Rain details

1. Construction

] We obtained 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.(BBa_K649303)

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°C for 7 min, increase to 280°C at rate of 10°C min^-1, 280°C for 5 min. The mass spectrometer worked in SIM mode, m/z 67.

Fig. 2 GC-MS

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. 3). If X (x=logX) represents the area of isoprene's peak 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. 3 calibration curve

3. Assay Method by E. coli

Fig. 4 constraction for assay

Each bacterial sample was grown in a 500 mL flask containing 100 mL LB media. Cultures were grown at 37°C and then induced by 0.5 mM IPTG when OD₆₀₀ reached 0.6. After 4 hours of induction, 50 mL of headspace gas was taken by absorbing material (mini-PAT including Tenax: Japan Analytical Industry Co., Ltd) and injected into GC-MS.

Fig. 5 Assay Method

We calculated the amount of isoprene production, using the calibration data we obtained above.

Fig. 6 The amount of isoprene detected in E. coli extract.

4. Make it rain!

The ozone-isoprene reaction was carried out in 10 L teflon bag as follows. Into the bag were added, firstly a certain mount of ozone, secondly isoprene, and lastly water. To facilitate the reaction, ultraviolet radiation was used. 20 min after the reaction stared, formation of aerosol was confirmed by using a laser as shown the photos below.

Isoprene -	Isoprene +

Fig. 7 aerosol conformation

The picture on the left shows that when isoprene was not present no aerosol was detected even when air, water and ozone were put together under reaction conditions. On the other hand, the picture on the right shows that when isoprene was used, it formed an aerosol (this became evident because the trajectory of the laser light was visible).