Part:BBa_K343003
NpSopII-NpHtrII-StTar (M-fusion)
Sensory Rhodopsin II bluelight receptor fused to its transducer, HtrII, with a 27 BP linker region. This is fused to Salmonella enterica serovar typhimurium chemotaxis protein Tar, so that the protein effectively couples the input from the receptor to the chemotaxis pathway. The fusion between HtrII and Tsr is an M-fusion in the HAMP domain, which is supposed to give it maximum activity. Sequencing confirmed that the sequence is identical to the found in the article by Jung, Spudich E, Trivedi and Spudich J in the article: An Archaeal Photosignal-Transducing Module Mediates Phototaxis in Escherichia coli. (1)
Usage and Biology
When exposed to bluelight, the sensory rhodopsin II will undergo a change in ultrastructure, which is being transduced through HtrII on to the Tar domain. This influences the cell's normal chemotaxis pathway, so that it will decrease the amount of phosphorylated CheY and thereby decrease the tumbling frequency of the cell.
The part requires retinal to work in E.Coli. This can be achieved through adding retinal to the liquid growth medium and/or the plates. Currently we are doing experiments on wether the part also functions with an internal retinal source, ie retinal synthesis in E. Coli.
Results: Bacteria containing this part will exhibit a lowered tumbling rate when exposed to blue light (wavelengths around 350nm - 450nm). This was analysed with the help of video microscopy and the open source software [http://db.cse.ohio-state.edu/CellTrack/ "CellTrack"]. The individual cells trajectory was tracked and their speed measured. The tracking results are as follows:
(Blue dots show the location of the cell in the given frame, so the number of dots equals the number of frames from the sample.)
Trajectory of E.Coli containing the photosensor, when exposed to blue light:
Trajectory of E.Coli containing the photosensor, when exposed to red light:
Trajectory of E.Coli Mg1655, when exposed to blue light:
The phototaxic bacteria move more in a straight line when exposed to bluelight, as can be seen when comparing the trajectories of the thee bacteria given earlier. These were taken from a batch of 10 cells tracked per sample.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 1783
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 73
Illegal NgoMIV site found at 331
Illegal AgeI site found at 1585 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1027
Illegal BsaI.rc site found at 1300
Illegal SapI site found at 801
Illegal SapI.rc site found at 1801
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