Difference between revisions of "Team:Tokyo Tech/AmiE Assay"
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<h3 class="link"><a href="#introduction">1. Introduction</a></h3> | <h3 class="link"><a href="#introduction">1. Introduction</a></h3> | ||
| − | <h3 class="link"><a href="#summary">2. Summary of the | + | <h3 class="link"><a href="#summary">2. Summary of the experiment</a></h3> |
<h3 class="link"><a href="#results">3. Results</a></h3> | <h3 class="link"><a href="#results">3. Results</a></h3> | ||
<h3 class="link"><a href="#discussion">4. Discussion</a></h3> | <h3 class="link"><a href="#discussion">4. Discussion</a></h3> | ||
| − | <h3 class="link"><a href="#methods">5. Materials and | + | <h3 class="link"><a href="#methods">5. Materials and methods</a></h3> |
<h3 class="link"><a href="#construction"><font size="2.7"> 5-1. Construction</font></a></h3> | <h3 class="link"><a href="#construction"><font size="2.7"> 5-1. Construction</font></a></h3> | ||
| − | <h3 class="link"><a href="#protocol"><font size="2.7"> 5-2. Assay | + | <h3 class="link"><a href="#protocol"><font size="2.7"> 5-2. Assay protocol</font></a></h3> |
<h3 class="link"><a href="#reference">6. Reference</a></h3> | <h3 class="link"><a href="#reference">6. Reference</a></h3> | ||
</div><!-- /contents_menu --> | </div><!-- /contents_menu --> | ||
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</div><!-- /_header --> | </div><!-- /_header --> | ||
<div id="introduction_contents" class="container_contents"> | <div id="introduction_contents" class="container_contents"> | ||
| − | <p class="normal_text">The Prince <span style="font-style : italic">coli</span> expresses AmiE protein, and Snow White <span style="font-style : italic">coli</span> recovers from its apparent death and | + | <p class="normal_text">The Prince <span style="font-style : italic">coli</span> expresses AmiE protein, and Snow White <span style="font-style : italic">coli</span> recovers from its apparent death and opens her eyes by the action of AmiE.</p> |
<p class="normal_text">We tested the function of AmiE protein that influences the end of the story. | <p class="normal_text">We tested the function of AmiE protein that influences the end of the story. | ||
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<div id="summary" class="container"> | <div id="summary" class="container"> | ||
<div id="id_header" class="container_header"> | <div id="id_header" class="container_header"> | ||
| − | <h2><span>2. Summary of the | + | <h2><span>2. Summary of the experiment</span></h2> |
</div><!-- /_header --> | </div><!-- /_header --> | ||
<div id="summary_contents" class="container_contents"> | <div id="summary_contents" class="container_contents"> | ||
| − | <p class="normal_text">The objective of this experiment is to characterize | + | <p class="normal_text">The objective of this experiment is to characterize AmiE protein. We prepared three samples shown below to see the degradation ability of AmiE,and the results showed that C4 was not degraded by AmiE, but C12 was degraded by AmiE. The transfomants used in this study are indicated below(Figs.3-3-1-2-1– 3-3-1-2-3):</p><br><br> |
| − | <p class="normal_text">A. PBAD/araC | + | <p class="normal_text">A. PBAD/araC ‐ <span style="font-style : italic">rbs ‐ amiE</span> (<a href="http://parts.igem.org/Part:BBa_K1949052">K1949052</a>) (pSB6A1)</p><br> |
<div align="center"><img src="https://static.igem.org/mediawiki/2016/b/b0/Fig.3-3-1-2-1.png" height="150"> <br></div> | <div align="center"><img src="https://static.igem.org/mediawiki/2016/b/b0/Fig.3-3-1-2-1.png" height="150"> <br></div> | ||
| − | <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-3-1-2-1 </span>PBAD | + | <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-3-1-2-1 </span>PBAD ‐ <i>rbs ‐ amiE</i> (pSB6A1) |
</p></div> | </p></div> | ||
| − | <p class="normal_text">B. Ptet | + | <p class="normal_text">B. Ptet ‐ <span style="font-style : italic">rbs ‐ luxR</span> ‐ <i>tt</i> ‐ Plux ‐ <span style="font-style : italic">rbs ‐ gfp</span> (pSB6A1)</p><br> |
<div align="center"><img src="https://static.igem.org/mediawiki/2016/b/b3/Fig.3-3-1-2-2.png" height="150"><br></div> | <div align="center"><img src="https://static.igem.org/mediawiki/2016/b/b3/Fig.3-3-1-2-2.png" height="150"><br></div> | ||
| − | <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-3-1-2-2 </span>Pcon | + | <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-3-1-2-2 </span>Pcon ‐ <i>rbs ‐ rhlR ‐ tt</i> ‐ Prhl ‐ <i>rbs ‐ gfp</i> (pSB6A1) |
</p></div> | </p></div> | ||
| − | <p class="normal_text">C. Ptet - <span style="font-style : italic">rbs | + | <p class="normal_text">C. Ptet - <span style="font-style : italic">rbs ‐ luxR</span> ‐ <i>tt</i> ‐ Plux ‐ <span style="font-style : italic">rbs ‐ gfp</span> (pSB6A1) |
