Difference between revisions of "Part:BBa K1379000:Design"
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===Design Notes=== | ===Design Notes=== | ||
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<b>Identifying the Possible Promoter Regions</b> | <b>Identifying the Possible Promoter Regions</b> | ||
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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. | 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. | ||
Revision as of 21:17, 11 October 2014
PcelA
- 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
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.