Device

Part:BBa_K786001

Designed by: Leung Wai Tak   Group: iGEM12_Hong_Kong-CUHK   (2012-09-21)

Sensory rhodopsin II (SRII) with HtrII & Tsr, sensitive to blue-green light

Sensory Rhodopsin II have been fused with HtrII with the a linker peptide, only the membrane-proximal cytoplasmic domain have been kept, while the cytoplasmic domains have been replaced by that of Tsr. Once the fusion protein was triggered, the histidine kinase (CheA) will be up regulated, leading to longer tumbling period(Negative phototaxis), sensitive to blue-green light, with sensing spectra covering from 400-500 nm. We used it for sensing blur-green 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 140
    Illegal NgoMIV site found at 398
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1139
    Illegal SapI.rc site found at 1919
    Illegal SapI site found at 913


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].

     Bgg1.png</p>
       

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.

 

Negative Phototactic construct for blue light detection 
BBa_K786001

Bgg4.png

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.

Bgg5.png

 

Biobrick DNA length verification

In order to check the inserted biobrick DNA length, PCR was performed with standard biobrick vector sequencing primers (BBa_G00100 and BBa_G00101) .

1. BBa_K786001

Chara1.png

Reference

[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.

[edit]
Categories
//cds/membrane/receptor
//chassis/prokaryote/ecoli
//classic/signalling/sender
//function/sensor/light
//plasmid/expression
Parameters
device_type
function
protein