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'''<br>
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'''Identifying the Possible Promoter Regions'''
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<br>
 
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.
 
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.
  

Revision as of 12:37, 9 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
Since the sequence of the entire PcelA 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.

Source

- PcelA promoter were cloned from genomic DNA of Streptococcus pneumoniae NCTC7465 strain

References