Regulatory

Part:BBa_K174001:Design

Designed by: The Newcastle 2009 iGEM team   Group: iGEM09_Newcastle   (2009-09-26)

215 bp long bindingsite_promoter_bindingsite_RBS 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 wildtype Bacillus subtilis.

Forward primer used: GATCTG-GAATTCGCGGCCGCTTCTAGAG-CACAGCGTTCTTTGTAAG (Clamp sequence - Standard Biobrick prefix - first 18 base from the Biobrick)

Reverse primer used: TGTGAC-ACTAGTA-GCCCTCCCGAATGTTGAG (Clamp sequence - SpeI site - last 18 base from the Biobrick)

Construction

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

Newcastle 2009 ara 1.png

The backbone and the ara insert were then ligated

Newcastle 2009 ara 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.