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

From 2011.igem.org

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<p>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.  
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<h3 id="Em-isp-result">Result</h3>
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Peak area was about one thousandth of expectable area by calibration data at experiment No.1-4, drip isoprene.
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It was the peak in the same retention time as that of isoprene at experiment No.5 (no isoprene, LB medium only),
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though the area was much less than those of No.1-4.<br />
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<img alt="Fig.3 - Peak Area" />
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<h3 id="Em-isp-discus">Discussion</h3>
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We wondered whether the adsorbing material was saturated with isoprene at experiment No.1-4
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and isoprene still existed in silicon tube at experiment No.5.<br />
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So, firstly the tube needed to be used only once and then thrown away.
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Secondly, Headspace gas of dilution series of liquid isoprene diluted in chloroform
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in water needed to be sampled.
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50ml of headspace gas samples were taken using absorbing material  
50ml of headspace gas samples were taken using absorbing material  
(mini-PAT including Tenax: Japan Analytical Industry Co., Ltd) and injected into GC-MS.
(mini-PAT including Tenax: Japan Analytical Industry Co., Ltd) and injected into GC-MS.
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<h3 id="Assay-result">Result</h3>
 
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Unfortunately, the GC-MS instrument got broken just before the wiki freeze deadline
 
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so we were not able to conclude our experiments and report assay results.
 
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But before the World Championship Jamboree, we are able to use the GC-MS again and report
 
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our result.
 
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<h3 id="Assay-discus">Discussion</h3>
 
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<img alt="Fig.4 - Isoprene by E.coli and to rain" /><br />
 
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Fig.4  On the left and middle, isoprene expectable production by different engineered
 
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<span class="name">E.coli</span> (LT21) strains(positive control:pSB3K-placIQ-ispS, negative control:pSB3K-placIQ)
 
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(Zao Y et al.2011). After 5 hours' induction in 100ml LB media, 50ml of headspace gas was
 
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sampled. On the right, Isoprene requirement to make aerosol is 0.22-1.8[&micro;g] in 400ml
 
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headspace (Tadeusz E et al. 2007).
 
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Revision as of 10:25, 4 October 2011

Tokyo Tech 2011

Tokyo Tech 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)


Fig.2 - Calibration Data
Fig.2 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.
Table.1 various experiments: We sampled headspace gas of solvent including isoprene or LB media with E.coli or none.
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.

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