Difference between revisions of "Part:BBa K1031710"

Revision as of 16:30, 26 September 2013

PaaX-Terminator

For detailed information concerning PaaX, please visit 2013 Peking iGEM Biosensor PaaX

Characterization

PaaX is a repressor of 316-amino acid. As a member of GntR family, it contains a stretch of 25 residues that is similar with the helix-turn-helix motif functioning in DNA recognition and binding. PaaX contacts with palindrome sequence located at its cognate promoter, Pa, inhibiting the promoter at the absence of the ligand. Unlike other sensors in E. coli, PaaX detects phenylacetic acid-CoA (PA-CoA), which is the first intermediate in the PA degradation pathway. The first step is catalyzed by PaaK. There are three operons in paa clusters, paaZ, paaABCDEFGHIJK and paaXY. (Fig.1) The promoters regulated by PaaX, Pz and Pa, are located at the intergenic region between paaZ and paaA. They possess a palindromic sequence respectively for binding to the repressor.

Fig 1 Structure of the paa cluster. The arrows indicate the direction of transcription of each gene. Pz, Pa, Px, the promoters controlling cognate clusters. 3-hydroxyphenylacetic will derepress the promoters, Pz and Pa, repressed by PaaX. Enzymes coded by the operons catalyze the degradation of PAA to intermediates in TCA cycle.

Sequence and Features

Sequence and Features

Construction of biosensor circuit

Pa/PaaX biosensor circuit is constructed (Fig 2). The coding sequence of PaaX was obtained via PCR from E.coli.

Fig 2 Schematic diagram of PaaX biosensor circuit. Orange arrowheads represent promoters. RBS is shown as green oval and red square stands for B0015 terminator.