Rain details

1.Construction

We obtaind the gene ispS on pMK from Gene Arts.

fig.1 construction for ispS parts

It was difficult to excise ispS gene from pMK by cutting between EcoRI and PstI site, EcoRI and SpeI site, or XbaI and PstI site, because the length of pMK from which ispS was excised were as long as the length of ispS. So, we cut the pMK at NcoI site to make different length and ligated into the pSB3K3 including lacIQ promoter. Finally, we cut out PlacIQ-ispS and ligated it into pSB1C3 backbone vector.

2.Assay Preparation

To measure the amount of isoprene produced by our E. coli, we used electron-ionization Gas Chromatography-Mass Spectrometry equipment (GC-MS, QP-2010, SHIMADZU, Japan). Analytes were separated by 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).

We made dilution series of liquid isoprene (Wako Pure Chemical Industries, Ltd, Japan) diluted in chloroform(diluted 10²,10³,10⁴,10⁵,10⁶,10⁷-fold). The undiluted isoprene solution 1 µL is 0.654 mg. We injected diluted isoprene into GC-MS, and draw a calibration curve (Fig.2). If X (x=logX)represents the area and Y (y=logY) represents the amount of isoprene [mg], the calibration curve is described by the equation " Y = 10^-7.9 × X^0.89 ".

Fig.2 calibration curve

3.Assay Method by E. coli

fig.3 constraction for assay

Each E. coli are grown in 500 mL flasks containing 100 mL LB media. Cultures are grown at 37℃ and then induced by 0.5 mM IPTG when OD600 reached 0.6. After 4 hours of induction, 50 mL gas samples from headspace are taken by absorbing material (mini-PAT including Tenax: Japan Analytical Industry Co., Ltd) and injected into GC-MS.

We calculated the amount of isoprene by calibration date we obtained.

4.Make it rain!

To do the ozone-isoprene reaction, we used 10 L teflon bag as a container for the reaction. Injecting a certain amount of ozone in to the bag, then inject isoprene, at last some water. Ultraviolet radiation helps the reaction. 20 min after the reaction we saw some foggy things formed in the bag, the laser helped us see our results clearly.

Isoprene +	Isoprene -

Fig.4 aerosol conformation

Photo on the left side shows that isoprene in the condition of air, water and ozone formed bigger molecule aerosol. The photo on the right side shows that without isoprene, even put them in the reaction condition no aerosol can`t be detected.