Coding

Part:BBa_K649400:Experience

Designed by: Maiko Hayashi   Group: iGEM11_Tokyo_Tech   (2011-09-19)

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Applications of BBa_K649400

To characterize this part, we constructed the composite part, BBa_K649303. BBa_K649303 is composed of PlacIQ(BBa_I14032) and RBS-ispS(BBa_K649400).

we confirmed that E. coli introduced ispS produced isoprene, by means of using electron-ionization Gas Chromatography-Mass Spectrometry equipment.

isoprene synthesized by E. coli BL21 introduced ispS gene.
This work is done by Yuto Sugiuchi.

[Sample]
1. PlacIQ on pSB3K3
2. PlacIQ-RBS-ispS on pSB3K3


[Experimental method]
Each bacterial sample was grown in a 500 mL flask containing 100 mL LB media. Cultures were grown at 37℃ 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.


[Calculation method]
We calculated the amount of isoprene by [http://2011.igem.org/Team:Tokyo_Tech/Projects/making-rain/GC-Assay#AP calibration date we obtained]. X represents the area and Y represents the amount of isoprene [mg]. The calibration curve is described by the equation,
Y = 10-7.9 × X0.89.


[Results]
According to the calibration curve, we detected 4.1×10-5 mg/L isoprene produced by E. coli BL21 (DE3) introduced ispS, while negative control (PlacIQ) produced one eighth of our new E. coli.

IspS table2.png

[Mechanism of GC-MS]
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).


If you are interested in the project related to this parts, please see [http://2011.igem.org/Team:Tokyo_Tech/Projects/making-rain/index.htm our project page in Tokyo_Tech 2011 wiki].


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