Difference between revisions of "Part:BBa J64997"
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===Characterization ''in vivo''=== | ===Characterization ''in vivo''=== | ||
This part has been characterized for GFP expression in the pCDF plasmid by the 2015 LASATX iGEM team. | This part has been characterized for GFP expression in the pCDF plasmid by the 2015 LASATX iGEM team. | ||
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| + | We placed the T7 promoter in front of the sfGFP gene (BBa_K1624004). The experimental construct was placed in a pCDFDuet-1 vector and transformed into BL21(DE3) cells. We used BL21(DE3) cells without the plasmid containing T7 as our negative control. Cells were grown overnight. A 1:100 dilution was made, and cultures were grown with 20% glucose to 0.6 OD. At 0.6 OD, cells were resuspended in IPTG and induced for 2 hours. After induction, cells were washed and resuspended in PBS buffer. Fluorescence was then measured at 600 nm for 25 flashes. | ||
<center>https://static.igem.org/mediawiki/2015/f/fd/T7characterization.jpg</center> | <center>https://static.igem.org/mediawiki/2015/f/fd/T7characterization.jpg</center> | ||
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| + | The sfGFP gene placed downstream of the T7 promoter was successfully expressed, with fluorescence from the experimental construct significantly higher than the control. | ||
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Revision as of 00:33, 19 September 2015
T7 consensus -10 and rest
Characterization in vivo
This part has been characterized for GFP expression in the pCDF plasmid by the 2015 LASATX iGEM team.
We placed the T7 promoter in front of the sfGFP gene (BBa_K1624004). The experimental construct was placed in a pCDFDuet-1 vector and transformed into BL21(DE3) cells. We used BL21(DE3) cells without the plasmid containing T7 as our negative control. Cells were grown overnight. A 1:100 dilution was made, and cultures were grown with 20% glucose to 0.6 OD. At 0.6 OD, cells were resuspended in IPTG and induced for 2 hours. After induction, cells were washed and resuspended in PBS buffer. Fluorescence was then measured at 600 nm for 25 flashes.
The sfGFP gene placed downstream of the T7 promoter was successfully expressed, with fluorescence from the experimental construct significantly higher than the control.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]