Team:Tokyo Tech/Projects/making-rain/GC-Assay

From 2011.igem.org

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<ol style="list-style-type: decimal;">
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<ul>
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<li><a href="#Const">Construction</a></li>
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<li><a href="#const">1. Construction</a></li>
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<li><a href="#AP">Assay Preparation</a></li>
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<li><a href="#AP">2. Assay Preparation</a></li>
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<li><a href="#Em-isp">Isoprene in Solvent</a></li>
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<li><a href="#Em-isp">3. Assay Method by <i>E. coli</i></a></li>
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<li><a href="#Assay">Assay Method by <span class="">E. coli</span></a></li>
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<li><a href="#aerosol">4. Aerosol formation</a></li>
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                <li><a href="#results">Results</a></li>
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<h1> Rain details  </h1>
<h1> Rain details  </h1>
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<h2 id="const"> 1.Construction </h2>
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<h2 id="const"> 1. Construction </h2>
          
          
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<p>We obtaind the gene <span class="gene">ispS</span> on pMK from Gene Arts. </p><br />
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<p>
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We obtained the gene <span class="gene">ispS</span> (on pMK) from Gene Arts.  
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</p>
<div align="center">
<div align="center">
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<img src="https://static.igem.org/mediawiki/2011/6/60/Rain_constcution_third_layer.png" alt="construction" />
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<img src="https://static.igem.org/mediawiki/2011/f/f1/IspS1.png" alt="construction" width="600"/>
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        <center>Fig. 1 construction for <span class="gene">ispS</span> parts</center>
<p>
<p>
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It was difficult to excise <span class="gene">ispS</span> gene from pMK by cutting between EcoRI and PstI site, EcoRI and SpeI site, or XbaI and PstI site, because the length of pMK from which <span class="gene">ispS</span> was excised were as long as the length of <span class="gene">ispS</span>. So, we cut the pMK at NcoI site to make different length and ligated into the pSB3K3 including lacIQ promoter. Finally, we cut PlacIQ-<span class="gene">ispS</span> and ligated into pSB1C3 backbone vector.
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It was difficult to excise <span class="gene">ispS</span> gene from pMK by cutting between EcoRI and PstI site, EcoRI and SpeI site, or XbaI and PstI site, because the length of pMK from which <span class="gene">ispS</span> was excised were as long as the length of <span class="gene">ispS</span>. So, we cut the pMK at NcoI site to make different length and ligated into the pSB3K3 including lacIQ promoter. Finally, we cut out PlacIQ-<span class="gene">ispS</span> and ligated it into pSB1C3 backbone vector.(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K649303">BBa_K649303</a>)
</p><br />
</p><br />
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<h2 id="AP">2.Assay Preparation </h2>
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<h2 id="AP">2. Assay Preparation </h2>
<p>
<p>
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In order to assay the amount of isoprene produced by our <span class="name">E. coli</span>, we use the Gas chromatography-mass spectrometry (GC-MS, QP-2010, SHIMADZU, Japan). The MS takes an electron ionization method and quadrupole. Analytes were separated by a nonpolar column (Rtx-1MS: Length 30 m, ID 0.25 mm film thickness 0.5 &micro;m, USA) working in a constant flow mode (2.99 mL min<sup>-1</sup>). The temperature program was chosen as follows: 40℃ for 7 min, increase to 280℃ at rate of 10℃ min<sup>-1</sup>, 280℃ for 5 min. The mass spectrometer worked in SIM mode, m/z 67. The retention time of isoprene is very short (about 1.06-1.10 min). But thanks to MS, isoprene could be identified.
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To measure the amount of isoprene produced by our <span class="name">E. coli</span>, we used electron-ionization Gas Chromatography-Mass Spectrometry equipment (GC-MS, QP-2010, SHIMADZU, Japan). Analytes were separated by a nonpolar column (Rtx-1MS: Length 30 m, ID 0.25 mm film thickness 0.5 &micro;m, USA) working in a constant flow mode (2.99 mL min<sup>-1</sup>). The temperature program was chosen as follows: 40&deg;C for 7 min, increase to 280&deg;C at rate of 10&deg;C min<sup>-1</sup>, 280&deg;C for 5 min. The mass spectrometer worked in SIM mode, m/z 67.
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</p><br />
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<table style="float:right;">
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        <center><img src="https://static.igem.org/mediawiki/2011/3/3d/111028GCMS.JPG" alt="assay"width="300" height="200" /></center><br />
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<tr>
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<center>Fig. 2 GC-MS</center>
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<td><img src="https://static.igem.org/mediawiki/2011/6/6b/Rain_assay_Fig_1.png" alt="assay" /></td>
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        </p>
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<tr>
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<td style="text-align:center;">Fig.1  Dilution series of liquid isoprene diluted in chloroform were injected into GC-MS.<br />
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        Let area be the vertical axis, and amount of isoprene itself ([mg]) the horizontal axis.
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<p>
<p>
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We firstly made dilution series of liquid isoprene (Wako Pure Chemical Industries, Ltd, Japan) diluted in chloroform(diluted 10<sup>2</sup>,10<sup>3</sup>,10<sup>4</sup>,10<sup>5</sup>,10<sup>6</sup>,10<sup>7</sup> times). The undiluted isoprene solution 1 &micro;L is 0.654 mg. We injected diluted isoprene into GC-MS, and draw a calibration curve (Fig.1).
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We made dilution series of liquid isoprene (Wako Pure Chemical Industries, Ltd, Japan) diluted in chloroform(diluted 10<sup>2</sup>,10<sup>3</sup>,10<sup>4</sup>,10<sup>5</sup>,10<sup>6</sup>,10<sup>7</sup>-fold). The undiluted isoprene solution 1 &micro;L is 0.654 mg. We injected diluted isoprene into GC-MS, and draw a calibration curve (Fig. 3). If X (x=logX) represents the area of isoprene's peak and Y (y=logY) represents the amount of isoprene [mg], the calibration curve is described by the equation &ldquo;Y = 10<sup>-7.9</sup> &times; X<sup>0.89</sup>&rdquo;.
</p>
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<p style="clear:both">
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Since we obtained data with high variability, the calibration curve could not be drawn precisely. We gave up the idea of quantitative analysis and decided to analyze at the level of an order of magnitude.
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</p>
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        <p>       
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        <center><img src="https://static.igem.org/mediawiki/2011/d/de/Isp-kenryou.jpg" alt="assay" /></center><br />
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<h2 id="Em-isp">3.Isoprene in Solvent</h2>
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<center>Fig. 3 calibration curve</center>
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        </p>
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<h2 id="Em-isp">3. Assay Method by <span class="name">E. coli</span></h2>
   
