37°C, 180 rpm for 12 h, so that the final concentration of glucose becomes 0.2%.<br><br>
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                                        2. Incubate all samples at 37&deg;C, 180 rpm for 12 h.<br><br>
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3. Dilute the overnight culture of A so that the OD600 becomes about 0.05, and begin fresh culture at 37°C, 180 rpm.<br><br>
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3. Dilute the overnight culture of A so that the turbidity becomes about 0.05.<br><br>
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4. Add arabinose into test tube ① and ② so that the final concentration becomes 0.2% when the OD600 reaches 0.6 to 0.7.<br><br>
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                                        4. Incubate at 37&deg;C, 180 rpm so that turbidity becomes 0.6 to 0.7.<br><br>
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5. 2 h after addition arabinose, add 60 µL of 500 µM C12AHL into test tube ① and ③, add 200 µL of 500 µM C12AHL into test tube ② and ④.<br><br>
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5. Add arabinose into test tube (&#8560;) and (&#8561;) so that the final concentration becomes 0.2%.<br><br>
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6. 5 h after adding AHL, dispense the fresh cultures into 1.5 mL tubes, and centrifuge in duplicate for 2 min at 18000x g. Transfer it to other 1.5 mL tube.<br><br>
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6. 2 h after addition arabinose, add 60 microL of 3OC12HSL (500 microM) into test tube (&#8560;) and (&#8562;), add 200 microL of 3OC12HSL (500 microM) into test tube (&#8561;) and (&#8563;).<br><br>
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7. Dilute the overnight cultures of B and C, and begin fresh culture at 37°C, 180 rpm. (Add LB + ampicillin 800 µL into 10 µL overnight culture.)<br><br>
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7. 5 h after adding AHLs, dispense the fresh cultures into 1.5 mL tubes, and centrifuge in duplicate for 2 min at 18000G.<br><br>
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8. Prepare the overnight cultures containing LB medium and 1000 µL ampicillin in a 1.5 mL tube for control.<br><br>
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                                        8. Transfer supernatant to other 1.5 mL tube.<br><br>
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9. After 1.5 h, add 200 µL supernatants of A ①, ②, ③ and ④, and add 4 µL C12AHL into tube e, 13.3 µL C4AHL into tube f, 4 µL DMSO into tube g, 13.3 µL DMSO into tube h for control.<br><br>
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9. Add 10 microL overnight cultures of B and C into 800 microL LB medium containing ampicillin (50 microg / mL). Also, add 10 microL overnight cultures of B and C into 1 mL LB medium containing ampicillin (50 microg / mL) for controls.<br><br>
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10. Measure RFU of GFP / Turbidity and the optical density after incubation at 37°C, 180 rpm for 4 h.</p>
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10. After 1.5 h, add 200 µL supernatants of A into (&#8560;), (&#8561;), (&#8562;) and (&#8563;), and add 4 µL 3OC12HSL into tube e, 13.3 µL C4HSL into tube f, 4 µL DMSO into tube g, 13.3 µL DMSO into tube h for controls.<br><br>
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                                        11. Incubate at 37&deg;C, 180 rpm for 4 h.<br><br>
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12. Measure RFU of GFP / Turbidity and the turbinity.</p>
  
 
</div><!-- /protocol_contents -->
 
</div><!-- /protocol_contents -->

Revision as of 14:25, 18 October 2016

3-3 AmiE assay

1. Introduction

The Prince coli expresses AmiE protein, and Snow White coli recovers from its apparent death and wakes up again.

We tested the function of AmiE protein that influences the end of the story.

2. Summary of the Experiment

The objective of this experiment is to characterize the function of AmiE protein. We prepared three samples shown below. When the AmiE degradation ability with these samples was analyzed, the results showed that C4HSL was not degraded by AmiE, but 3OC12HSL was degraded by AmiE.

