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

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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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<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>The gene ispS with RBS on pMK was obtained 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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<img src="https://static.igem.org/mediawiki/2011/6/60/Rain_constcution_third_layer.png" alt="construction" /></div><br />
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<img src="https://static.igem.org/mediawiki/2011/f/f1/IspS1.png" alt="construction" width="600"/>
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</div><br />
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        <center>Fig. 1 construction for <span class="gene">ispS</span> parts</center>
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<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 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>)
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</p><br />
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<h2 id="AP">2. Assay Preparation </h2>
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<p>
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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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        <p>       
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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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<center>Fig. 2 GC-MS</center>
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        </p>
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<p>
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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;.
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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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<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>
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<p>
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        <div align="center">
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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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        <center>
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                Fig. 4 constraction for assay
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        </center>
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<p>It was difficult to extract ispS gene from backbone vector by cutting between neither EcoRI and PstI site, nor EcoRI and SpeI site, nor XbaI and PstI site because the rest of the genes were as long as ispS. So, we cut the pMK at NcoI site to make linear plasmid. Then this was cut and ligated into the pSB3K3 including lacIQ promoter. Finally, we cut PlacIQ-RBS-ispS and ligated into pSB1C3 backbone vector.</p><br />
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        </p>
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<h2 id="AP">2.Assay Preparation </h2>
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        <p>
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<p>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) for measurement. The MS uses an electron ionization method and quadrupole. Analytes were separated using 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.</p><br />
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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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                <div align="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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                        <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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<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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                        <img src="https://static.igem.org/mediawiki/2011/2/2f/111028sampling.JPG" alt="isoprene_ex" width="180" height="240" />
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Let area be the vertical axis, and amount of isoprene itself ([mg]) the horizontal axis.</td>
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</tr>
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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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</table>
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<p>We firstly made dilution series (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) of liquid isoprene (Wako Pure Chemical Industries, Ltd, Japan) diluted in chloroform. 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).</p><br />
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                </div>
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                        <center>Fig. 5 Assay Method
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                                <br /> </center>
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                </p>      
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<p>
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<p style="clear:both">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.</p><br />
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We calculated the amount of isoprene production, using the calibration data we obtained above.
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</p>
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<h2 id="Em-isp">3.Isoprene in Solvent</h2>
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<div align="center">
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<p>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.</p><br />
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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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<table border="1" style="text-align:center;">
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Fig. 6 The amount of isoprene detected in <span class="name">E. coli</span> extract.   
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<th>number</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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<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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<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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<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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<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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<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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<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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<p>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><br />
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<table style="float:left;">
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<tr><td><img src="https://static.igem.org/mediawiki/2011/9/98/Peak_Area_11titech.jpg" alt="assay" width="436px" /></td></tr>
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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.</p> <br />
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<p>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.</p><br />
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<h2 style="clear:both" id="Assay">4.Assay Method by <span class="">E. coli</span>(unoperation)</h2>
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<h2 id="aerosol"> 4.Aerosol formation</h2>
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<p>Both E.coli are grown in separate 500 mL flasks containing 100 mL LB media. Cultures are grown at 37℃ and then induced using 0.5 mM IPTG at OD<sub>600</sub> of 0.6 reached. After 34 hours of induction, 50 mL of headspace gas samples are taken using absorbing material (mini-PAT including Tenax: Japan Analytical Industry Co., Ltd) and injected into GC-MS.</p><br/>
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<p>
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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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<div align="center">
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<img src="https://static.igem.org/mediawiki/2011/8/82/Ozone.JPG" width="450px" />
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</div>
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<br/>
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<center>
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Fig. 7 Method of aerosol formation experiment
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</center>
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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.


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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”.

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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