Part:BBa_K1031410

HcaR-Terminator

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

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.

Figure 1. The ph promoter and the degradation pathway carried out by the hca gene cluster. (a) The ph is a σ70-dependent promoter. The HcaR protein will bind to the DNA operator centered at -40 when the aromatic inducer are present; it will subsequently recruit the RNAP and initiate transcription. (b) The enzymes that catalyze each step of the pathway are indicated; PPA and CnA will finally be degraded into 2,3-DHPPA and 2,3-DHCnA, respectively.

The cognate promoter of HcaR, ph, is quite regular: it is σ70-dependent and functions via contacting the α-unit of RNAP. The presence of aromatic effectors will cause the HcaR to dimerize and to bind to sequence-specific DNA operator in the ph promoter (Fig. 1a).

According to these properties of HcaR, we could design an HcaR biosensor that is supposed to detect 3-phenylpropionic acid, cinnamic acid and their derivatives. It aromatics-sensing profile is quite narrow, supposed to be 3-phenylpropionic acid (PPA) and cinnamic acid (CnA) only, thus to guarantee the detection specificity of the biosensor.

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).

Figure.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.

Based on the design frame of biosensors we've discussed in the Biosensor Introduction section, we constructed a HcaR biosensor using Ph/HcaR pair obtained from the genome of E. coli strain K12. The constitutive promoter (Pc) to control the expression of HcaR is BBa_J23106 and the RBS preceding sfGFP is BBa_B0034.

This primary construct, however, did not work (Fig. 3). Therefore, we used a library of combinations of Pc promoters and RBS sequences to tune the performance of HcaR biosensor. Experimental measurement using Test Protocol 1 showed that HcaR performed the best using the Pc promoter BBa_J23106 and RBS BBa_B0032 (Fig. 3).

The best HcaR biosensor was then subjected to the ON/OFF Test using overall 78 aromatics. Results showed that the HcaR biosensor worked as a specific sensor for PPA (CnA is not an aromatic compound, thus not taken into consideration) (Fig.4).

Pc and RBS Library

Figure 3. RBS and Pc constitutive promoter library for HcaR biosensor. X-axis stands for different construction of biosensor HcaR. Y-axis denotes induction ratios. The HcaR biosensor with J23106 is a strong constitutive promoter, and B0032 is a weak RBS, and this construction performed well, which showed the induction ratio higher than 25 folds.

ON/OFF test

When tested HcaR biosensor adopting Pc J23106 and RBS B0032 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.4).

Figure 4. ON/OFF Test to evaluate the induction ratios of all 78 aromatic compounds in the aromatics spectrum. (For the detailed information about the 78 compounds, Click Here ). (a) The induction ratios of various aromatic species. HcaR could respond to only 1 out of 78 aromatics (PPA, 1000 μM) with the induction ratio higher than 25. (b) The aromatics-sensing profile of HcaR biosensor.The aromatic species that can elicit strong responses of NahR biosensor is highlighted in purple in the aromatics spectrum. The structural formula of PPA is also listed. The induction ratio was calculated by dividing the fluorescence intensity of biosensor exposed to object inducers by the basal fluorescence intensity of the biosensor itself.

Dose Response

Furthermore, the dose-response curves of optimized HcaR biosensor (J23106-B0032) was experimentally measured using gradient concentrations of inducers ranging from 10 μM to 1 mM followingTest Protocol 1 (Fig. 5). 30-fold induction can be obtained using PPA even at micro-molar concentration. Notably, the HcaR biosensor specifically gives response to PPA, making it a robust and convenient biosensor for the presence of PPA in water.

Figure 5. Dose-response curves of HcaR biosensor induced by PPA. The optimized version of HcaR biosensor (J23106-B0032) exhibited an induction ratio higher than 25. The HcaR biosensor in the original version (J23106-B0034) was also tested as a control to show the necessity of our fine-tuning. The induction ratio was calculated by dividing the fluorescence intensity of biosensor exposed to object inducers by the basal fluorescence intensity of the biosensor itself.

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] Ferrández, A., García, J. L., & Díaz, E. (1997). Genetic characterization and expression in heterologous hosts of the 3-(3-hydroxyphenyl) propionate catabolic pathway of Escherichia coli K-12. Journal of bacteriology, 179(8), 2573-2581.

[3] 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.