Difference between revisions of "Part:BBa K649303:Experience"

Line 8: Line 8:
 
[[Image:ispS_table.png|thumb|left|400px|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.]]
 
[[Image:ispS_table.png|thumb|left|400px|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.]]
 
<div style="clear:both;"></div>
 
<div style="clear:both;"></div>
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].If X represents the area and Y represents the amount of isoprene [mg], the calibration curve is described by the equation,<br> '''Y = 10<sup>-7.9</sup> &times; X<sup>0.89</sup>.'''
+
We calculated the amount of isoprene by [http://2011.igem.org/Team:Tokyo_Tech/Projects/making-rain/GC-Assay#AP calibration date we obtained].If X represents the area and Y represents the amount of isoprene [mg], the calibration curve is described by the equation,<br> '''Y = 10<sup>-7.9</sup> &times; X<sup>0.89</sup>.'''<br>
 +
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''.
 +
 
  
 
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).
 
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).

Revision as of 05:47, 28 October 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 synthesize isoprene.

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

User Reviews

UNIQf4308b640d3ba4c7-partinfo-00000000-QINU UNIQf4308b640d3ba4c7-partinfo-00000001-QINU