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Team:Tokyo Tech/Projects/making-rain/GC-Assay
From 2011.igem.org
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<h2>Method </h2> | <h2>Method </h2> | ||
| - | <p>In order to assay the amount of isoprene produced by our E.coli, 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 30m, ID 0.25mm film thickness 0.5µm, USA) working in a constant flow mode (2.99ml 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). But thanks to MS, isoprene could be identified.</p> | + | <p>In order to assay the amount of isoprene produced by our E.coli, 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 30m, ID 0.25mm film thickness 0.5µm, USA) working in a constant flow mode (2.99ml 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). But thanks to MS, isoprene could be identified.</p><br /> |
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| + | <p>We firstly made dilution series (diluted 102,103,104,105,106,107 times) of liquid isoprene (Wako Pure Chemical Industries, Ltd, Japan) diluted in chloroform. The undiluted isoprene solution 1[µl] is 0.654[mg]. We injected diluted isoprene into GC-MS, and draw a calibration curve (Fig.2)</P><br /> | ||
<img alt="Fig.2 - Calibration Data" /><br /> | <img alt="Fig.2 - Calibration Data" /><br /> | ||
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Let area be the vertical axis, and amount of isoprene itself ([mg]) the horizontal axis. | Let area be the vertical axis, and amount of isoprene itself ([mg]) the horizontal axis. | ||
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Revision as of 10:20, 4 October 2011
Rain details
Method
In order to assay the amount of isoprene produced by our E.coli, 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 30m, ID 0.25mm film thickness 0.5µm, USA) working in a constant flow mode (2.99ml 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). But thanks to MS, isoprene could be identified.
<Quantitative Analysis>
Method
We made dilution series (deluted 102,103,104,105,106,107 times) of liquid isoprene (Wako Pure Chemical Industries, Ltd, Japan) diluted in chloroform. The undiluted isoprene solution 1[µl] is 0.654[mg]. We injected diluted isoprene into GC-MS. We tried to draw a calibration curve (Fig.2).
We firstly made dilution series (diluted 102,103,104,105,106,107 times) of liquid isoprene (Wako Pure Chemical Industries, Ltd, Japan) diluted in chloroform. The undiluted isoprene solution 1[µl] is 0.654[mg]. We injected diluted isoprene into GC-MS, and draw a calibration curve (Fig.2)
| number | container | solvent | isoprene[mg] | sampling[ml] | Condition from dripping isoprene to sampling |
|---|---|---|---|---|---|
| 1 | 15ml centrifuge tube | None | 6.54 | 15 | room temperature, 20minutes |
| 2 | 500ml flask | Water 100ml | 13.1 | 50 | room temperature, 20minutes |
| 3 | 500ml flask | Water 100ml | 13.1 | 50 | 37℃, 20minutes |
| 4 | 500ml flask | LB medium 100ml | 13.1 | 50 | 37℃, 20minutes |
| 5 | 500ml flask | LB medium 100ml | 0 | 50 | 37℃, culture, 6hours |
| 6 | 500ml flask | LB medium 100ml | 0 | 50 | 37℃, 6hours + E.coli(LT-21) |
Result
Peak area was about one thousandth of expectable area by calibration data at experiment No.1-4, drip isoprene.
It was the peak in the same retention time 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.
Discussion
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, 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.
<Assay Isoprene from E.coli>
Method
E. coli BL21 (DE3) with positive control pSB3K-placIQ-ispS constructed, and other with negative control pSB3K-placIQ were grown in separate 500 ml flasks containing 100ml LB media. Cultures were grown at 37℃ and then induced using 0.5mM IPTG when OD600 of 0.6 was reached. After 5 hours of induction, 50ml of headspace gas samples were taken using absorbing material (mini-PAT including Tenax: Japan Analytical Industry Co., Ltd) and injected into GC-MS.
Result
Unfortunately, the GC-MS instrument got broken just before the wiki freeze deadline so we were not able to conclude our experiments and report assay results. But before the World Championship Jamboree, we are able to use the GC-MS again and report our result.
