Difference between revisions of "Part:BBa K1031510"

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HpaR-Terminator

Characterization

HpaR is of 17,235 Da (149 amino acids) that belongs to MarR family [4]. It performs as a repressor of the hpa cluster consisting of hpaGEDFHI genes (Fig.1), which participates in the catabolic pathway of 4-hydroxyphenylacetic acid (4HPAA) (Fig.2). HpaR derepress the downstream genes when contacting with ligands, including 4HPAA, 3-hydroxyphenylacetic acid (3HPAA) and 3, 4-dihydroxyphenylacetic acid (3,4-DHPAA).

Fig 1 The hpa cluster map in genome. The arrows inside the squares show the transcriptional direction of genes.Pr, Pg, Px, Pa and Pbc, represent the promoter controlling cognate clusters. Addition of 3-hydroxyphenylacetic acid can derepress the promoters inhibited by HpaR (for Pr and Pg activation) or HpaA (for Pbc activation). Finally the enzymes in these clusters will degrade the 3-HPAA to the intermediates in TCA cycle.

hpa cluster consists of three operons. The regulator gene, hpaR, is transcribed in the divert direction to other genes under Pr promoter. The adjacent promoter, Pg, initiates the transcription of the functional hpaGEDFHI operon. Pr and Pg are both regulated by HpaR and located in the intergenic region between the hpaR and hpaG (Fig.1).

Fig 2 The degradation pathway of 3HPAA. The letters on the arrows are the names of genes in the clusters. eg. HpaB,C means the step 1 is catalyzed by the product of hpaB and hpaC.

Sequence and Features

Sequence and Features

Construction of biosensor circuit

Pg/HpaR biosensor circuit is constructed. The coding sequence of HpaR was obtained via PCR from E.coli.(Fig 3).

Fig 3 Schematic diagram of HpaR biosensor circuit. Orange arrowheads represent promoters. RBS is shown as green oval and red square stands for B0015 terminator.