Difference between revisions of "Team:Tokyo Tech/AHL Assay/Only Assay"
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| − | <p class="normal_text"> This section is | + | <p class="normal_text"> This section is an extension of the previous section (3-2-1 AHL reporter assay), and the point of Quorum Sensing was further evaluated. Specifically, production of AHLs and AHL-mediated communication between the Snow White <span style="font-style : italic">coli</span> and Queen <span style="font-style : italic">coli</span> were analyzed. |
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| − | <p class="normal_text"> The reporter (Queen-like <i>coli</i> and Snow White-like <i>coli</i>) and sender (Queen-like <i>coli</i>) <i>E. coli</i> cells were prepared which carried <i>rhlR</i> and <i>luxR</i> genes, respectively. Then,(a) C4 was added into Queen-like <span style="font-style : italic">coli</span>, and (b) C4 was added into a co-culture of Queen-like <span style="font-style : italic">coli</span> and Snow White-like <span style="font-style : italic">coli</span>. The Relative Fluorescent Unit (RFU) of GFP | + | <p class="normal_text"> The reporter (Queen-like <i>coli</i> and Snow White-like <i>coli</i>) and sender (Queen-like <i>coli</i>) <i>E. coli</i> cells were prepared which carried <i>rhlR</i> and <i>luxR</i> genes, respectively. Then,(a) C4 was added into Queen-like <span style="font-style : italic">coli</span>, and (b) C4 was added into a co-culture of Queen-like <span style="font-style : italic">coli</span> and Snow White-like <span style="font-style : italic">coli</span>. The Relative Fluorescent Unit (RFU) of GFP was measured. The results indicated that Prhl(<a href="http://parts.igem.org/Part:BBa_I14017">BBa_I14017)</a> activity was so low that our final genetic circuits would not work with a wild type Prhl.(<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071)</a> |
</p> | </p> | ||
<br> | <br> | ||
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<p class="normal_text"> -Plasmids | <p class="normal_text"> -Plasmids | ||
| − | <p class="normal_text">A. Pcon-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">rhlR</span>-<span style ="font-style : italic">LVA</span>(<a href="http://parts.igem.org/Part:BBa_C0071">BBa_C0071)</a>(pSB6A1), Prhl(<a href="http://parts.igem.org/Part:BBa_I14017">BBa_I14017)</a>-<i>rbs</i>-<span style ="font-style : italic">lasI</span>(<a href="http://parts.igem.org/Part:BBa_C0078">BBa_C0078)</a>-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">gfp</span>-<span style ="font-style : italic">LVA</span> (pSB3K3) (Fig. 3-2-2- | + | <p class="normal_text">A. Pcon-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">rhlR</span>-<span style ="font-style : italic">LVA</span>(<a href="http://parts.igem.org/Part:BBa_C0071">BBa_C0071)</a>(pSB6A1), Prhl(<a href="http://parts.igem.org/Part:BBa_I14017">BBa_I14017)</a>-<i>rbs</i>-<span style ="font-style : italic">lasI</span>(<a href="http://parts.igem.org/Part:BBa_C0078">BBa_C0078)</a>-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">gfp</span>-<span style ="font-style : italic">LVA</span> (pSB3K3) (Fig. 3-2-2-2-1) |
</p><br> | </p><br> | ||
<div align="center"><img src="https://static.igem.org/mediawiki/2016/6/6d/Fig.3-2-2-5-1.png"" height ="150"><br></div> | <div align="center"><img src="https://static.igem.org/mediawiki/2016/6/6d/Fig.3-2-2-5-1.png"" height ="150"><br></div> | ||
| − | <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-2- | + | <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-2-2-1 </span> Pcon-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">rhlR</span>-<span style ="font-style : italic">LVA</span>(pSB6A1), Prhl-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">lasI</span>-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">gfp</span>-<span style ="font-style : italic">ssrA</span> (pSB3K3) |
| − | </p></div> | + | </p></div><br> |
| − | <p class="normal_text">B. Pcon-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">luxR</span>(<a href="http://parts.igem.org/Part:BBa_C0062">BBa_C0062)</a>(pSB6A1), Plux(<a href="http://parts.igem.org/Part:BBa_R0062">BBa_R0062)</a>-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">rhlI</span>(<a href="http://parts.igem.org/Part:BBa_I19026">BBa_I19026)</a>-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">rfp</span>-<span style ="font-style : italic">ssrA</span> (pSB3K3) (Fig. 3-2-2- | + | <p class="normal_text">B. Pcon-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">luxR</span>(<a href="http://parts.igem.org/Part:BBa_C0062">BBa_C0062)</a>(pSB6A1), Plux(<a href="http://parts.igem.org/Part:BBa_R0062">BBa_R0062)</a>-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">rhlI</span>(<a href="http://parts.igem.org/Part:BBa_I19026">BBa_I19026)</a>-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">rfp</span>-<span style ="font-style : italic">ssrA</span> (pSB3K3) (Fig. 3-2-2-2-2)</p><br> |
