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Team:Tokyo Tech/Projects/making-rain/GC-Assay
From 2011.igem.org
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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 µL is 0.654 mg. We injected diluted isoprene into GC-MS, and draw a calibration curve (fig. | + | 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 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><img src="https://static.igem.org/mediawiki/2011/d/de/Isp-kenryou.jpg" alt="assay" /></center><br /> | ||
| - | <center>fig. | + | <center>fig.3 calibration curve</center> |
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| - | fig. | + | fig.4 constraction for assay |
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| - | <center> | + | <center>fig.5 isoprene detected by GC-MS |
<br />(a)up:negative control(PlacIQ), middle:sample(PlacIQ-rbs-<span class="gene">ispS</span>), down:reference material <br />(b)The amount of isoprene detected in <span class="name">E. coli</span> extract. | <br />(a)up:negative control(PlacIQ), middle:sample(PlacIQ-rbs-<span class="gene">ispS</span>), down:reference material <br />(b)The amount of isoprene detected in <span class="name">E. coli</span> extract. | ||
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| - | <center>fig. | + | <center>fig.6 aerosol conformation</center> |
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Revision as of 10:29, 28 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.
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 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.
(a)up:negative control(PlacIQ), middle:sample(PlacIQ-rbs-ispS), down:reference material
(b)The amount of isoprene detected in E. coli extract.
We calculated the amount of isoprene by calibration date we obtained.
4.Make it rain!
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 isoprene, and lastly 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.
| Isoprene - | Isoprene + |
|---|---|
|
|
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.
