Difference between revisions of "Part:BBa K203110:Design"

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We performed [http://2009.igem.org/Team:Heidelberg/Project_Synthetic_promoters RA-PCR] with oligos containing binding sites for some well known generally activating transcription factors (Sp1, Ap1, CREB, NF-Y) which we identified from literature search [1],[2],[3]. We also added NF-κB responsive oligos as NF-κB has non-specific activity and is therefore used by a variety of viral constitutive promoters, e.g. the HIV promoter [4]. We then cloned the construct into [[Part:BBa_K203112]] by SpeI and HindIII to obtain a core promoter. We picked 24 colonies, two of which we dismissed after a test digest. This promoter corresponds to clone S5.  
 
We performed [http://2009.igem.org/Team:Heidelberg/Project_Synthetic_promoters RA-PCR] with oligos containing binding sites for some well known generally activating transcription factors (Sp1, Ap1, CREB, NF-Y) which we identified from literature search [1],[2],[3]. We also added NF-κB responsive oligos as NF-κB has non-specific activity and is therefore used by a variety of viral constitutive promoters, e.g. the HIV promoter [4]. We then cloned the construct into [[Part:BBa_K203112]] by SpeI and HindIII to obtain a core promoter. We picked 24 colonies, two of which we dismissed after a test digest. This promoter corresponds to clone S5.  
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[[Image:Rapcr.jpg|thumb|none|600px]]
 
[[Image:Rapcr.jpg|thumb|none|600px]]
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 +
{|  class="wikitable float-left" border="1"
 +
|-
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!Oligo name !! Oligo Sequence !! µL of Oligo used
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|-
 +
|NFkB responsive forward 1 || GCGATCGGCAGATCAGGGGACTTTGCCGGGTGACGGGTTCA || 0,5
 +
|-
 +
|NFkB responsive forward 2 || GCGATCGGCAGATCAGGGGRWYYCCCGGGTGACGGGTTCA  || 0,5
 +
|-
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|NFkB responsive reverse|| TGATCTGCCGATCGCCCGTTTCAGGGGTGAACCCGTCACCC  || 1
 +
|-
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|Spacer reverse || TGATCTGCCGATCGCNNNNNNNNNNTGAACCCGTCACCC  || 3
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|-
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|Sp1 responsive || GCGATCGGCAGATCAGGGGCGGGGCGGGTGACGGGTTCA  || 2
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|-
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|Ap1 responsive || GCGATCGGCAGATCATGACTCAGGGTGACGGGTTCA  || 1
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|-
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|CREB responsive || GCGATCGGCAGATCABNBVNTGACGTCAGGGTGACGGGTTCA  || 1
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|-
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|NF-Y responsive || GCGATCGGCAGATCANNCCAATNNGGGTGACGGGTTCA  || 1
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|-
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|Stop 5' || CAGTACTAGTGGGTGACGGGTTCA  || 1
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|-
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|Stop 3' || TGACAAGCTTAGTGAACCCGTCACCC  || 1
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|}
  
 
===Source===
 
===Source===
  
−
Generated in our laboratory.
+
Generated in our laboratory.[5]
  
 
===References===
 
===References===
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[3] Tornoe, J. Generation of a synthetic mammalian promoter library by modification of sequences spacing transcription factor binding sites. Gene 297, 21-32 (2002). <br>
 
[3] Tornoe, J. Generation of a synthetic mammalian promoter library by modification of sequences spacing transcription factor binding sites. Gene 297, 21-32 (2002). <br>
 
[4] Rattner, A. NF-kappa B activates the HIV promoter in neurons. EMBO 12, 4261–4267 (1993). <br>
 
[4] Rattner, A. NF-kappa B activates the HIV promoter in neurons. EMBO 12, 4261–4267 (1993). <br>
 +
[5] RA-PCR, a method for the generation of randomized promoter libraries. igem 2009 Heidelberg team wiki. Available online at http://2009.igem.org/Team:Heidelberg/Project_Synthetic_promoters#Results

Latest revision as of 13:32, 20 October 2009

Constitutive promoter; 0.4 REU


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

We performed [http://2009.igem.org/Team:Heidelberg/Project_Synthetic_promoters RA-PCR] with oligos containing binding sites for some well known generally activating transcription factors (Sp1, Ap1, CREB, NF-Y) which we identified from literature search [1],[2],[3]. We also added NF-κB responsive oligos as NF-κB has non-specific activity and is therefore used by a variety of viral constitutive promoters, e.g. the HIV promoter [4]. We then cloned the construct into Part:BBa_K203112 by SpeI and HindIII to obtain a core promoter. We picked 24 colonies, two of which we dismissed after a test digest. This promoter corresponds to clone S5.

Rapcr.jpg
Oligo name Oligo Sequence µL of Oligo used
NFkB responsive forward 1 GCGATCGGCAGATCAGGGGACTTTGCCGGGTGACGGGTTCA 0,5
NFkB responsive forward 2 GCGATCGGCAGATCAGGGGRWYYCCCGGGTGACGGGTTCA 0,5
NFkB responsive reverse TGATCTGCCGATCGCCCGTTTCAGGGGTGAACCCGTCACCC 1
Spacer reverse TGATCTGCCGATCGCNNNNNNNNNNTGAACCCGTCACCC 3
Sp1 responsive GCGATCGGCAGATCAGGGGCGGGGCGGGTGACGGGTTCA 2
Ap1 responsive GCGATCGGCAGATCATGACTCAGGGTGACGGGTTCA 1
CREB responsive GCGATCGGCAGATCABNBVNTGACGTCAGGGTGACGGGTTCA 1
NF-Y responsive GCGATCGGCAGATCANNCCAATNNGGGTGACGGGTTCA 1
Stop 5' CAGTACTAGTGGGTGACGGGTTCA 1
Stop 3' TGACAAGCTTAGTGAACCCGTCACCC 1

Source

Generated in our laboratory.[5]

References

[1] Edelmann, G.M. et al. Synthetic promoter elements obtained by nucleotide sequence variation and selection for activity. PNAS 97, 3038-43 (2000).
[2] Ogawa, R. Construction of strong mammalian promoters by random cis-acting element elongation. Biotechniques 42, 628-632 (2007).
[3] Tornoe, J. Generation of a synthetic mammalian promoter library by modification of sequences spacing transcription factor binding sites. Gene 297, 21-32 (2002).
[4] Rattner, A. NF-kappa B activates the HIV promoter in neurons. EMBO 12, 4261–4267 (1993).
[5] RA-PCR, a method for the generation of randomized promoter libraries. igem 2009 Heidelberg team wiki. Available online at http://2009.igem.org/Team:Heidelberg/Project_Synthetic_promoters#Results