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="name">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>  
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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 out PlacIQ-<span class="gene">ispS</span> and ligated it 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>)
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</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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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 µm, 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).
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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.
</p><br />
</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>
<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.2). If X (x=logX) represents the area and Y (y=logY) represents the amount of isoprene [mg], the calibration curve is described by the equation " Y = 10<sup>-7.9</sup> &times; X<sup>0.89</sup> ".
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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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         <center><img src="https://static.igem.org/mediawiki/2011/d/de/Isp-kenryou.jpg" alt="assay" /></center><br />
         <center><img src="https://static.igem.org/mediawiki/2011/d/de/Isp-kenryou.jpg" alt="assay" /></center><br />
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<center>fig.2 calibration curve</center>
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<center>Fig. 3 calibration curve</center>
         </p>
         </p>
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<h2 id="Em-isp">3.Assay Method by <span class="name">E. coli</span></h2>
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<h2 id="Em-isp">3. Assay Method by <span class="name">E. coli</span></h2>
   
   
<p>
<p>
         <div align="center">
         <div align="center">
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<img src="https://static.igem.org/mediawiki/2011/5/5e/Isoprene_sample.png" alt="construction" width="250px" />
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<img src="https://static.igem.org/mediawiki/2011/7/74/Isoprene_sample2.png" width="250px" />
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         <center>
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                 fig.3 constraction for assay
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                 Fig. 4 constraction for assay
         </center>
         </center>
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         </p>
         <p>
         <p>
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Each bacterial sample was grown in a 500 mL flask containing 100 mL LB media. Cultures were grown at 37℃ 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.
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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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<p>We calculated the amount of isoprene by calibration date we obtained.
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<h2 id="rain">4.Make it rain!</h2>
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The ozone-isoprene reaction was carried out in 10 L teflon bag as follows. Into the bag were added, firstly a certain mount of ozone, secondly some isoprene, and lastly some water. To facilitate the reaction, ultraviolet radiation was used. 20 min after the reaction stared, formation of aerosol was confirmed by using a laser as shown the photos below.
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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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        <center>
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                </div>
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<table>
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                        <center>Fig. 5 Assay Method
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<tr>
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                                <br /> </center>
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<th>Isoprene -</th>
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                </p>      
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<th>Isoprene +</th>
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<img src="https://static.igem.org/mediawiki/2011/9/93/Aerosol2.png" alt="aerosol2" />
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<img src="https://static.igem.org/mediawiki/2011/archive/c/c0/20111027081411%21Aerosol1.png" alt="aerosol1" />
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</table>
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        </center>
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<center>fig.4 aerosol conformation</center>
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<p>
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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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<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>
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<h2 id="aerosol"> 4.Aerosol formation</h2>
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The photo on the left shows that isoprene formed aerosol under reaction conditions in the presence of air, water and ozone. On the other hand, the photo on the right shows that without isoprene, no aerosol could be detected even when air, water and ozone were put together under reaction conditions.  
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<p>
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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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<br/>
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<center>
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Fig. 7 Method of aerosol formation experiment
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</center>
<!-- ############ 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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