Generator

Part:BBa_K649303:Experience

Designed by: Goshi Sugano   Group: iGEM11_Tokyo_Tech   (2011-09-19)
Revision as of 12:43, 28 October 2011 by Takuya 1613 (Talk | contribs)

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K649303

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


[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 table.png


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].

User Reviews

UNIQ4d5de61facb1ba97-partinfo-00000000-QINU UNIQ4d5de61facb1ba97-partinfo-00000001-QINU