Difference between revisions of "Part:BBa K649303:Experience"
 
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===Applications of BBa_K649303===
 
===Applications of BBa_K649303===
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we confirmed that E. coli introduced <i>ispS</i> produced isoprene, by means of using electron-ionization Gas Chromatography-Mass Spectrometry equipment. <br>
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We confirmed that ''E. coli'',introduced ''ispS'', produced isoprene, by means of using Gas Chromatography-Mass Spectrometry equipment. <br>
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[[Image:ispS_assay_result.png|thumb|center|400px|isoprene synthesized by ''E. coli ''BL21 introduced ''ispS'' gene.<br>This work is done by Yuto Sugiuchi.]]
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[[Image:Rain-fig4-2.jpg|thumb|center|400px|isoprene synthesized by ''E. coli ''BL21 introduced ''ispS'' gene.<br>This work is done by Yuto Sugiuchi.]]
  
 
'''[Sample]'''<br>
 
'''[Sample]'''<br>
 
1. PlacIQ on pSB3K3<br>
 
1. PlacIQ on pSB3K3<br>
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2. PlacIQ-rbs-ispS<br>
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2. PlacIQ-RBS-''ispS'' on pSB3K3<br>
  
  
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'''[Results]'''<br>
 
'''[Results]'''<br>
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According to the calibration curve, we detected 4.1×10<sup>-5</sup> mg/L isoprene produced by ''E. coli'' BL21 (DE3) introduced ispS, while negative control (PlacIQ) produced one eighth of our new ''E. coli''.<br>
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According to the calibration curve, we detected 4.1×10<sup>-5</sup> mg/L isoprene produced by ''E. coli'' BL21 (DE3) introduced ''ispS'', while negative control (PlacIQ) produced one eighth of our new ''E. coli''.<br>
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[[Image:ispS_table.png|thumb|left|350px|]]
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[[Image:ispS_table2.png|thumb|left|350px|]]
 
<div style="clear:both;"></div>
 
<div style="clear:both;"></div>
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'''[Mechanism of GC-MS]'''<br>
 
'''[Mechanism of GC-MS]'''<br>

Latest revision as of 06:12, 1 November 2011

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 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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