Team:Hong Kong-CUHK/PROJECT BACKGROUND
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<p style="font-size:16px"><strong>The gene expression system</strong></p> | <p style="font-size:16px"><strong>The gene expression system</strong></p> | ||
<p><center><img src="https://static.igem.org/mediawiki/2012/4/40/Bg11.png" width="324" height="436" style="margin:15px" /></center>Other than controlling cellular movement by SR, we have also designed a gene expression system induced by SRs. <br /> | <p><center><img src="https://static.igem.org/mediawiki/2012/4/40/Bg11.png" width="324" height="436" style="margin:15px" /></center>Other than controlling cellular movement by SR, we have also designed a gene expression system induced by SRs. <br /> | ||
- | As explained previously, after light stimulation, SRs can control the autophosphorylation activity of CheA. While CheA can phosphorylate OmpR and phosphorylated OmpR in turn stimulates transcription from the otnpF promoter (part R0083) [6], we therefore built the construct BBa_K786010. Together with SR sensory systems, gene expression downstream of otnpF promoter can be controlled by different light source. </p> | + | As explained previously, after light stimulation, SRs can control the autophosphorylation activity of CheA. While CheA can phosphorylate OmpR and phosphorylated OmpR in turn stimulates transcription from the otnpF promoter (part R0083) [6], we therefore built the construct BBa_K786010. Together with SR sensory systems, gene expression downstream of otnpF promoter can be controlled by different light source. (Must be used in E.coli deficient in EnvZ.)</p> |
<p> </p> | <p> </p> | ||
<p>BBa_K786010</p> | <p>BBa_K786010</p> |
Revision as of 07:59, 26 September 2012
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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 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]. ![]()
HtrII
Tsr
Negative 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 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
![]() 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.
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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. ![]() 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 ![]() As explained previously, after light stimulation, SRs can control the autophosphorylation activity of CheA. While CheA can phosphorylate OmpR and phosphorylated OmpR in turn stimulates transcription from the otnpF promoter (part R0083) [6], we therefore built the construct BBa_K786010. Together with SR sensory systems, gene expression downstream of otnpF promoter can be controlled by different light source. (Must be used in E.coli deficient in EnvZ.)
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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