<div align="center"><img src="https://static.igem.org/mediawiki/2016/1/1f/Fig.3-2-2-5-2.png"" height ="150"><br></div> | <div align="center"><img src="https://static.igem.org/mediawiki/2016/1/1f/Fig.3-2-2-5-2.png"" height ="150"><br></div> | ||
| − | <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-2- | + | <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-2-2-2 </span> Pcon-<span style ="font-style : italic">rbs</span>-<span style="font-style : italic">luxI</span>(pSB6A1), Plux-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">rhlI</span>-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">rfp</span>-<span style ="font-style : italic">ssrA</span> (pSB3K3) |
| − | </p></div> | + | </p></div><br> |
| − | <p class="normal_text">C. pSB6A1, pSB3K3…Negative (Fig. 3-2-2- | + | <p class="normal_text">C. pSB6A1, pSB3K3…Negative (Fig. 3-2-2-2-3)</p><br> |
<div align="center"><img src="https://static.igem.org/mediawiki/2016/9/92/Fig._3-2-2-5-3.png" height ="150"><br></div> | <div align="center"><img src="https://static.igem.org/mediawiki/2016/9/92/Fig._3-2-2-5-3.png" height ="150"><br></div> | ||
| − | <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-2- | + | <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-2-2-3 </span> pSB6A1, pSB3K3</p></div> |
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<div id="results_contents" class="container_contents"> | <div id="results_contents" class="container_contents"> | ||
| − | <p class="normal_text"> RFU of GFP / Turbidity was almost the same | + | <p class="normal_text"> RFU of GFP / Turbidity was almost the same though adding C4 or DMSO (negative control; note that C4 was dissolved with DMSO) into Queen-like <span style="font-style : italic">coli</span> or the co-culture (Fig.3-2-2-3). The leak of Prhl(<a href="http://parts.igem.org/Part:BBa_I14017">BBa_I14017)</a> was high and the cause is explained Rhl system assay page.<!-- リンクを貼る --></p><br> |
<div align="center"><img src="URL"><br></div> | <div align="center"><img src="URL"><br></div> | ||
<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-2-3 </span> RFU of GFP / Turbidity of Only Assay | <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-2-3 </span> RFU of GFP / Turbidity of Only Assay | ||
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<div id="construction_contents"> | <div id="construction_contents"> | ||
| − | <p class="normal_text">-Strain | + | <p class="normal_text">-Strain<br> |
All the plasmids were prepared in XL1-Blue strain.<br> | All the plasmids were prepared in XL1-Blue strain.<br> | ||
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</div><!-- /_header --> | </div><!-- /_header --> | ||
<div id="methods_contents"> | <div id="methods_contents"> | ||
| − | <p class="normal_text"> The following experiments | + | <p class="normal_text"> The following experiments are performed at 37℃ unless otherwise stated.<br><br> |
1) Prepare the overnight cultures in LB medium AK with vigorous shaking.<br><br> | 1) Prepare the overnight cultures in LB medium AK with vigorous shaking.<br><br> | ||
2) Dilute the overnight cultures to 1 / 60 in fresh LB medium AK with vigorous shaking for 1 h.<br><br> | 2) Dilute the overnight cultures to 1 / 60 in fresh LB medium AK with vigorous shaking for 1 h.<br><br> | ||
| − | 3) Add C4 (100 microM) | + | 3) Add C4 (100 microM) to Queen-like <span style="font-style : italic">coli</span> and C12 into Snow White-like <span style="font-style : italic">coli</span>, and incubate them with vigorous shaking for 4 h.<br><br> |
4) Measure RFU of GFP at 490nm as an exciting wavelength, 525nm as a measurement wavelength. | 4) Measure RFU of GFP at 490nm as an exciting wavelength, 525nm as a measurement wavelength. | ||
Revision as of 17:37, 18 October 2016
3-2-2 AHL Only of Final genetic circuits Assay
Contents
1. Introduction
This section is an extension of the previous section (3-2-1 AHL reporter assay), and the point of Quorum Sensing was further evaluated. Specifically, production of AHLs and AHL-mediated communication between the Snow White coli and Queen coli were analyzed.
