Difference between revisions of "Part:BBa K1379000:Design"
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===Design Notes===
 
===Design Notes===
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'''Identifying the Possible Promoter Regions'''
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<b>Identifying the Possible Promoter Regions</b>
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Since the sequence of the entire P<sub>celA</sub> length is not yet clearly defined, we had to locate the promoter region based on the relevant information we obtained from a wide range of research papers. Among the many conditions taken into account for identifying the promoter region, we first searched for the consensus sequence “TACGAATA” in the genomic DNA of Streptococcus Pneumoniae of R6, D39, ATCC7699, as well as NCTC7465 strain which the genomic DNA was available particularly in our laboratory. Many of the researches regarding the study of combox promoters were also based on the R6 strain as well. We used the online software Basic Local Alignment Search Tool (BLAST) from NCBI for searching the gene sequences. In the genomic DNA sequences we obtained, many of the genes were identified and highlighted as red or green regions. We hypothesized that the combox promoter would be in the non-highlighted genes as no promoters were highlighted in the gene sequences. Among the list of all the regions of the genomic DNAs reading TACGAATA, we filtered for the regions that were upstream of late competence genes, such as celA. This is because the promoter is believed to be activating the transcription of the late competence genes. Among the many sequences identified, we observed that 4 strains, R6, ATCC7699, D39, and NCTC7465 had a consensus on 67 base-pair sequences that were upstream of late competence genes and were also positioned in between identified genes. We then hypothesized that this region would contain the combox promoter. However, the gene sequence gap between the identified genes containing the 67 base pair sequence varied from 67 to as large as 200. Since we were not sure that the combox promoter would be shorter or longer than the 67 base pair sequence, we decided to experiment different lengths of the combox promoter, to determine the exact length of the combox promoter. In view of this, we planned to make 6 identical constructs (combox promoter-RBS-GFP-Double Terminator) with 6 combox promoters truncated at different lengths (67, 100, 150, 180, 249, 300) in each construct.
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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.  
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The procedure adopted was as follow:
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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===
  
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- P<sub>celA</sub> promoter were cloned from genomic DNA of Streptococcus pneumoniae NCTC7465 strain<br>
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iGEM 2014 Hong_Kong_HKUST Team has cloned P<sub>celA</sub> from S. pneumoniae strain NCTC7465 by PCR, followed by restriction-ligation into [[Part:pSB1C3|pSB1C3]] backbone.
  
 
===References===
 
===References===
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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
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<br><br>
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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
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<br><br>

Latest revision as of 21:23, 11 October 2014

PcelA


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

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, followed by restriction-ligation into pSB1C3 backbone.

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