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.