Fig. 3-2-2-1 Our final genetic circuits
2. Summary of the Experiment
The reporter (Queen-like coli and Snow White-like coli) and sender (Queen-like coli) E. coli cells were prepared which carried rhlR and luxR genes, respectively. Then,(a) C4 was added into Queen-like coli, and (b) C4 was added into a co-culture of Queen-like coli and Snow White-like coli. The Relative Fluorescent Unit (RFU) of GFP was measured. The results indicated that Prhl(BBa_I14017) activity was so low that our final genetic circuits would not work with a wild type Prhl.(BBa_R0071)
-Plasmids
A. Pcon-rbs-rhlR-LVA(BBa_C0071)(pSB6A1), Prhl(BBa_I14017)-rbs-lasI(BBa_C0078)-rbs-gfp-LVA (pSB3K3) (Fig. 3-2-2-2-1)
Fig. 3-2-2-2-1 Pcon-rbs-rhlR-LVA(pSB6A1), Prhl-rbs-lasI-rbs-gfp-ssrA (pSB3K3)
B. Pcon-rbs-luxR(BBa_C0062)(pSB6A1), Plux(BBa_R0062)-rbs-rhlI(BBa_I19026)-rbs-rfp-ssrA (pSB3K3) (Fig. 3-2-2-2-2)
Fig. 3-2-2-2-2 Pcon-rbs-luxI(pSB6A1), Plux-rbs-rhlI-rbs-rfp-ssrA (pSB3K3)
C. pSB6A1, pSB3K3…Negative (Fig. 3-2-2-2-3)
Fig. 3-2-2-2-3 pSB6A1, pSB3K3
3. Results
RFU of GFP / Turbidity was almost the same though adding C4 or DMSO (negative control; note that C4 was dissolved with DMSO) into Queen-like coli or the co-culture (Fig.3-2-2-3). The leak of Prhl(BBa_I14017) was high and the cause is explained Rhl system assay page.
Fig. 3-2-2-3 RFU of GFP / Turbidity of Only Assay
4. Discussion
The expression level of Prhl(BBa_I14017) was so weak and the leak was so high that Queen-like coli could not influence Snow White-like coli; in other words, our final genetic circuits did not work. Taken together the results, we again recognized that we have to improve Prhl activity to operate our final circuits.
5. Materials and Methods
5-1. Construction
-Strain
All the plasmids were prepared in XL1-Blue strain.
-Medium
LB medium AK:
LB medium containing ampicillin (50 microg/ mL) and kanamycin (50 microg/ mL)
5-2. Assay Protocol
The following experiments are performed at 37℃ unless otherwise stated.
1) Prepare the overnight cultures in LB medium AK with vigorous shaking.
2) Dilute the overnight cultures to 1 / 60 in fresh LB medium AK with vigorous shaking for 1 h.
3) Add C4 (100 microM) to Queen-like coli and C12 into Snow White-like coli, and incubate them with vigorous shaking for 4 h.
4) Measure RFU of GFP at 490nm as an exciting wavelength, 525nm as a measurement wavelength.
6. Reference
(1)
Bo Hu et al. (2010) An Environment-Sensitive Synthetic Microbial Ecosystem. PLoS ONE 5(5): e10619
(2) Chen M. Zhang et al (2014) Distributed implementation of the genetic double-branch structure
in Escherichia coli. Chinese Science Bulletin 59: 4625-4630