   
<p>
<p>
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We then experimented in various conditions to make sure that isoprene can emit from water and LB medium. Headspace gas was sampled though an adsorbing material (mini-PAT including Tenax: Japan Analytical Industry Co., Ltd) and injected into GC-MS.     
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        <div align="center">
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</p>
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<img src="https://static.igem.org/mediawiki/2011/7/74/Isoprene_sample2.png" width="250px" />
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</div><br />
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<table border="1" style="text-align:center;">
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        <center>
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<caption>
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                Fig. 4 constraction for assay
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</caption>
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        </center>
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<tr class="topline">
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<th>number</th>
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<th>container</th>
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<th>solvent</th>
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<th>isoprene[mg]</th>
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<th>sampling[mL]</th>
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<th>Condition from dripping isoprene　to sampling</th>
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</tr>
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<tr>
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<th>1</th>
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<td>15 mL centrifuge tube</td>
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<td>None</td>
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<td>6.54</td>
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<td>15</td>
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<td>room temperature, 20 minutes</td>
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</tr>
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<tr>
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<th>2</th>
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<td>500 mL flask</td>
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<td>Water 100 mL</td>
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<td>13.1</td>
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<td>50</td>
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<td>room temperature, 20 minutes</td>
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</tr>
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<tr>
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<th>3</th>
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<td>500 mL flask</td>
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<td>Water 100 mL</td>
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<td>13.1</td>
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<td>50</td>
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<td>37℃, 20 minutes</td>
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</tr>
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<tr>
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<th>4</th>
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<td>500 mL flask</td>
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<td>LB medium 100 mL</td>
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<td>13.1</td>
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<td>50</td>
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<td>37℃, 20 minutes</td>
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</tr>
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<tr>
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<th>5</th>
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<td>500 mL flask</td>
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<td>LB medium 100 mL</td>
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<td>0</td>
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<td>50</td>
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<td>37℃, culture, 6 hours</td>
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</tr>
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<tr>
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<th>6</th>
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<td>500 mL flask</td>
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<td>LB medium 100 mL</td>
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<td>0</td>
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<td>50</td>
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<td>37℃, 6 hours + <span class="name">E.coli</span>(BL-21)</td>
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</tr>
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</table>
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<p>
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Table.1 Different conditions that used in samples` headspace gas of solvent including isoprene or LB media with <span class="name">E. coli</span> or none. The condition is from dripping to sampling.  
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</p>
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<table style="float:left;">
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        </p>
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<tr>
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        <p>
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<td>
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Each bacterial sample was grown in a 500 mL flask containing 100 mL LB media. Cultures were grown at 37&deg;C and then induced by 0.5 mM IPTG when OD<sub>600</sub> reached 0.6. After 4 hours of induction, 50 mL of headspace gas was taken by absorbing material (mini-PAT including Tenax: Japan Analytical Industry Co., Ltd) and injected into GC-MS.
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<img src="https://static.igem.org/mediawiki/2011/9/98/Peak_Area_11titech.jpg" alt="assay" width="436px" />
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</td>
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</tr>
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</table>
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<p>
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As results, the peak area was about one thousandth of expectable area by calibration data at experiment No.1-4, drip isoprene. The peak area’s retention time was same as that of isoprene at experiment No.5 (no isoprene, LB medium only), though the area was much less than those of No.1-4.
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</p>
</p>
<p>
<p>
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We wondered whether the adsorbing material was saturated with isoprene at experiment No.1-4 and isoprene still existed in silicon tube at experiment No.5. So we considered that firstly the tube needed to be used only once and then thrown away. Secondly, Headspace gas of dilution series of liquid isoprene diluted in chloroform in water needed to be sampled.
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                <div align="center">
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</p>
