Difference between revisions of "Part:BBa K1519125"

Revision as of 02:33, 18 October 2014

pBad with araC

This part is identical in sequence (minus one 3' cytosine nucleotide [does not impact function]) to the I0500 part, which is the complete pBAD inducible system. This includes the araC repressor gene and the pBAD promoter in its wild type configuration. Previous teams, including the 2012 Michigan iGEM team, have been unable to use this part or any of its duplicates. They have either been severely mutated or missing entirely from the registry. This part was amplified from the pGLO plasmid, which contains GFP downstream of the I0500 sequence. We have sequenced this part and are confident that this part is correct.

Repression of the pBAD promoter occurs when two araC dimers bind to O2 and I1 operator sites within the araC gene and pBAD promoter respectively, causing a DNA loop. The spacing between these sites is crucial. Inserting or deleting 5bp between these sites abolishes AraC mediated repression of the PBAD promoter.[1] This spacing requirement arises from the double helix nature of DNA, in which a complete turn of the helix is about 10.5 nucleotides. Inserting/deliting 5bp between these operator sites rotates the helix ~180 degrees. This reverses the direction that the O2 operator faces when the DNA is looped and prevents dimerization.

Whereas many pBAD promoters within the registry lack these crucial operator sites and/or correct spacing, this part has all the required operator sites and correct spacing between them (being identical to the native DNA.

1. Schleif R. AraC protein, regulation of the L-arabinose operon in Escherichia coli, and the light switch mechanism of AraC action. FEMS Microbiol Rev (2010) 1–18.

Sequence and Features