Team:Hong Kong-CUHK/PROJECT BACKGROUND

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BACKGROUND

Sensory Rhodopsins

Sensory Rhodopsins (SRs) were well-known in playing a crucial role for the survival of many strains of archaea. They are seven-helix transmembrane receptors, whose structures and functions are similar to human visual pigments [1]. These receptors serve as light sensors that mediate positive and negative phototaxis [1]. When exposed to light with wavelength longer than 520 nm, Sensory Rhodopsin I (SRI) coupled with its transducer protein HtrI is stimulated to mediate positive phototaxis. Another SR, Sensory Rhodopsin II (SRII), couples with its transducer protein HtrII to be stimulated by wavelength shorter than 520 nm for triggering negative phototaxis [1]. These phototatic mechanisms allow archaea to obtain useful light source for ATP generation while prevent near-UV light from causing harm [2].

SRs bind with all-trans retinal, a chromophore which binds in the middle of the seven-transmembrane helix. Upon activation by photons, the trans-cis photoisomerization of the retinal chromophores will be triggered, switching the histidine kinase (CheA) on for negative phototaxis, and off for positive phototaxis. CheA is able to phosphorylate CheY, where phosphorylated CheY is a switch factor for the flagella motor. High level of phosphorylated CheY favours tumbling, whereas a low level favours running motion [3, 4].

HtrI is the transducer protein of SRI and belong to the Methyl-accepting chemotaxis protein-Like Protein (MLP) family, containing HAMP domain mediates signal transduction to flagella motor [8].

HtrII

HtrII is the transducer protein of SRII and belong to the Methyl-accepting chemotaxis protein-Like Protein (MLP) family, containing HAMP domain mediates signal transduction to flagella motor [8].

Tsr and Tar
Tsr and Tar are a methyl-accepting chemotaxis protein found in E. coli, which are responsible for detecting serine and aspartate respectively. [7]. Once triggered, the histidine kinase (CheA) will be regulated, and thus regulating CheY, a switch factor for the flagella motor.

In our CUHK iGEM team this year, we incorporated the SR systems into E. coli. We did it by fusing SR and the transducer part with a flexible linker (GSASNGASA). The transducer part consists of the Htr membrane-proximal cytoplasmic fragment and the cytoplasmic domains of the E. coli chemotaxis receptor Tsr or Tar. Domain determination was done by using the Pfam protein database [5]. By using these principles, we have designed and made three different constructs.

Negative Phototactic construct for blue light detection 
BBa_K786001

SRII was fused with HtrII with a linker peptide, where only the membrane-proximal cytoplasmic domain of the native HtrII was kept, while the cytoplasmic domains were replaced by that of Tsr. Once the fusion protein was triggered, the histidine kinase (CheA) will be up-regulated, leading to a longer tumbling period and achieving negative phototaxis.

Positive Phototactic construct for blue light detection 
BBa_K786002

SRII was fused with HtrII with a linker peptide, where only the membrane-proximal cytoplasmic domain of the native HtrII was kept, while the cytoplasmic domains were replaced by that of Tar. Once the fusion protein was triggered, the histidine kinase (CheA) will be down-regulated, leading to longer running period and achieving positive phototaxis.

Phototactic construct for orange light detection 

BBa_K786003

Red light sensing construct

Other than SRs, we have also explored the possibilities of using other sensory proteins to achieve phototaxis. By using biobrick part BBa_I15010- the Cph8 protein, which consists of two domains, namely the light responsive domain and a EnvZ histidine kinase domain. In the absence of red light, the EnvZ domain will be switched on. As described by Igo et al. [6], the EnvZ/OmpR system and Che systems exhibit cross specificity. EnvZ histidine kinase could alter the level of flagella regulator CheY and thus mediates positive phototaxis.

The gene expression system

BBa_K786010

Co-transformation of BBa_K786010 with BBa_K786001

With the stimulation of blue light, fusion protein SRII-HtrII-Tsr would increase the autophosphorylation of kinase CheA, thus phosphorylating OmpR and activating downstream genes of R0083.

Co-transformation of BBa_K786010 with BBa_K786002

With the stimulation of blue light, fusion protein SRII-HtrII-Tar would switch off CheA and thus suppress gene expression downstream of R0083.

[1] Hoff WD, Jung KH, Spudich JL (1997). Molecular mechanism of photosignaling by archaeal sensory rhodopsins. Annu Rev Biophys Biomol Struct. 26: 223-258.

[2] Spudich JL, Yang CS, Jung KH, Spudich EN (2000). Retinylidene proteins: structures and functions from archaea to humans. Annu Rev Cell Dev Biol. 16: 365-392.

[3] Welch M, Oosawa K, Aizawa S, Eisenbach M (1993). Phosphorylation-dependent binding of a signal molecule to the flagellar switch of bacteria. Proc Natl Acad Sci U S A. 90: 8787-8791.

[4] Barak R, Eisenbach M (1992). Correlation between phosphorylation of the chemotaxis protein CheY and its activity at the flagellar motor. Biochemistry. 31: 1821-1826.

[5] Trivedi VD, Spudich JL (2003). Photostimulation of a sensory rhodopsin II/HtrII/Tsr fusion chimera activates CheA-autophosphorylation and CheY-phosphotransfer in vitro. Biochemistry. 42: 13887-13892.

[6] Igo MM, Ninfa AJ, Stock JB, Silhavy TJ (1989). Phosphorylation and dephosphorylation of a bacterial transcriptional activator by a transmembrane receptor. Genes Dev. 3: 1725-1734.

[7] Sourjik V (2004). Receptor clustering and signal processing in E. coli chemotaxis. Trends Microbiol. 12: 569-576.

[8] Suzuki D, Irieda H, Homma M, Kawagishi I, Sudo Y (2010). Phototactic and chemotactic signal transduction by transmembrane receptors and transducers in microorganisms. Sensors (Basel). 10: 4010-4039.

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