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                        <img src="https://static.igem.org/mediawiki/2011/4/41/111028zairyo_rain.JPG" alt="isoprene_ex" width="320" height="240"/></a>
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                        <img src="https://static.igem.org/mediawiki/2011/2/2f/111028sampling.JPG" alt="isoprene_ex" width="180" height="240" />
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                        <img src="https://static.igem.org/mediawiki/2011/2/24/111028gaschunyu.JPG" alt="isoprene_ex" width="180" height="240" />
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<h2 style="clear:both" id="Assay">4.Assay Method by <span class="">E. coli</span></h2>
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                </div>
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<p>
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                        <center>Fig. 5 Assay Method
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Each <span class="name">E. coli</span> are grown in 500 mL flasks containing 100 mL LB media. Cultures are grown at 37℃ and then induced by 0.5 mM IPTG until OD600 reached 0.6. After 4 hours of induction, 50 mL gas samples from headspace gas are taken by absorbing material (mini-PAT including Tenax: Japan Analytical Industry Co., Ltd) and injected into GC-MS.
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                                <br /> </center>
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</p>
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                </p>      
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<h2 id="results">5.Results</h2>
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<p>
 +
We calculated the amount of isoprene production, using the calibration data we obtained above.
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</p>
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<div align="center">
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<img src="https://static.igem.org/mediawiki/2011/e/e4/Rain-fig4-2.jpg" alt="isprene-graph" width="450px" />
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</div>
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<center>
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Fig. 6 The amount of isoprene detected in <span class="name">E. coli</span> extract.  
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</center>
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<p>We calculated amount of isoprene from area and calibration data (Fig.1).</p>
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<h2 id="aerosol"> 4.Aerosol formation</h2>
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<p>
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<table border="1" style="text-align:center;">
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To do the ozone-isoprene reaction, we used teflon bag as a container for the reaction. Firstly ozone was produce by the ozone generator as the following photo. After measured the concentration of ozone, we used big syringe, injected a certain amount of ozone into the bag. Then using small size of syringe we injected isoprene and water into the bags. We could see that isoprene evaporated immediately. Finally because ultraviolet radiation helps the reaction, we put the teflon bags into clean bench and turned the UV on.
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</p>
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<tr class="topline">
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<div align="center">
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<th>Designation</th>
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<img src="https://static.igem.org/mediawiki/2011/8/82/Ozone.JPG" width="450px" />
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<th>Arear</th>
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</div>
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<th>retention time [min]</th>
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<br/>
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<th>isoprene[ng] (50ml sampling)</th>
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<center>
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</tr>
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Fig. 7 Method of aerosol formation experiment
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<tr>
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</center>
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<td>PlacIQ</td>
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<td>29</td>
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<td>1.170</td>
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<td>0.2</td>
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</tr>
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<tr>
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<td>PlacIQ-isp</td>
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<td>303</td>
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<td>1.118</td>
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<td>2</td>
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</tr>
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</table>
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<h2 id="rain">4.Make it rain!</h2>
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<p>
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To do the ozone-isoprene reaction, we used 10 L teflon bag as a container for the reaction. Injecting a certain amount of ozone in to the bag, then inject isoprene, at last some water. Ultraviolet radiation helps the reaction. 20 min after the reaction we saw some foggy things formed in the bag, the lazer helped us see our results clearly.  
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</p> 
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<table>
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<tr>
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<th>aerosol +</th>
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<th>aerosol -</th>
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</tr>
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<td>
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<img src="https://static.igem.org/mediawiki/2011/archive/c/c0/20111027081411%21Aerosol1.png" alt="aerosol1" />
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</td>
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<td>
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<img src="https://static.igem.org/mediawiki/2011/9/93/Aerosol2.png" alt="aerosol2" />
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</td>
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</table>
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<center>Fig.3 aerosol conformation</center>
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<p>
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Photo on the left side shows that isoprene in the condition of air, water and ozone formed bigger molecule aerosol. The photo on the right side shows that without isoprene, even put them in the reaction condition no aerosol can`t be detected.   
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</p>
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<!-- ############ End of main contents ############ -->
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Latest revision as of 03:46, 29 October 2011

