Difference between revisions of "Part:BBa K422001"

Revision as of 20:42, 31 October 2010

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

In order to characterize the archeal light receptor fusion, BBa_K422001 was cloned into plasmid pACT3 (ori p15A, chmloramphenicol resistance) under the control of an IPTG-inducable tac promotor. This construct will be referred to as pACT3-BBa_K422001. For cloning and plasmid details visit the ETH iGEM 2010 team's wiki page at http://2010.igem.org/Team:ETHZ_Basel.

Testing expression and integration of the archeal light receptor fusion into the outer membrane

Figure 1: Membrane extraction after expression of the archeal light receptor fusion. A: Suspension of cells after expressing the archeal light receptor (left) or of control cells (right), harbouring the empty vector. cells were washed twice with 50 mM Tris-HCl of cells. B: Sedimented membranes of cells after expressing the archeal light receptor (left) or of control cells (right).C: Resuspended membranes that were used for OD500 measurements. Left: Cells after expressing the archeal light receptor. Right: control cells.

All-trans retinal binds via Schiff-base formation to a lysin residue in the extracellular part of the archeal light receptor to form the actual light absorbing chromophore. If the archeal light receptir is expressed and integrated properly into the outer membrane of E. coli, all-trnas retinal present in the growth medium can bind to the receptor. Membranes extracted from these cells should thus show an increase in light absorbance at 500 nm when compared to membranes, extracted from cells that have not been expressing the archeal light receptor.

Cells harboring pACT3-BBa_K422001 and cells harboring only the empty vector pACT3 were therefore grown in 1 L of LB media at 30 °C. Expression was induced with 1 mM IPTG and 20 uM all-trans retinal at an OD600 of 0.3. All-trans retinal was also added to the control cells only harboring the empty vector pACT3. After 3.5 hours of expression cells were harvested by centrifugation for 20 min at 3000 x g and 4 °C. The cell pellet was washed twice with 50 ml 50 mM Tris-HCl, pH 8 before the cells were disrupted in a fast fluid homogenizer. Residual intact cells and cell debris were spinned down at 10 000 x g for 10 min. Membranes were then sedimented at 100 000 x g and 4 °C for 1 hour. Membranes were washed once with 1 M NaCl in 50 mM Tris-HCl, pH 8. For OD measurement membranes were resuspended in 100 mM NaCl in 50 mM Tris-HCl, pH 8. The total amount of protein present in the membrane suspension was determined by a general Bradford assay and proteins amounts were adjusted to 0.15 mg/ml.

Figure 2: Absorption of membranes at 500 nm. Absorption at 500 nm of membranes extracted from cells after expressing the archeal light receptor or from control cells harboring the empty vector.

Already during membrane preparation, the difference in the binding of all-trans retinal to the bacterial membrane between cells expressing the archeal light receptor and control cells, harboring the empty vector gets visible (figure 1). Membranes binding all-trans retinal due to receptor insertion get deep red. The increase in absorption of light at 500 nm of membranes extracted from cells after expressing the archeal light receptor is a further indicator for the expression and proper insertion of our light receptor fusion into the outer mebrane of E. coli.

Chemotaxis assay

Video 1: E. lemming in action.
The unprocessed microscope images are available here.

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. 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 .

Figure 3: Angles of the E. lemming in video 1. White background: blue light off. Light blue background: blue light on.

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 3. 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