Team:Tokyo-NoKoGen/rhodopsin

From 2013.igem.org

Team:Tokyo-NoKoGen - 2013.igem.org



Improving a BioBrick part-Rhodopsin







Sensory Rhodopsin fused with EnvZ histidine kinase (BBa_K769000)

From Tokyo-NoKoGen 2012 http://2012.igem.org/Team:Tokyo-NoKoGen



Last year's team member ofTokyo-NoKoGen2012 have been working on developing a light sensor by combining the sensory domain of rhodospin derived from N. pharaonis, and the histidine kinase domain of EnvZ from Escherichia coli.


Halophilic archaea, such as Halobacterium salinarum and Natronobacterium pharaonis (N. pharaonis) are known to show phototaxis by the illumination of light by using receptors called sensory rhodopsin I and II (SRI and SRII). The SR proteins are seven-transmembrane retinylidene photoreceptors, which transmits blue light signal (λmax 487 nm) to their corresponding transducers HtrI and HtrII respectively. This two-component system regulates cells’ flagellar motors for phototaxis (Ref. 1, 2).



Fig.1 Sensory rhodopsin II (SRII) (orange) from N. pharaonis, fused to cognate transducer protein HtrII (green) by a 9 amino acid linker, fused to E. coli chemotaxis receptor Tsr (blue) in the cytoplasmic region. (Ref.4)




It has been previously reported that chimeric rhodopsin can be constructed inside E. coli. Kwang-Hwan et al. constructed chimeric transducer proteins, by exchanging the Histidine-kinase domain of the bacteriorhodopsin from N. pharaonis with homologous chemotaxis transducers Tsr and Tar of E. coli and Salmonella enterica, (chemotaxis transducers of serine and aspartate respectively).They observed a that E. coli succesfully responded to light (Ref.3). It was also previously reported, that the sensory domain of Tar protein fused with histidine kinase domain of EnvZ protein of E. coli, functioned as a sensor protein to transmit signal received by the Tar sensory domain to the EnvZ two-component system (Ref.5). The histidine kinase domain of Tar protein and EnvZ protein are homologous. So last year, Tokyo-NoKoGen 2012 have deduced that sensory domain of sensory rhodopsin fused with EnvZ protein should also function to transmit signal downstream of the two-component system.






Fig.2 Construct from Tokyo-NoKogen 2012. EnvZ hisidine kinase domain was obtained from cph8, cut at NdeI and SpeI and fused together.


We experssed the following construct inside E. coli (ΔEnvZ) and measured GFP fluorescence.



This is the result.


Fig.5 Comparison of normalized GFPuv fluorescence under white light condition and dark condition.
Sensory rhodopsin : E.coli MG1655(ΔEnvZ)/pSB1C3-Pconst.-SRII-HtrII-EnvZ-PompC-GFP
Negative control : E.coli MG1655(ΔEnvZ)/pSB1A3-PompC-GFP
Positive control : E.coli BL21(DE3)/pSB1A3-PompC-GFP




As you can see from the result, we obtained a sensory rhodopsin fused by EnvZ that enhanced the down stream expression in the dark and repressed expression under light. For this year's project, we wanted to combine this light sensor with our Hammerhead ribozyme (HHR), so we decided to construct our genetic device. But however, during the process we realized that the downstream element in EnvZ, about 30 bp before the stop codon of EnvZ, was missing in BBa_K769000. Member from last year did not realize this because they were using the sequence of cph8 for sequence analysis, and did not search against the whole EnvZ sequence. We realized this mid-September, so we quickly re-constructed our sensory-rhodopin fused with EnvZ by designing the following primers:



The obtained PCR product was ligated into pSB1A3. Pconst(J23100) and RBS(B0034) were added infront of our new sensory rhodopin fused with EnvZ, and double terminator was added (B0015). This plasmid, together with pSB1C3-PompC-RBS-GFP-doubleTerm. was used to transform E. coli (ΔEnvZ). The colony was confirmed by colony PCR, and was pre-cultured for 12 hours. They were then inoculated into 2 mL LB medium. Half of the samples prepared were covered by aluminium foil and the other half were subjected to light for 12 hours.

After 12 hours of culturing under light or wrapped in aluminium foil, the OD at 595 nm and GFP fluorescence was measured. The result is shown below:


Fig.4 Graph showing the result of light illuminated or non-illuminated culture. E. coli (ΔEnvZ) harboring two plasmids: pSB1A3-Pconst(H)-RBS-rhodopsinEnvZ-doubleTerm and pSB1C3-PompC-RBS-GFP-doubleTerm.




As you can see, under the light sensoryrhodopsin-EnvZ enhanced GFP expression and under the dark, it repressed GFP expression. This is a light sensor that can enhance gene expression under light suppress it under dark. However, in practice we do not want high background, meaning that we do not want gene expression leakage in the dark.

The graph below shows GFP expression comparison with E. coli (ΔEnvZ) harboring ONLY pSB1C3-PompC-RBS-GFP-doubleTerm, without sensory rhodopsin.


Fig.5 Graph showing the result of E. coli (ΔEnvZ) harboring pSB1A3-Pconst(H)-RBS-rhodopsinEnvZ-doubleTerm and pSB1C3-PompC-RBS-GFP-doubleTerm under light or dark, and E. coli (ΔEnvZ) harboring only pSB1C3-PompC-RBS-GFP-doubleTerm.



E. coli (ΔEnvZ) harboring only pSB1C3-PompC-RBS-GFP-doubleTerm is showing a very high background, which is an indication that probably EnvZ histidine kinase is activated by OmpR from another factor inside the cell. If we want to try and strictly control the gene expression, we might have to use E. coli (ΔEnvZ) with OmpR delted mutation.




Reference

[1] Xue-Nong Zhang et al. (1999) The specificity of interaction of archaeal transducers with their cognate sensory rhodopsins is determined by their transmembrane helices, Proc. Natl. Acad. Sci. USA
[2] Wouter D. Hoff et al. (1997) Molecular mechanism of photosignaling by archaeal sensory rhodopsin, Anmu. Rev. Biophys. Biomol. Struct.
[3] Kwang-Hwan Jung et al. (2001) An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli, Journal of bacteriology
[4] Vishwa D. et al. (2003) Photostimulation of a sensory rhodopsin II/HtrII/Tsr fusion chimera activates CheA-autophosphorylation and CheY-phototransfer in vitro, Biochemistry
[5] Yoshida T et al., (2007) The design and development of Tar-EnvZ chimeric receptors, Methods Enzymol.