Part:BBa_K1031410

HcaR-Terminator

For detailed information concerning HcaR, please visit [http://2013.igem.org/Team:Peking/Project/BioSensors/HcaR#Mileston1]

Introduction

HcaR is a 32,838 Da (296 amino acids) protein, which belongs to LysR family. Its’N-terminal domain functions in DNA binding via a helix-turn-helix motif, while C-terminal domain functions in multimerization. As an activator, HcaR activates the expression of hca cluster when exposed to ligands. It detects limited profile of ligands, including 3-phenylpropionic acid (PPA) and cinnamic acid (CnA) ^[1]. Another gene cluster mhp locates downstream hca cluster. hca and mhp clusters are involved in the catabolism of PPA and CnA in E.coli (Fig.1). The enzymes encoded by hca cluster degrade PPA and CnA to 2,3-DHPPA and 2,3-DHCnA respectively, which serve as the substrates of the mhp cluster. The enzymes in mhp cluster function in the cleavage of aromatic ring.

Fig.1 The degradation pathway of PPA and CnA.

Compared with the sole 2,3-DHPPA, the special induction effect of PPA and 2,3-DHPPA is obtained, although PPA doesn't behave as inducer when alone. Based on the result and the observation of different binding site of PPA with MhpR, it is deduced that PPA and 2,3-DHPPA have synergistic effect to the activation of MhpR expression ^[2]. (That is to say, PPA enhances the activation effect as a cooperator of 2,3-DHPPA instead of a ligand.) The same effect is observed in 3-HPPA along with PPA.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 849
  Illegal AgeI site found at 522
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization of Biosensor

Construction and tunning

Ph/HcaR biosensor circuit is constructed. The coding sequence of HcaR was obtained from the genome of E. coli K12 via PCR. A Pc library of constitutive promoters at different intensities are constructed to fine-tune the biosensor, including BBa_J23113, J23109, J23114 and J23106 (Fig 2).

Fig.2 Construction of biosensor HcaR circuit.The orange arrowheads represent promoters. RBSs are shown as green ovals. Squares in dark red refers to terminators B0015.

ON/OFF test

When tested biosensor adopting J23106 to initiate transcription of HcaR with 78 aromatic compounds following Test Protocol 1[http://2013.igem.org/Team:Peking/Team/Notebook/Protocols], ON/OFF test via microplate reader showed that HcaR worked as a specific sensor to PPA (Fig.3).

Fig 3 Results of On/Off test of biosensor HcaR adopting circuit Ph/J23106-HcaR. HcaR specifically responds to PPA (1000 μM) with the induction ration over 2. Horizontal axis represents different kinds of inducers. Vertical axis stands for induction ratio calculated from fluorescence intensity.

Dose-response curve

PPA was used to test dose-response curve of Ph/J23106-HcaR.(Fig 4)

Fig.4 Dose-response curve for HcaR with constitutive promoter Pc J23106. Horizontal axis represents for PPA at the concentration of 1µM, 5µM, 10µM, 50µM, 100µM, 500µM and 1000µM respectively. Vertical axis stands for induction ratio calculated from fluorescence intensity.

Reference

[1] Díaz, E., Ferrández, A., & García, J. L. (1998). Characterization of the hca Cluster Encoding the Dioxygenolytic Pathway for Initial Catabolism of 3-Phenylpropionic Acid in Escherichia coliK-12. Journal of bacteriology, 180(11), 2915-2923.

[2] Manso, I., Torres, B., Andreu, J. M., Menéndez, M., Rivas, G., Alfonso, C., ... & Galán, B. (2009). 3-Hydroxyphenylpropionate and phenylpropionate are synergistic activators of the MhpR transcriptional regulator from Escherichia coli. Journal of Biological Chemistry, 284(32), 21218-21228.