Team:KULeuven/Discussion

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Sound/vibration sensor system for E.coli

--Erwinswinnen 10:06, 8 June 2011 (CDT)

Presence of mechanosensors MscL/MscS in E.coli: could they function as sensors?

Jonas: general info on mechanosensitive channels (like MscL etc): http://en.wikipedia.org/wiki/Mechanosensitive_channels, Question for any engineer or physics student: can we generate, by sound, enough force on the membrane of e coli (possibly spheroblasts, so without cell wall, to make them more sensitive) to force channel opening? We would need about 5.5 dyne/cm for MscS or so. Is this possible with sound?

Jochem: as for the channels in e.coli, 5.5 dyne/cm^2 is apparently 5.5 N/m^2, thus 5.5 Pa which translated to about 110 dB (20 log(P/Pref)) But this is in air, I don't know the attenuation coefficient for sound in water, but it might be quite high... However, from what I understand from the article it works on pressure difference over the membrane, to compensate for too high osmotic pressure and release some osmolytes. Which means that it requires a higher pressure inside the cell compared to outside, not possible with sound. Also it appears to already be in e.coli and many other organisms to compensate for osmotic pressure (which can be quite high).

Jonas: In the end, the channel is activated by lateral tension in the membrane. Would this not be induced (on-off) in the bacterial membrane when it is hit by soundwaves (perhaps amplified and transferred into the medium of a liquid culture)? Is this provable with a sonicator on very low intensity (so no bacterial cell lysis)? The channels (MscL/M/L) are indeed present in e.coli etc. But might be interesting to biobrick/overexpress/make hypersensitive (mutants described)/ ... as they can apparently also be removed without compromising viability of the culture under normal conditions)

Jochem:I am not sure how a membrane would respond to a higher or lower pressure... I guess it would have some effect in the same way as temperature can induce phase changes in the membrane. But I am unsure if it would result in any lateral tension keep in mind that sound waves are orders of magnitude larger then bacteria, but ultrasonic sound wave can be in the order of a bacteria and might result in some memrbane tension? Maybe it is an idea to tether the bacteria at two sides to two paralel flexible surfaces (see improvised picture below, 0=bacteria), which are then allowed to vibrate due to sound (away and towards each other); sort of stretching and squeezing the bacteria? (that should result in lateral tension ^^) or maybe this 'piezophilic' bacteria can be of use? http://www.ncbi.nlm.nih.gov/pubmed/10941789 I forgot what was in the slides of the guys who presented this idea, what did they find again/are the slides somewhere? :-) Also, is the wiki up yet?

Bart B: Maybe you could attach the bacteria on a speaker diaphragm. That way, the vibrations in the diaphragm cause stretching of the cell membrane and you still have sound within the audible range.

The speed of sound in water is almost 5 times as high as in air therefore the air-water interface is almost a perfect reflector. To induce membrane stretch in suspended cells you will have to generate the sound underwater. I do not know if the wavelength has to be in the range of the length of the cells, maybe the pressure difference before and after the sound wave are enough to provoke a response. If it is necessary to have a wavelength in the micrometer range you need a frequency in the range of 100 MHz in water, way beyond the audible range.

Use of human hair cells?

Koen: Is it recommended to work with E.coli? We can also use human hair cells, then we could make a direct sound sensor in stead of a pressure sensor. Good websites with the basics of hearing: http://www.ks.uiuc.edu/Research/hearing/ and http://www.beyonddiscovery.org/content/view.page.asp?I=259 In the ear the crucial point is converting the mechanical stimulation of the hair cells into a membrane electric potential which leads to neural signalling. Maybe it is possible to visualize this electic potential and make a correlation between the frequency of the sound and the colour we use. For example a blue colour is generated when the frequency of the sound is X MHz. Another advantage of working with human hair cells is having much more previous research literature. Now we only need the time to explore it ... ;-) It is impossible to detect sound with only one hair cell (like proposed in my previous mail), of course you need the whole ear organ. However we can still use hair cells for detecting pressure pulses but E. coli is also possible.

Jonas: The advantage of e. coli is that it's easy to work with + the registry is mainly aimed at this bacterium.

