Sensory rhodopsin II (SRII) with HtrII & Tar, 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the a linker peptide, only the membrane-proximal cytoplasmic domain have been kept, while the cytoplasmic domains have been replaced by that of Tar. Once the fusion protein was triggered, the histidine kinase (CheA) will be down regulated, leading to longer running period(Positive 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
- 10Illegal SpeI site found at 37
- 12Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal SpeI site found at 37
- 21Illegal BamHI site found at 785
- 23Illegal SpeI site found at 37
- 25Illegal SpeI site found at 37
Illegal NgoMIV site found at 140
Illegal NgoMIV site found at 398
Illegal AgeI site found at 1703
- 1000Illegal BsaI.rc site found at 1139
Illegal SapI site found at 913
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 . These receptors serve as light sensors that mediate positive and negative phototaxis . 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. These phototatic mechanisms allow archaea to obtain useful light source for ATP generation while prevent near-UV light from causing harm .
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].
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 .
Tsr and Tar
Tar are a methyl-accepting chemotaxis protein found in E. coli, which is responsible for detecting aspartate. . 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 . By using these principles, we have designed this constructs.
Positive Phototactic construct for blue light detection
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.
Biobrick DNA length verification
The inserted biobrick DNA length has been checked by PCR with standard biobrick vector sequencing primers (BBa_G00100 and BBa_G00101)
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 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.
 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.
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