Difference between revisions of "Part:BBa K174000:Design"
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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.
 

Revision as of 22:22, 26 September 2009

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

Forward primer used: GATCTG-GAATTCGCGGCCGCTTCTAG-ATGGATTTGTCACAGCTAAC (Clamp sequence - Standard Biobrick prefix - first 20 base from the Biobrick)

Reverse primer used: TGTGAC-ACTAGTA-TTACTTCACAACGCGTAATGC (Clamp sequence - SpeI site - last 21 base from the Biobrick)

Construction

Plasmid part BBa_J04450 (pSB1AT3) and our construct were treated with EcoRI and SpeI

Newcastle 2009 sspB 1.png

The backbone and the sspB insert were then ligated

Newcastle 2009 sspB 2.png