Jochem: Like Jonas said, the advantage of E.coli is the ease of work and the immensive amount of literature there is for e.coli. But it is not forbidden to work with anything else... However the main problem in working with human hair cells is the difficulty in growing this (btw afaik the rest of the ear is only there for amplification, so might be ignored). I do think the mechanism of the hair cells is mechanosensitive channelsl as well.

Koen: While learning about nanomotors, my course mentioned a protein, prestin, which is a motor protein in the inner ear. Its shape apparently responds to changes in the electrical potential across a membrane. Because of this, it can amplify sounds up to a 1000 fold. Maybe it could be useful since it amplifies sound (maybe we can implement it so that the bacteria detects small of changes in environmental sound)? This link gives a small introduction of the protein: http://en.wikipedia.org/wiki/Prestin

Coupling an output to the sound/vibration input signal

Coupling mechanism will depend on the signal generated by the sensor. If, for instance, the bacterial MscL/MscS proteins would be sound sensitive, how are we going to detect this? In order words, how are we going to couple activation (channel opening) to a measurable output? --Erwinswinnen 10:05, 8 June 2011 (CDT)

Dual function for E. coli, anti-freeze and ice-nucleation

Hi,

I like to open a discussion also about the ice-forming bacteria. If the sound project is not feasable, we should already have a good alternative.

So what i was thinking: we can make a bacteria that can perform 2 useful functions in winter using two different stimuli:

- If you want to induce ice-formation using Ice Nucleating Proteins (INP), then you can use the bacteria in lakes to make your ice more strong and maybe we can have again a "11-stedentocht" in the Netherlands. Also, it would decrease ice-melting of gletzers, thereby efectively slow down global warming.

- If you want to induce anti-freeze using Anti Freeze Proteins (AFP), then you can use the bacteria as anti-freeze biofilms on roads, which would help the roads become ice and snow free in winters.

We can make use of the N and C terminal domain of the INP as anchors to hang to protein extracellulary at the plasma membrane. We change the middle domain by a ice nucleating protein activity or a anti freeze protein activity, depending on the external stimuli. We would give the stimulus when growing the bacteria so we can separate both functions. Secondly, one condition should effectively inhibit the other function.

To make it more nice, we would let the bacteria also produce a certain pigment depending on the stimulus, i.e. green or red, that you can also distinguish with the naked eye and also see if the activation has occured. For the streets and hence anti-freeze function, a dark colour is preferred. For the INP, however, we can use a luminiscent colour that can only be seen in the dark and this would make ice-skating at night more safe.

Also, we should have some kind of kill-switch. The bacteria will not grow in ice anymore, but when deforsting, they would. So it is possible to induce the kill-switch when subjected to cold temperature (cold shock operon).

Two interesting review about these topics are: Hew et al. (1992): protein interaction with ice and Cochet and Wildehem (2000): Ice crystallization by Pseudomonas syringae + wikipedia link for anti-freeze http://en.wikipedia.org/wiki/Antifreeze_protein; http://microbewiki.kenyon.edu/index.php/Bacterial_nucleation_in_pseudomonas_syringae

What do you all think about this? Please let us know.

Yours sincerely, Instructor Ruben Ghillebert

Koen: Both ideas sound very attractive to me, especially the second idea because of the green impact of it.

Another property of this anti-freeze proteins is suppressing ice melting up to a certain point, although the protein's ability to suppress ice growth is much stronger. But on the road it is useful to have small cristals, it doesn't matter much if they last till +0,5 degrees for a couple of hours.[http://www.physorg.com/news186669694.html http://www.physorg.com/news186669694.html]-

Is it really a good alternative for making roads snow-free in winter, what are the possible disadvantages of it? Has it yet been tried to use anti-freeze proteins (with of without bacteria) as alternative for salt? (edited on 16 june at 18:21)

Ruben: I see I made a mistake. The green impact of gletscher melting is of the ice-nucleating proteins and not of the anti-freeze proteins, which is changed now. I think it is more efficient to decrease the melting temperature of the ice then make more stable small crystals.

I think it would be possible to use it as an alternative because the effects of this on the environment are not as worse as using salt. Also, as you noticed the last 2 years, we don't have enough salt:)

Hanne: A cool application would be to put the bacteria in a sort of holder where you can put eppendorfs in to defrost or eppendorfs that have to remain cold, like enzymes.

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