<div align="center"><img src="https://static.igem.org/mediawiki/2016/a/a8/Fig.3-3-1-2-3.png" height="150"><br></div> | <div align="center"><img src="https://static.igem.org/mediawiki/2016/a/a8/Fig.3-3-1-2-3.png" height="150"><br></div> | ||
| − | <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-3-1-2-3 </span>Pcon | + | <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-3-1-2-3 </span>Pcon ‐ <i>rbs ‐ rhlR ‐ tt</i> ‐ Prhl ‐ <i>rbs ‐ gfp</i> (pSB6A1) |
</p></div> | </p></div> | ||
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</div><!-- /_header --> | </div><!-- /_header --> | ||
<div id="results_contents" class="container_contents"> | <div id="results_contents" class="container_contents"> | ||
| − | <p class="normal_text"> | + | <p class="normal_text">The AmiE degradation activity against C4 or C12 is shown below. </p><br> |
<div align="center"><img src="https://static.igem.org/mediawiki/2016/9/96/Fig.3-3-1-3-2.png"height="300"><br></div> | <div align="center"><img src="https://static.igem.org/mediawiki/2016/9/96/Fig.3-3-1-3-2.png"height="300"><br></div> | ||
<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-3-1-3-1 </span>AmiE degrades C12 | <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-3-1-3-1 </span>AmiE degrades C12 | ||
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<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-3-1-3-2 </span>AmiE barely degrades C4 | <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-3-1-3-2 </span>AmiE barely degrades C4 | ||
</p></div> | </p></div> | ||
| − | <p class="normal_text"> | + | <p class="normal_text">The cultures of the AHL degrader (Fig. 3-3-1-2-1) were prepared in the presence or absence of arabinose. Then, AHLs (C4 or C12) were added to the cultures and incubated further to gain enough time to degrade AHLs. The supernatant of the cultures were collected and added to the cultures of corresponding reporter cells (Figs. 3-3-1-2-2 and 3-3-1-2-3).The RFU of GFP / turbidity of each supernatant was measured. The results are shown in the graph above.</p> |
| − | <p class="normal_text">First | + | <p class="normal_text">First, let us look at the results of C4 degradation by AmiE; the RFU of GFP / turbidity was almost the same in all the samples, indicating no C4 degradation.</p> |
| − | <p class="normal_text">However, in the case of the degradation of C12, the RFU of GFP / | + | <p class="normal_text">However, in the case of the degradation of C12, the RFU of GFP / turbidity of the reporter decreased to approximately 1/10 when C12 was pre-treated with AmiE-expressing cells. Based on the results above, we concluded that AmiE does not degrade C4 but degrades C12.</p> |
</div><!-- /results_contents --> | </div><!-- /results_contents --> | ||
</div><!-- /results --> | </div><!-- /results --> | ||
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</div><!-- /_header --> | </div><!-- /_header --> | ||
<div id="discussion_contents" class="container_contents"> | <div id="discussion_contents" class="container_contents"> | ||
| − | <p class="normal_text">AmiE is the protein that degrades AHLs that have acyl chains longer than six carbons. This experiment showed that C4 was not degraded and C12 was degraded | + | <p class="normal_text">AmiE is the protein that degrades AHLs that have acyl chains longer than six carbons. This experiment showed that C4 was not degraded and C12 was degraded, showing good agreement with the AmiE function written in the literature. </p> |
| − | <p class="normal_text">We | + | <p class="normal_text">We conclude that AmiE could be expressed in the Prince coli and degradation of C12 was cased as we desired. Also, it is expected that the Prince coli degrades the poisoned apple and makes Snow White <span style="font-style : italic">coli</span> recover from its apparent death. |
</p> | </p> | ||
</div><!-- /discussion_contents --> | </div><!-- /discussion_contents --> | ||
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<div id="methods" class="container"> | <div id="methods" class="container"> | ||
<div id="methods_header" class="container_header"> | <div id="methods_header" class="container_header"> | ||
| − | <h2><span>5. Materials and | + | <h2><span>5. Materials and methods</span></h2> |
</div><!-- /_header --> | </div><!-- /_header --> | ||
<div id="methods_contents" class="container_contents"> | <div id="methods_contents" class="container_contents"> | ||
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All the samples were XL1‐Blue strain.<br><br> | All the samples were XL1‐Blue strain.<br><br> | ||
<p class="normal_text">-Plasmids<br> | <p class="normal_text">-Plasmids<br> | ||
| − | A， PBAD/araC | + | A， PBAD/araC ‐ <span style="font-style : italic">rbs</span> ‐ <span style="font-style : italic">amiE</span> (pSB6A1)<br> |