・PBAD/araC - rbs - amiE (pSB6A1)
・Ptet - rbs - luxR - tt - Plux - rbs - gfp (pSB6A1)
・Ptet - rbs - luxR - tt - Plux - rbs - gfp (pSB6A1)

3. Results

We examined that the AmiE degradation activity of C4 and C12. The results are shown below.



Fig. 3-3- title


Fig. 3-3- title

This graph shows that the result of RFU of GFP / Turbidity of the supernatant centrifuged after several hours of the E. coli solution both induced and did not induce AmiE expression.

From the results of the AmiE degradation of C4, RFU of GFP / Turbidity of the reporter with the supernatant of the solution induced the AmiE expression is almost the same as the one with the supernatant of the one not induced the AmiE expression.

However, in the case of C12, RFU of GFP / Turbidity of the reporter added the solution which induced the AmiE expression is about 1/10 of which did not induce AmiE expression. Based on the above result, we concluded that AmiE does not degrade C4 but degrades C12.

4. Discussion

AmiE is the protein that degrades AHLs which have acyl chains longer than six carbons. This experiment showed that C4 was not degraded and C12 is degraded. This result is consistent with the AmiE function written in the literature.

We thought that we could express AmiE in the Prince coli and cause degradation of C12 as we desired. Also, it is expected that the Prince coli degrades the poisoned apple and makes Snow White coli recover from its apparent death.

5. Materials and Methods

5-1. Construction

-Strain
All the sample were XL1‐Blue strain.

-Plasmids
A， PBAD/araC - rbs - amiE (pSB6A1)
B， Ptet - rbs - luxR - tt - Plux - rbs - gfp (pSB6A1)
C， Ptet - rbs - rhlR - tt - Prhl - rbs - gfp (pSB6A1)






5-2. Assay Protocol

A. Fresh Culture
4 test tubes



Fig. 3-3- title

B. Fresh Culture
6 of 1.5 mL tubes



Fig. 3-3- title

control



Fig. 3-3- title


1. Inoculate A into 3 mL LB medium containing ampicillin (50 microg / mL), and add glucose so that the final concentration becomes 0.02%.
Inoculate B and C into 3 mL LB culture containing ampicillin (50 microg / mL).

2. Incubate all samples at 37°C, 180 rpm for 12 h.

3. Dilute the overnight culture of A so that the turbidity becomes about 0.05.

4. Incubate at 37°C, 180 rpm so that turbidity becomes 0.6 to 0.7.

5. Add arabinose into test tube (ⅰ) and (ⅱ) so that the final concentration becomes 0.2%.

6. 2 h after addition arabinose, add 60 microL of 3OC12HSL (500 microM) into test tube (ⅰ) and (ⅲ), add 200 microL of 3OC12HSL (500 microM) into test tube (ⅱ) and (ⅳ).

7. 5 h after adding AHLs, dispense the fresh cultures into 1.5 mL tubes, and centrifuge in duplicate for 2 min at 18000G.

8. Transfer supernatant to other 1.5 mL tube.

9. Add 10 microL overnight cultures of B and C into 800 microL LB medium containing ampicillin (50 microg / mL). Also, add 10 microL overnight cultures of B and C into 1 mL LB medium containing ampicillin (50 microg / mL) for controls.

10. After 1.5 h, add 200 µL supernatants of A into (ⅰ), (ⅱ), (ⅲ) and (ⅳ), and add 4 µL 3OC12HSL into tube e, 13.3 µL C4HSL into tube f, 4 µL DMSO into tube g, 13.3 µL DMSO into tube h for controls.

11. Incubate at 37°C, 180 rpm for 4 h.

12. Measure RFU of GFP / Turbidity and the turbinity.

6. Reference


[1]Ochiai S, Yasumoto S, Morohoshi T, Ikeda T. AmiE, a Novel N-Acylhomoserine
Lactone Acylase Belonging to the Amidase Family, from the Activated-Sludge Isolate Acinetobacter sp. Strain Ooi24. 2014 Nov;80(22):6919-25.