Difference between revisions of "Team:Tokyo Tech/AmiE Assay"
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Revision as of 17:49, 18 October 2016
3-3 AmiE assay
Contents
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. 3-3-3-1 title
Fig. 3-3-3-2 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. 3-3-5-1 title
B. Fresh Culture
8 of 1.5 mL tubes
Fig. 3-3-5-2 title
control
Fig. 3-3-5-3 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 microL supernatants of A into (ⅰ), (ⅱ), (ⅲ) and (ⅳ), and add 4 microL 3OC12HSL into tube e, 13.3 microL C4HSL into tube f, 4 microL DMSO into tube g, 13.3 microL 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.
