Difference between revisions of "Part:BBa K203119:Design"

(→‎Design Notes)
(→‎Design Notes)
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[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 [[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,.
 
[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 [[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,.
  
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[[Image:HD09_nfkb.png|thumb|none|500px|'''Fig. 1: Screening of putatively NF-κB inducible promoters''']]  
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[[Image:HD09_nfkb.png|thumb|none|600px|'''Fig. 1: Screening of putatively NF-κB inducible promoters''']]  
  
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[[Image:Image:Rapcr.jpg|thumb|none|500px|'''Fig. 2: The method of LAM-PCR.''' For a detailled description, see [http://2009.igem.org/Team:Heidelberg/Project_Synthetic_promoters our wiki] ]]
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[[Image:Rapcr.jpg|thumb|none|600px|'''Fig. 2: The method of LAM-PCR.''' For a detailled description, see [http://2009.igem.org/Team:Heidelberg/Project_Synthetic_promoters our wiki] ]]
  
 
===Source===
 
===Source===

Revision as of 14:28, 16 October 2009

NfKB Responsive promoter


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Unknown
  • 12
    INCOMPATIBLE WITH RFC[12]
    Unknown
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE 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 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,.

Fig. 1: Screening of putatively NF-κB inducible promoters
Fig. 2: The method of LAM-PCR. For a detailled description, see [http://2009.igem.org/Team:Heidelberg/Project_Synthetic_promoters our wiki]

Source

To be updated

References