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 22:21, 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


Spacing between the RBS and downstream CDSs

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.