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

From 2011.igem.org

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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 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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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> times). The undiluted isoprene solution 1 µL is 0.654 mg. We injected diluted isoprene into GC-MS, and draw a calibration curve (Fig.1).
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<p style="clear:both">
<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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<br />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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<h2 id="Em-isp">3.Isoprene in Solvent</h2>
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<h2 id="Em-isp">3.Assay Method by <span class="">E. coli</h2>
   
   
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<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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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 when 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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</p>
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<p>We calculated the amount of isoprene by calibration date we obtained.
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<table border="1" style="text-align:center;">
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<caption>
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</caption>
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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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<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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<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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<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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</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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<table style="float:left;">
 
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<td>
 
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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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</table>
 
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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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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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<h2 style="clear:both" id="Assay">4.Assay Method by <span class="">E. coli</span></h2>
 
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<p>
 
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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.
 
</p>
</p>
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<h2 id="results">5.Results</h2>
 
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<p>We calculated amount of isoprene from area and calibration data (Fig.1).</p>
 
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<table border="1" style="text-align:center;">
 
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<tr class="topline">
 
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<th>Designation</th>
 
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<th>Arear</th>
 
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<th>retention time [min]</th>
 
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<th>isoprene[ng] (50 mL sampling)</th>
 
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</tr>
 
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<tr>
 
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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>
 
<h2 id="rain">4.Make it rain!</h2>
<h2 id="rain">4.Make it rain!</h2>

Revision as of 11:41, 27 October 2011

Tokyo Tech 2011

Rain details

1.Construction

We obtaind the gene ispS on pMK from Gene Arts.


construction

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 PlacIQ-ispS and ligated into pSB1C3 backbone vector.


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℃ for 7 min, increase to 280℃ at rate of 10℃ min-1, 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).


assay
Fig.1 Dilution series of liquid isoprene diluted in chloroform were injected into GC-MS.
Let area be the vertical axis, and amount of isoprene itself ([mg]) the horizontal axis.

We made dilution series of liquid isoprene (Wako Pure Chemical Industries, Ltd, Japan) diluted in chloroform(diluted 102,103,104,105,106,107 times). The undiluted isoprene solution 1 µL is 0.654 mg. We injected diluted isoprene into GC-MS, and draw a calibration curve (Fig.1).


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.

3.Assay Method by E. coli

Each E. coli are grown in 500 mL flasks containing 100 mL LB media. Cultures are grown at 37℃ and then induced by 0.5 mM IPTG when 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.

We calculated the amount of isoprene by calibration date we obtained.

4.Make it rain!

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.

aerosol + aerosol -
aerosol1 aerosol2
Fig.3 aerosol conformation

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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