Tokyo Tech 2011

Rain details

1. Construction

We obtained the gene ispS (on pMK) from Gene Arts.

construction

Fig. 1 construction for ispS parts

It was difficult to excise ispS gene from pMK by cutting between EcoRI and PstI site, EcoRI and SpeI site, or XbaI and PstI site, because the length of pMK from which ispS was excised were as long as the length of ispS. So, we cut the pMK at NcoI site to make different length and ligated into the pSB3K3 including lacIQ promoter. Finally, we cut out PlacIQ-ispS and ligated it into pSB1C3 backbone vector.(BBa_K649303)


2. Assay Preparation

To measure the amount of isoprene produced by our E. coli, we used electron-ionization Gas Chromatography-Mass Spectrometry equipment (GC-MS, QP-2010, SHIMADZU, Japan). Analytes were separated by a nonpolar column (Rtx-1MS: Length 30 m, ID 0.25 mm film thickness 0.5 µm, USA) working in a constant flow mode (2.99 mL min-1). The temperature program was chosen as follows: 40°C for 7 min, increase to 280°C at rate of 10°C min-1, 280°C for 5 min. The mass spectrometer worked in SIM mode, m/z 67.


assay

Fig. 2 GC-MS

We made dilution series of liquid isoprene (Wako Pure Chemical Industries, Ltd, Japan) diluted in chloroform(diluted 102,103,104,105,106,107-fold). The undiluted isoprene solution 1 µL is 0.654 mg. We injected diluted isoprene into GC-MS, and draw a calibration curve (Fig. 3). If X (x=logX) represents the area of isoprene's peak and Y (y=logY) represents the amount of isoprene [mg], the calibration curve is described by the equation “Y = 10-7.9 × X0.89”.

assay

Fig. 3 calibration curve

3. Assay Method by E. coli


Fig. 4 constraction for assay

Each bacterial sample was grown in a 500 mL flask containing 100 mL LB media. Cultures were grown at 37°C and then induced by 0.5 mM IPTG when OD600 reached 0.6. After 4 hours of induction, 50 mL of headspace gas was taken by absorbing material (mini-PAT including Tenax: Japan Analytical Industry Co., Ltd) and injected into GC-MS.

isoprene_ex isoprene_ex isoprene_ex
Fig. 5 Assay Method

We calculated the amount of isoprene production, using the calibration data we obtained above.

isprene-graph
Fig. 6 The amount of isoprene detected in E. coli extract.

4.Aerosol formation

To do the ozone-isoprene reaction, we used teflon bag as a container for the reaction. Firstly ozone was produce by the ozone generator as the following photo. After measured the concentration of ozone, we used big syringe, injected a certain amount of ozone into the bag. Then using small size of syringe we injected isoprene and water into the bags. We could see that isoprene evaporated immediately. Finally because ultraviolet radiation helps the reaction, we put the teflon bags into clean bench and turned the UV on.


Fig. 7 Method of aerosol formation experiment

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