Difference between revisions of "Part:BBa K203119:Design"
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===Design Notes=== | ===Design Notes=== | ||
− | [http://2009.igem.org/Team:Heidelberg/Project_Synthetic_promoters RA-PCR] (Fig. 1) was conducted with Oligos containing a NF-κB binding site, plus a small number of "general activators" (NF-Y, Sp1, Ap1, CREB). 33 clones were picked, miniprepped and transfected. NF-κB was then induced by the addition of TNF-&alpha (2.5µM) for 10 hours, and left uninduced as a control. The plate was then scanned by TECAN, an automated fluorescence plate reader. TECAN is very imprecise on eukaryotic cells, and the arbitrary fluorescence we meausred is not proportional to [ | + | [http://2009.igem.org/Team:Heidelberg/Project_Synthetic_promoters RA-PCR] (Fig. 1) was conducted with Oligos containing a NF-κB binding site, plus a small number of "general activators" (NF-Y, Sp1, Ap1, CREB). 33 clones were picked, miniprepped and transfected. NF-κB was then induced by the addition of TNF-&alpha (2.5µM) for 10 hours, and left uninduced as a control. The plate was then scanned by TECAN, an automated fluorescence plate reader. TECAN is very imprecise on eukaryotic cells, and the arbitrary fluorescence we meausred is not proportional to [http://2009.igem.org/Team:Heidelberg/Project_Measurement REU] or another precise measure of mammalian promoter activity, but it can serve as a rough indicator of promoter induction. The result (Fig. 2) shows that most clones appear not to be induced by NF-κB, whereas others are induced at varying levels of strength. We picked clone 31 for submission to the registry and detailled charscterization,. |
[[Image:HD09_nfkb.png|thumb|none|600px|'''Fig. 1: Screening of putatively NF-κB inducible promoters''']] | [[Image:HD09_nfkb.png|thumb|none|600px|'''Fig. 1: Screening of putatively NF-κB inducible promoters''']] |
Revision as of 14:29, 16 October 2009
NfKB Responsive promoter
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
[http://2009.igem.org/Team:Heidelberg/Project_Synthetic_promoters RA-PCR] (Fig. 1) was conducted with Oligos containing a NF-κB binding site, plus a small number of "general activators" (NF-Y, Sp1, Ap1, CREB). 33 clones were picked, miniprepped and transfected. NF-κB was then induced by the addition of TNF-&alpha (2.5µM) for 10 hours, and left uninduced as a control. The plate was then scanned by TECAN, an automated fluorescence plate reader. TECAN is very imprecise on eukaryotic cells, and the arbitrary fluorescence we meausred is not proportional to [http://2009.igem.org/Team:Heidelberg/Project_Measurement REU] or another precise measure of mammalian promoter activity, but it can serve as a rough indicator of promoter induction. The result (Fig. 2) shows that most clones appear not to be induced by NF-κB, whereas others are induced at varying levels of strength. We picked clone 31 for submission to the registry and detailled charscterization,.
Source
To be updated