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Team:Tokyo Tech/Projects/making-rain/GC-Assay
From 2011.igem.org
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.1).
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.1)
| 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) |
Table.1 Different conditions that used in samples` headspace gas of solvent including isoprene or LB media with E.coli or none. The condition is from dripping to sampling.
<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.
