Difference between revisions of "Part:BBa K649303:Experience"
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===Applications of BBa_K649303===
 
===Applications of BBa_K649303===
 
We confirmed that ''E. coli'' introduced ispS synthesize isoprene. <br>
 
We confirmed that ''E. coli'' introduced ispS synthesize isoprene. <br>
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[[Image:ispS_table.png|thumb|right|300px|]]
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[[Image:ispS_table.png|thumb|right|400px|]]
 
[[Image:ispS_assay_result.png|thumb|right|400px|isoprene synthesized by ''E. coli ''BL21 introduced ''ispS'' gene.<br>This work is done by Yuto Sugiuchi.]]
 
[[Image:ispS_assay_result.png|thumb|right|400px|isoprene synthesized by ''E. coli ''BL21 introduced ''ispS'' gene.<br>This work is done by Yuto Sugiuchi.]]
  
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'''[Method]'''<br>
 
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.]]
 
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.]]
  

Revision as of 12:18, 28 October 2011

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Applications of BBa_K649303

We confirmed that E. coli introduced ispS synthesize isoprene.

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

[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.]]


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.
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.


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).

For more information, see [http://2011.igem.org/Team:Tokyo_Tech/Projects/making-rain/index.htm our work in Tokyo_Tech 2011 wiki].

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