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| − | 498 bp long sspB CDS is amplified by PCR with dangling end primers with EcoRI-NotI-XbaI restriction site at 5’ and SpeI at 3’ and inserted into a Biobrick compatible vector. The sequence is taken from E. coli strain DH5 alpha. Genbank accession number for E. coli MG1655 strain is NC_000913.2
| + | The sequence is taken from E. coli strain DH5 alpha. Genbank accession number for E. coli MG1655 strain is NC_000913.2 |
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| − | Forward primer used: GATCTG-GAATTCGCGGCCGCTTCTAG-ATGGATTTGTCACAGCTAAC (Clamp sequence - Standard Biobrick prefix - first 20 base from the Biobrick)
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| − | Reverse primer used: TGTGAC-ACTAGTA-TTACTTCACAACGCGTAATGC (Clamp sequence - SpeI site - last 21 base from the Biobrick)
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| | ==Construction== | | ==Construction== |
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| | [[Image:Newcastle_2009_sspB_2.png]] | | [[Image:Newcastle_2009_sspB_2.png]] |
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| − | ==Spacing between the RBS and downstream CDSs==
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| − | To make the RBS efficient, 7 bases are needed between the RBS and CDS. The scar between the biobricks will already have the 6 bases and we left one base after the rbs to total it to 7 bases.
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Latest revision as of 22:36, 21 October 2009
The sequence is taken from E. coli strain DH5 alpha. Genbank accession number for E. coli MG1655 strain is NC_000913.2
Construction
Plasmid part BBa_J04450 (pSB1AT3) and our construct were treated with EcoRI and SpeI
The backbone and the sspB insert were then ligated
