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Team:Tokyo Tech/Projects/making-rain/GC-Assay
From 2011.igem.org
Rain detail method/result
<GC-MS>
Gas chromatography (GC)-mass spectrometry (MS) (GC-MS QP-2010, SHIMADZU, Japan) was performed to assay amount of isoprene.
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 too short (about 1.06-1.10 min)(Fig.1). 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).
Result
Discussion
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.
<Emission of Isoprene in Water>
Method
In various conditions, we experimented in order to make sure that isoprene in water or LB medium emit into air and to know background of LB media or E.coli BL21 (DE3) not constructed (Table.1). Headspace gas was sampled though an adsorbing material (mini-PAT including Tenax: Japan Analytical Industry Co., Ltd) and injected into GC-MS.
| 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℃, culture, 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.
Discussion
We calculated the amount of isoprene E. coli can produced based on a paper by Zao Y et al. (2011).
According to our calculation results, E. coli BL21 (DE3) with positive control constructed will
accumulate around 9.6×10 [µg/L] (microgram total amount of isoprene per liter of broth) isoprene,
while E. coli with negative control will produce very little amounts of isoprene about 9.2 [µg/L].
The amount of isoprene require to form secondary organic aerosols is
0.2-1.6[ppm] (=0.56-4.5 [µg/(air-l)])[3]. Note that both the positive control and the negative control
produce amounts of isoprene greater than that required to form the aerosols
when cultivated in 100ml of LB media (0.92[µg] and 9.6[µg], respectively),
we can therefore conclude that, at least in principle, it is possible to make an E. coli
that can make induce the formation of these secondary aerosols.
How to calculate: Both the negative control and positive control can produce 9 and 94 [µg/l] isoprene
under the condition of OD600 of 140 and 34 hours induction. Because the fact of the matter is
that OD600 of 1 and induction time is 5hours, production of isoprene will be 1/140 × 1/7.
