Difference between revisions of "Part:BBa K343003"

Revision as of 14:34, 16 October 2010

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 and reduces the amount of phosphorylated CheY, which results in a lowered tumbling frequency. 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)

Background

The mechanism of bacterial motility

Phototaxis is naturally not present in E.Coli, so that the wildtype does not change it's motility in response to light. The bacteria do move in response to different chemical gradients, so called chemotaxis. By this ability the cell can choose whether it wants tu "run" or "tumble", which are the 2 modes of bacterial propulsion known. When running the bacteria just go in a straight line. When the bacteria tumble they will randomly reorientate themselves, until the mode is switched back to run and they run off into another direction. The cells use these mechanisms so that they will increase the rate of tumbling in an unfavorable environment, so that they tumble a lot and thereby get away from the unfavorable environment. In a favorable environment the bacteria will reduce their tumbling frequency so as they won't leave the good environment they are in at the moment.

The bacterial propulsion works with the help of flagellae, small bacterial motors that rotate. The 6 - 8 flagella of E. Coli bacteria will rotate counter clockwise when being in "run-mode" and three of them will bundle up to create one big flagellum. This results in a smooth-straight line pattern of movement. When the bacteria tumble the flagellae rotate clockwise, which breaks the bigger bundle up and makes the flagella whip around randomly instead of their normal rotation.

Molecular mechanism of the photosensor

The fusion,chimera-protein coupled to the chemotaxis pathway. Figure taken from Trivedi et al.(2)

The photosensor acts directly on the tumbling frequency by effecting E. Coli's normal chemotaxis pathway. When exposed to bluelight, the sensory rhodopsin II will absorb the photons and undergo a change in ultrastructure, which is being transduced through HtrII on to the Tar domain. This effects decreases the autophosphorylation of the CheA protein, which in turn again decreases the amount of phosphorylated CheY, which just means that less of it will get phosphorylated. If there is less of the CheY-P, then there is a smaller chance of one of these molecules binding to the flagellar motor and making it turn clockwise, thereby inducing a lowered tumbling frequency in the system. The photosensor can also act in the opposite way, inducing a higher tumbling rate in the bacteria. Which effect the sensor will have depends on where NpHtrII and StTar are fused in the HAMP domain. If the fusion contains 20 more basepair of the HtrII domain and 20 less of the Tar domain, the photosensor would have the opposite effect and would be increasing the autophosphorylation of CheA.

Usage and parameters

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. On top of adding retinal the cells will have to be grown in the dark for at least two hours after the addition of retinal to the growth medium if you want to see an effect. This is so that the photosensor is not exposed to light before the experiments and will result in maximum output if exposed to blue light.

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:

From left to right, trajectory of: E.Coli with photosensor exposed to blue light, E.Coli with photosensor exposed to red light and E.Coli Mg1655 Wildtype exposed to blue light: (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.)

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