Rain details

Construction

The gene ispS with RBS on pMK was obtained from Gene Arts.

It was difficult to extract ispS gene from backbone vector by cutting between neither EcoRI and PstI site, nor EcoRI and SpeI site, nor XbaI and PstI site because the rest of gene ispS was as long as ispS. So, we cut the pMK at NcoI site to make linear plasmid. Then this was cut and ligated into the pSB3K3 including lacIQ promoter. Finally, we cut PlacIQ-RBS-ispS and ligated into pSB1C3 backbone vector.

Assay Preparation

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 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℃ 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.

Fig.1 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.

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).

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.

Isoprene in Solvent

We then experimented in various conditions to make sure that isoprene can emit from water and LB medium. 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	15 mL centrifuge tube	None	6.54	15	room temperature, 20 minutes
2	500 mL flask	Water 100 mL	13.1	50	room temperature, 20 minutes
3	500 mL flask	Water 100 mL	13.1	50	37℃, 20 minutes
4	500 mL flask	LB medium 100 mL	13.1	50	37℃, 20 minutes
5	500 mL flask	LB medium 100 mL	0	50	37℃, culture, 6 hours
6	500 mL flask	LB medium 100 mL	0	50	37℃, 6 hours + E.coli(BL-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.

As results, the peak area was about one thousandth of expectable area by calibration data at experiment No.1-4, drip isoprene. The peak area’s retention time was same 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.

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 we considered that 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 Method (unoperation)

Both E.coli are grown in separate 500ml flasks containing 100ml LB media. Cultures are grown at 37℃ and then induced using 0.5mM IPTG at OD600 of 0.6 reached. After 34 hours of induction, 50ml of headspace gas samples are taken using absorbing material (mini-PAT including Tenax: Japan Analytical Industry Co., Ltd) and injected into GC-MS.