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

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<p class="normal_text">AmiE is the protein that degrades AHLs which have acyl chains longer than eight carbons. This experiment shows that C4AHL is not degraded, and C12AHL is degraded. This result is consistent with the AmiE function written in a paper. We thought that we can express AmiE in the Prince <span style="font-style : italic">E. coli</span> and make it degrade 3OC12HSL.<br>
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<p class="normal_text">AmiE is the protein that degrades AHLs which have acyl chains longer than eight carbons. This experiment shows that C4HSL is not degraded, and 3OC12HSL is degraded. This result is consistent with the AmiE function written in a paper. We thought that we can express AmiE in the Prince <span style="font-style : italic">E. coli</span> and make it degrade 3OC12HSL.<br>
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From the results, it is expected that the Prince E. coli degrades the poisoned apple and make Snow White E. coli recover from its apparent death.
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From the results, it is expected that the Prince <span style="font-style : italic">E. coli</span> degrades the poisoned apple and make Snow White <span style="font-style : italic">E. coli</span> recover from its apparent death.
 
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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">1. Inoculate A into 2997 µL LB culture containing 3 µL ampicillin, and incubate at 37°C, 180 rpm for 12 h, so that the final concentration of glucose becomes 0.2%. <br><br>
 
<p class="normal_text">1. Inoculate A into 2997 µL LB culture containing 3 µL ampicillin, 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. Inoculate B and C into 2997 µL LB culture containing 3 µL ampicillin, 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. Inoculate B and C into 2997 µL LB culture containing 3 µL ampicillin, 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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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 OD600 becomes about 0.05, and begin fresh culture at 37°C, 180 rpm.<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. 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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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. 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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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. 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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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>
+
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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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. 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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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. 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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10. Measure the fluorescence intensity and the optical density after incubation at 37°C, 180 rpm for 4 h.
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10. Measure the fluorescence intensity and the optical density after incubation at 37°C, 180 rpm for 4 h.</p>
  
 
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<p class="normal_text">[1]Ochiai S, Yasumoto S, Morohoshi T, Ikeda T. AmiE, a Novel N-Acylhomoserine<br>
 
<p class="normal_text">[1]Ochiai S, Yasumoto S, Morohoshi T, Ikeda T. AmiE, a Novel N-Acylhomoserine<br>
−
Lactone Acylase Belonging to the Amidase Family, from the Activated-Sludge Isolate Acinetobacter sp. Strain Ooi24. 2014 Nov;80(22):6919-25.
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Lactone Acylase Belonging to the Amidase Family, from the Activated-Sludge Isolate Acinetobacter sp. Strain Ooi24. 2014 Nov;80(22):6919-25.
  
 
   
 
   

Revision as of 20:03, 13 October 2016

3-3 AmiE assay

1. Introduction

The Prince E. coli expresses AmiE protein, and Snow White E. 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

Our objective is to characterize the function of AmiE protein. We prepared three samples shown below. When we tested the AmiE degradation ability with these samples, the results show that C4HSL is not degraded by AmiE, but 3OC12HSL is 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 C4HSL and 3OC12HSL. The results are shown below.



Fig. 3-3- title


Fig. 3-3- title

This graph shows that the result of the fluorescence intensity 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 C4HSL, the fluorescence intensity 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 3OC12HSL, the fluorescence intensity 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 C4HSL but degrades 3OC12HSL.

4. Discussion

AmiE is the protein that degrades AHLs which have acyl chains longer than eight carbons. This experiment shows that C4HSL is not degraded, and 3OC12HSL is degraded. This result is consistent with the AmiE function written in a paper. We thought that we can express AmiE in the Prince E. coli and make it degrade 3OC12HSL.
From the results, it is expected that the Prince E. coli degrades the poisoned apple and make Snow White E. coli recover from its apparent death.

5. Materials and Methods

5-1. Construction

-Strain All the sample were XL1-Blue strain.
-Plasmids
A， Para-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 2997 µL LB culture containing 3 µL ampicillin, and incubate at 37°C, 180 rpm for 12 h, so that the final concentration of glucose becomes 0.2%.

2. Inoculate B and C into 2997 µL LB culture containing 3 µL ampicillin, and incubate at 37°C, 180 rpm for 12 h, so that the final concentration of glucose becomes 0.2%.

3. Dilute the overnight culture of A so that the OD600 becomes about 0.05, and begin fresh culture at 37°C, 180 rpm.

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.

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.

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.

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.)

8. Prepare the overnight cultures containing LB medium and 1000 µL ampicillin in a 1.5 mL tube for control.

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.

10. Measure the fluorescence intensity and the optical density after incubation at 37°C, 180 rpm for 4 h.

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.