| − | B， Ptet | + | B， Ptet ‐ <span style="font-style : italic">rbs ‐ luxR</span> ‐ <i>tt</i> ‐ Plux ‐ <span style="font-style : italic">rbs ‐ gfp</span> (pSB6A1)<br> |
| − | C， Ptet | + | C， Ptet ‐ <span style="font-style : italic">rbs ‐ rhlR</span> ‐ <i>tt</i> ‐ Prhl ‐ <span style="font-style : italic">rbs ‐ gfp</span> (pSB6A1) |
</p><br> | </p><br> | ||
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<div id="protocol"> | <div id="protocol"> | ||
<div id="protocol_header"> | <div id="protocol_header"> | ||
| − | <h3><span>5-2. Assay | + | <h3><span>5-2. Assay protocol</span></h3> |
</div><!-- /_header --> | </div><!-- /_header --> | ||
<div id="methods_contents"> | <div id="methods_contents"> | ||
| − | <p class="normal_text">1) Fresh | + | <p class="normal_text">1) Fresh culture<br> |
4 test tubes</p><br> | 4 test tubes</p><br> | ||
<div align="center"><img src="https://static.igem.org/mediawiki/2016/f/f5/Fig.3-3-1-5-1.jpg" height="300"><br></div> | <div align="center"><img src="https://static.igem.org/mediawiki/2016/f/f5/Fig.3-3-1-5-1.jpg" height="300"><br></div> | ||
<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-3-1-5-1 </span> | <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-3-1-5-1 </span> | ||
</p></div> | </p></div> | ||
| − | <p class="normal_text">2) Fresh | + | <p class="normal_text">2) Fresh culture<br> |
8 of 1.5 mL tubes</p><br> | 8 of 1.5 mL tubes</p><br> | ||
<div align="center"><img src="https://static.igem.org/mediawiki/2016/9/92/Fig.3-3-1-5-2.jpg" height="200"><br></div> | <div align="center"><img src="https://static.igem.org/mediawiki/2016/9/92/Fig.3-3-1-5-2.jpg" height="200"><br></div> | ||
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<p class="normal_text"><br> | <p class="normal_text"><br> | ||
<!--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--> | <!--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--> | ||
| − | 1. Inoculate A in 3 mL LB medium containing ampicillin (50 microg / mL), and add glucose so that the final concentration becomes 0.2%.<br>Inoculate B and C | + | 1. Inoculate A in 3 mL LB medium containing ampicillin (50 microg / mL), and add glucose so that the final concentration becomes 0.2%.<br>Inoculate B and C in 3 mL LB culture containing ampicillin (50 microg / mL).<br><br> |
2. Incubate all the samples at 37°C, 180 rpm for 12 h.<br><br> | 2. Incubate all the samples at 37°C, 180 rpm for 12 h.<br><br> | ||
3. Dilute the overnight culture of A so that the turbidity becomes about 0.05.<br><br> | 3. Dilute the overnight culture of A so that the turbidity becomes about 0.05.<br><br> | ||
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6. 2 h after adding 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).<br><br> | 6. 2 h after adding 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).<br><br> | ||
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> | 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> | ||
| − | 8. Transfer supernatant to | + | 8. Transfer supernatant to another 1.5 mL tube.<br><br> |
9. Add each 10 microL overnight culture for B and C into 800 microL LB medium containing ampicillin (50 microg / mL). Also, add each 10 microL overnight culture for B and C into 1 mL LB medium containing ampicillin (50 microg / mL) for controls. (B: a, c, e, g) (C: b, d, f, h)<br><br> | 9. Add each 10 microL overnight culture for B and C into 800 microL LB medium containing ampicillin (50 microg / mL). Also, add each 10 microL overnight culture for B and C into 1 mL LB medium containing ampicillin (50 microg / mL) for controls. (B: a, c, e, g) (C: b, d, f, h)<br><br> | ||
10. After 1.5 h, add 200 microL supernatants of A into (ⅰ), (ⅱ), (ⅲ) and (ⅳ), and add 4 of microL 3OC12HSL into tube (e), 13.3 of microL C4HSL into tube (f), 4 of microL DMSO into tube (g), 13.3 microL of DMSO into tube (h) for controls.<br><br> | 10. After 1.5 h, add 200 microL supernatants of A into (ⅰ), (ⅱ), (ⅲ) and (ⅳ), and add 4 of microL 3OC12HSL into tube (e), 13.3 of microL C4HSL into tube (f), 4 of microL DMSO into tube (g), 13.3 microL of DMSO into tube (h) for controls.<br><br> | ||
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</div><!-- /reference --> | </div><!-- /reference --> | ||
| + | |||
| + | <div id="next_page" class="container"> | ||
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| + | <p class="normal_text">Jump to <a href="https://2016.igem.org/Team:Tokyo_Tech/Promoter_Assay/Pcold">Pcold assay</a> page.</p> | ||
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Latest revision as of 12:18, 6 November 2016
3-3 AmiE assay
Contents
1. Introduction
The Prince coli expresses AmiE protein, and Snow White coli recovers from its apparent death and opens her eyes by the action of AmiE.
