Difference between revisions of "Part:BBa K1379005:Design"
 
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===Design Notes===
 
===Design Notes===
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None
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 +
 
 +
<b>Identifying the Possible Promoter Regions</b>
 +
 
 +
To date (12 Oct 2014), the core/minimal promoter region of P<sub>celA</sub> has not yet been experimentally defined. In locating the promoter region required to initiate transcription, iGEM 2014 Hong_Kong_HKUST team attempted in making educated guesses based on relevant information available from the literature.
 +
 
 +
The procedure adopted was as follow:
 +
 
 +
The Com-Box promoter consensus sequence “TACGAATA” was BLASTed for targets in the <i>Streptococcus pneumoniae</i> genomes of strains R6, D39, ATCC7699 and NCTC7465 in the NCBI database. Genome of strain NCTC7465 was particularly given attention because its gDNA was available for manipulation. A list of loci with annotated genes returned.  &sigma;<sup>X</sup> was known to turn on late competence gene and therefore loci containing any of those genes documented were favored and filtered for. Those loci were then manually checked for consensus among the 4 genomes mentioned above. A sequence of 67 base pairs stood out as a promising target because it was 1) upstream of a late competence genes <i>celA</i> (encodes competence protein CelA), and 2) was identical across the 4 genomes. This 67 bp region has varying upstream sequences and the potential promoter region can reach as far as 200bp. Different truncations (67, 100, 150, 180, 249, 300 bp) were planned for deciding the minimal promoter region, but in the course of construction, only the 100bp version could be finished in time. It was tested to be functional and therefore submitted as P<sub>celA</sub>.
  
 
===Source===
 
===Source===
  
−
- Both sigmaX gene and PcelA promoter were cloned from genomic DNA of Streptococcus pneumoniae NCTC7465 strain<br>
+
iGEM 2014 Hong_Kong_HKUST Team has cloned P<sub>celA</sub> from S. pneumoniae strain NCTC7465 by PCR. Then, restriction-ligation with &sigma;<sup>X</sup> generator [[Part:BBa_K1379006|BBa_K1379006]] and [[Part:BBa_E0240|BBa_E0240]] in [[Part:pSB1C3|pSB1C3]] backbone was performed.
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- Constitutive promoter + RBS (BBa_K880005) was obtained from iGEM distribution kit 2014
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- Double terminator (BBa_B0015) was obtained from iGEM distribution kit 2013
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- GFP generator (BBa_E0240) was obtained from iGEM distribution kit 2014
+
  
 
===References===
 
===References===
 +
Luo P., & Morrison D. (2003).'' Transient Association of an Alternative Sigma Factor, ComX, with RNA Polymerase during the Period of Competence for Genetic Transformation in Streptococcus pneumoniae''. Journal of Bacteriology. doi:10.1128/JB.185.1.349-358.2003
 +
<br><br>
 +
Piotrowski A., Luo P., & Morrison D. (2009). ''Competence for genetic transformation in Streptococcus pneumoniae: termination of activity of the alternative sigma factor ComX is independent of proteolysis of ComX and ComW.'' Journal of Bacteriology. doi:10.1128/JB.01750-08
 +
<br><br>

Latest revision as of 05:00, 12 October 2014

σx Generator + PcelA-E0240


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 359
    Illegal BsaI.rc site found at 1490


Design Notes

Identifying the Possible Promoter Regions

To date (12 Oct 2014), the core/minimal promoter region of PcelA has not yet been experimentally defined. In locating the promoter region required to initiate transcription, iGEM 2014 Hong_Kong_HKUST team attempted in making educated guesses based on relevant information available from the literature.

The procedure adopted was as follow:

The Com-Box promoter consensus sequence “TACGAATA” was BLASTed for targets in the Streptococcus pneumoniae genomes of strains R6, D39, ATCC7699 and NCTC7465 in the NCBI database. Genome of strain NCTC7465 was particularly given attention because its gDNA was available for manipulation. A list of loci with annotated genes returned. σX was known to turn on late competence gene and therefore loci containing any of those genes documented were favored and filtered for. Those loci were then manually checked for consensus among the 4 genomes mentioned above. A sequence of 67 base pairs stood out as a promising target because it was 1) upstream of a late competence genes celA (encodes competence protein CelA), and 2) was identical across the 4 genomes. This 67 bp region has varying upstream sequences and the potential promoter region can reach as far as 200bp. Different truncations (67, 100, 150, 180, 249, 300 bp) were planned for deciding the minimal promoter region, but in the course of construction, only the 100bp version could be finished in time. It was tested to be functional and therefore submitted as PcelA.

Source

iGEM 2014 Hong_Kong_HKUST Team has cloned PcelA from S. pneumoniae strain NCTC7465 by PCR. Then, restriction-ligation with σX generator BBa_K1379006 and BBa_E0240 in pSB1C3 backbone was performed.


References

Luo P., & Morrison D. (2003). Transient Association of an Alternative Sigma Factor, ComX, with RNA Polymerase during the Period of Competence for Genetic Transformation in Streptococcus pneumoniae. Journal of Bacteriology. doi:10.1128/JB.185.1.349-358.2003

Piotrowski A., Luo P., & Morrison D. (2009). Competence for genetic transformation in Streptococcus pneumoniae: termination of activity of the alternative sigma factor ComX is independent of proteolysis of ComX and ComW. Journal of Bacteriology. doi:10.1128/JB.01750-08