Part:BBa_K786003

Sensory rhodopsin I (SRI) with HtrII & Tar, sensitive to orange light

Sensory Rhodopsin I have been fused with HtrI with the a linker peptide, only the membrane-proximal cytoplasmic domain have been kept, while the cytoplasmic domains have been replaced by that of Tar. Once the fusion protein was triggered, the histidine kinase (CheA) will be regulated, leading to phototactic effect, which is sensitive to orange light, with sensing spectra covering from 560-600 nm. We used it for sensing orange light to achieve phototaxis and switch for downstream gene expression.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 37
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal SpeI site found at 37
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 785
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 37
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 37
  Illegal NgoMIV site found at 614
  Illegal NgoMIV site found at 638
  Illegal NgoMIV site found at 742
  Illegal NgoMIV site found at 875
  Illegal AgeI site found at 144
  Illegal AgeI site found at 1616
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 977
  

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 are 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].

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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
Tar are a methyl-accepting chemotaxis protein found in E. coli, which is responsible for detecting aspartate. [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 HtrII 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 this constructs.

 

Phototactic construct for orange light detection 

BBa_K786003

SRI was fused with HtrI with a linker peptide, where only the membrane-proximal cytoplasmic domain of the native HtrI was kept, while the cytoplasmic domains were replaced by that of Tar. Once the fusion protein was triggered, the histidine kinase (CheA) will be regulated, leading to phototactic effect.

Reference

<p>[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.