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 AmiE protein. We prepared three samples shown below to see the degradation ability of AmiE,and the results showed that C4 was not degraded by AmiE, but C12 was degraded by AmiE. The transfomants used in this study are indicated below(Figs.3-3-1-2-1– 3-3-1-2-3):
A. PBAD/araC ‐ rbs ‐ amiE (K1949052) (pSB6A1)
Fig. 3-3-1-2-1 PBAD ‐ rbs ‐ amiE (pSB6A1)
B. Ptet ‐ rbs ‐ luxR ‐ tt ‐ Plux ‐ rbs ‐ gfp (pSB6A1)
Fig. 3-3-1-2-2 Pcon ‐ rbs ‐ rhlR ‐ tt ‐ Prhl ‐ rbs ‐ gfp (pSB6A1)
C. Ptet - rbs ‐ luxR ‐ tt ‐ Plux ‐ rbs ‐ gfp (pSB6A1)
Fig. 3-3-1-2-3 Pcon ‐ rbs ‐ rhlR ‐ tt ‐ Prhl ‐ rbs ‐ gfp (pSB6A1)
3. Results
The AmiE degradation activity against C4 or C12 is shown below.
Fig. 3-3-1-3-1 AmiE degrades C12
Fig. 3-3-1-3-2 AmiE barely degrades C4
The cultures of the AHL degrader (Fig. 3-3-1-2-1) were prepared in the presence or absence of arabinose. Then, AHLs (C4 or C12) were added to the cultures and incubated further to gain enough time to degrade AHLs. The supernatant of the cultures were collected and added to the cultures of corresponding reporter cells (Figs. 3-3-1-2-2 and 3-3-1-2-3).The RFU of GFP / turbidity of each supernatant was measured. The results are shown in the graph above.
First, let us look at the results of C4 degradation by AmiE; the RFU of GFP / turbidity was almost the same in all the samples, indicating no C4 degradation.
However, in the case of the degradation of C12, the RFU of GFP / turbidity of the reporter decreased to approximately 1/10 when C12 was pre-treated with AmiE-expressing cells. Based on the results above, we concluded that AmiE does not degrade C4 but degrades C12.
4. Discussion
AmiE is the protein that degrades AHLs that have acyl chains longer than six carbons. This experiment showed that C4 was not degraded and C12 was degraded, showing good agreement with the AmiE function written in the literature.
We conclude that AmiE could be expressed in the Prince coli and degradation of C12 was cased 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 samples 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
1) Fresh culture
4 test tubes
Fig. 3-3-1-5-1
2) Fresh culture
8 of 1.5 mL tubes
Fig. 3-3-1-5-2
control
Fig. 3-3-1-5-3
1. Inoculate A in 3 mL LB medium containing ampicillin (50 microg / mL), and add glucose so that the final concentration becomes 0.2%.
Inoculate B and C in 3 mL LB culture containing ampicillin (50 microg / mL).
2. Incubate all the 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 0.16 to 0.18.
5. Add arabinose into test tube (ⅰ) and (ⅱ) so that the final concentration becomes 0.2%.
6. 2 h after adding 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 another 1.5 mL tube.
9. Add each 10 microL overnight culture for B and C into 800 microL LB medium containing ampicillin (50 microg / mL). Also, add each 10 microL overnight culture for B and C into 1 mL LB medium containing ampicillin (50 microg / mL) for controls. (B: a, c, e, g) (C: b, d, f, h)
10. After 1.5 h, add 200 microL supernatants of A into (ⅰ), (ⅱ), (ⅲ) and (ⅳ), and add 4 of microL 3OC12HSL into tube (e), 13.3 of microL C4HSL into tube (f), 4 of microL DMSO into tube (g), 13.3 microL of DMSO into tube (h) for controls.
11. Incubate at 37°C, 180 rpm for 4 h.
12. Measure RFU of GFP and the Turbidity.
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.
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