Part:BBa_K422001
Archeal light receptor fused to bacterial chemotaxis transducer
Biological Background
Fusion of the Natronobacterium pharaonis Np seven-transmembrane retinylidene photoreceptor sensory rhodopsins II NpSRII and their cognate transducer HtrII to the cytoplasmic domain of the chemotaxis transducer EcTsr of Escherichia coli [1].
Rhodopsins are photoreactive, membrane-embedded proteins, which are found not only in archaea, but in eubacteria and microbes as well. In Natronobacterium pharaonis, the NpSRII contains a domain of seven membrane-spanning helices, which carry out two distinct functions: Firstly, they serve as photo-inducible ion-pumps and secondly, as actors in the chemotaxis signaling network [1].
All-trans retinal is needed for NpSRII to change it's conformation into an active light absorbing pigment. It can either be added to the growth media or produced by the organism. Phototactic stimuli can be delivered through a light pulse at 500 nm.
Design
De novo Synthesis by GeneArt.
Codon optimized.
Characterization
Chemotaxis assay
To observe chemotactic behaviour, cells were grown at 30 °C in Lysogeny Broth to on OD of 1.0. All-trans retinal has to be added to the media for NpSRII to change it's conformation into an active light absorbing pigment. The Archeal Receptor was subloned under the control of an IPTG inducible promoter. Phototactic stimuli were delivered through a light pulse at 500 nm and cells tracked.
The video shows the iGEM 2010 project of the ETH The E. lemming. The core idea was to control the swimming of E. coli cells by repressing or inducing tumbling by applying short blue light pulses. This movie shows a short sequence of the E. lemming as it changes its swimming behavior when sensing changes in the blue light signals. Please note that the natural adaptation system of E. coli downstream of the light receptor is active in this mutant, such that the swimming behavior only changes directly after the blue light is switched on or off, but is not necessarily different between long periods of light on or off. All cells but the E. lemming are visualized with a blue glowing. The E. lemming is glowing yellow, with its current movement direction visualized with a yellow light cone. The transfected E. coli cells were grown until a cell density of around 1.0 OD at 30 °C and imaged with a 20x light lense in a approximately 50 μm high flow channel. For more information please visit the ETH iGem 2010 team's wiki page at http://2010.igem.org/Team:ETHZ_Basel .
Angle change
The angle of the E. lemming during that measurement (see Video 1) as calculated from the central differences of its positions is depicted in figure 1. The estimated reaction times between the switching of the blue light and the reactions of the E. lemming are marked in the image. For the reaction delay between switch-on of the light and straight swimming, we obtained Δt1≈2.1s and Δt2≈3.0s. For the delay between the switch-off of the blue light and start of tumbling it was only possible to estimate the time delay for the second light pulse, Δt3≈2.4s. To characterize the change of swimming behavior when switching on or off the blue light signal, we estimated the angle of the E. lemming for each frame of Video 1. This was done by obtaining the positions (xi, yi) of the E. lemming from our cell detection and tracking algorithm. The angle φi of frame i was then calculated by central differences: tan(φi)=(yi+1-yi-1)/(xi+1-xi-1).
When plotting the angle over time (see Figure 1), one observes that during white light periods the angle is increasing with a nearly constant angular speed of about 27° per second (&asymp8° per frame). When switching on blue light, the angular speed decreases to nearly zero for several seconds after a delay between 2 and 3s.
For the first light pulse this decrease of angular speed lasted for about 10s until the return to pre-blue light behavior, for the second light pulse this effect only ended after the blue light was switched off again. In the latter, normal swimming behavior re-established after a delay of approximately 2.4s, which is nearly the same delay as the delay when switching the light on. Please note that the natural adaptation system of the chemotaxis pathway downstream of the light receptor is active in this mutant, such that the swimming behavior only changes directly after the blue light is switched on or off, but is not necessarily different between long periods of light on or off.
We furthermore noticed that the E. lemming seems to have the tendency to show a bigger tumble right before starting swimming straight when the blue light is switched on. However, if this behavior occurred by chance or if this is a general property of the E. lemming was yet not possible to show.
References
[1] Jung, Spudich, Trivedi and Spudich: An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli. Journal of bacteriology. 2001; 21.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 747
Illegal AgeI site found at 840 - 1000COMPATIBLE WITH RFC[1000]
| excitation | 500 nm |