Device

Part:BBa_K317028:Experience

Designed by: Yoshiyuki MURAKAMI   Group: iGEM10_Tokyo-NoKoGen   (2010-10-20)

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Applications of BBa_K317028

Experience

User Reviews

UNIQbe5424a51301bb30-partinfo-00000000-QINU This biobrick has been improved to BBa_K786002

Improvement

I. Biobrick Construction The Sensory Rhodopsin system works in E.coli by fusing SR and Htr with a flexible linker and joint the HtrII membrane-proximal cytoplasmic fragment with the cytoplasmic domain (Methyl-accepting chemotaxis protein (MCP) signalling domain) of the eubacterial chemotaxis receptor.[1] The fusion protein in BBa_K317028 and BBa_K317003 used the Tar gene from Synechocystis.typhimurium, have their construct design based on [2]. The previous study made three different junction constructs (P, G, M) based on studies of dimerization, yet no accurate tool was used for domain determination. Therefore, we made the following improvements.

1. Tar gene from E.coli K12 strain was used instead of that of S.typhimurium, as we believe E.coli can express and function more properly with its native genes and proteins. 2. We designed the construct by using an accurate protein domain determining tool [3], the whole E.coli Tar protein consist of three domains- Tar ligand binding domain (amino acid sequence 1-175), the HAMP domain (amino acid sequence 194-263) and the Methyl-accepting chemotaxis protein (MCP) signalling domain (amino acid sequence 264-553). As only the cytoplasmic domain is needed, the exact Methyl-accepting chemotaxis protein (MCP) signalling domain (amino acid sequence 264-553 of EcTar) was cut and fused with membrane-proximal cytoplasmic fragment part of HtrII (amino acid sequence 1- 133). 3. Restriction sites of HindIII and BamHI were added before and after the SRII gene respectively for enabling switching the sensory rhodopsin portion of the fusion protein. According to previous study. [2] A series of mutant sensory rhodopsins have been identified which covers a large variation of absorbing spectrum. These two restriction sites allow further switching of the sensing unit, so the light sensing system can be tuned for sensing different kinds of light source. Further integration of other peptides (e.g.: His-tag), for the construction of a larger fusion protein is also possible. (HindIII for N-terminal while, BamHI for C-terminal)

The improvements made were successful as the function (sense light for cell movement) of the above fusion proteins were tested with positive results.


II. Method of measurement According to previous studies on positive phototactic microorganisms, colonies of microorganisms should spread towards the light source. [4]

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We have received similar result for our improved SRII-HtrII-Tar positive phototactic device.

As our cells receive stimulation of blue light from all directions instead of unidirectional as what the paper used, therefore, they spread out in all directions after 12 hr exposure of light. Average diameters were measured by a electronic ruler with precision = ± 0.01 mm. Data of diameters from at least three independent clones were collected. Paired t-test was used to analyze the collected data. A significant difference was observed between the plates (*** indicates p < 0.001). The average diameters of three clones exposed under blue light are bigger than the counterparts put in dark. Blue light triggers a change in diameter of 180 ± 40 %, while there is no significant change in diameter (<10 % change) for those without our biobrick BBa_K786002.



[1] 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. [2] Jung KH, Spudich EN, Trivedi VD, Spudich JL (2001). An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli. J Bacteriol. 183: 6365-6371. [3] Finn RD, Mistry J, Tate J, et al. (2010). The Pfam protein families database. Nucleic Acids Res. 38: D211-222. [4] Masuda S, Ono TA (2004). Biochemical characterization of the major adenylyl cyclase, Cya1, in the cyanobacterium Synechocystis sp. PCC 6803. FEBS Lett. 577: 255-258.

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