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Team:Tokyo Tech/Projects/making-rain/GC-Assay
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| - | <li> | + | <li><a href="#const">1. Construction</a></li> |
| - | + | <li><a href="#AP">2. Assay Preparation</a></li> | |
| - | + | <li><a href="#Em-isp">3. Assay Method by <i>E. coli</i></a></li> | |
| - | <li> | + | <li><a href="#aerosol">4. Aerosol formation</a></li> |
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| - | <h1> Rain | + | <h1> Rain details </h1> |
| - | + | <h2 id="const"> 1. Construction </h2> | |
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<p> | <p> | ||
| - | < | + | 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/f/f1/IspS1.png" alt="construction" width="600"/> | ||
| + | </div><br /> | ||
| + | <center>Fig. 1 construction for <span class="gene">ispS</span> parts</center> | ||
<p> | <p> | ||
| - | < | + | 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 /> | |
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| - | + | <h2 id="AP">2. Assay Preparation </h2> | |
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| - | < | + | <p> |
| - | + | 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°C for 7 min, increase to 280°C at rate of 10°C min<sup>-1</sup>, 280°C for 5 min. The mass spectrometer worked in SIM mode, m/z 67. | |
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| - | + | </p><br /> | |
| - | + | <p> | |
| - | + | <center><img src="https://static.igem.org/mediawiki/2011/3/3d/111028GCMS.JPG" alt="assay"width="300" height="200" /></center><br /> | |
| - | + | <center>Fig. 2 GC-MS</center> | |
| - | + | </p> | |
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| - | + | <p> | |
| - | + | 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 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<sup>-7.9</sup> × X<sup>0.89</sup>”. | |
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| - | + | <center><img src="https://static.igem.org/mediawiki/2011/d/de/Isp-kenryou.jpg" alt="assay" /></center><br /> | |
| - | + | <center>Fig. 3 calibration curve</center> | |
| - | + | </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> | |
| - | + | <div align="center"> | |
| - | + | <img src="https://static.igem.org/mediawiki/2011/7/74/Isoprene_sample2.png" width="250px" /> | |
| - | + | </div><br /> | |
| - | + | <center> | |
| - | + | Fig. 4 constraction for assay | |
| - | + | </center> | |
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| - | + | </p> | |
| - | + | <p> | |
| - | + | 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 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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| + | <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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| + | </div> | ||
| + | <center>Fig. 5 Assay Method | ||
| + | <br /> </center> | ||
| + | </p> | ||
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<p> | <p> | ||
| - | < | + | We calculated the amount of isoprene production, using the calibration data we obtained above. |
| - | + | </p> | |
| - | + | <div align="center"> | |
| - | + | <img src="https://static.igem.org/mediawiki/2011/e/e4/Rain-fig4-2.jpg" alt="isprene-graph" width="450px" /> | |
| - | + | </div> | |
| - | + | <center> | |
| - | </ | + | Fig. 6 The amount of isoprene detected in <span class="name">E. coli</span> extract. |
| - | + | </center> | |
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| + | <h2 id="aerosol"> 4.Aerosol formation</h2> | ||
<p> | <p> | ||
| - | + | 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> | </p> | ||
| - | + | <div align="center"> | |
| - | < | + | <img src="https://static.igem.org/mediawiki/2011/8/82/Ozone.JPG" width="450px" /> |
| - | + | </div> | |
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| - | + | <center> | |
| - | + | Fig. 7 Method of aerosol formation experiment | |
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<!-- ############ End of main contents ############ --> | <!-- ############ End of main contents ############ --> | ||
Latest revision as of 03:46, 29 October 2011
Rain details
1. Construction
We obtained the gene ispS (on pMK) from Gene Arts.
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.
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”.
3. Assay Method by E. coli
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.
We calculated the amount of isoprene production, using the calibration data we obtained above.
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.
