Bench philosophy: Shock wave transformation

Blasting Bugs
by Steven Buckingham, Labtimes 02/2012



A recent paper in Analytical Biochemistry is surely vying for the most shocking attempt at violating bugs – introducing genes into bacteria using shock waves generated by an explosion.


Shot-firer ready to blast a coal face. Similar igniters may be used to transform bacteria.

A team of workers in Bangalore, India, led by Dipshikha Chakravortty, have shown that a shockwave can be used to impel DNA into bacteria and they have built a device to prove it (Prakash et al., Anal. Biochem., 419, 292-01). The device, which measures just a few centimetres across, creates this shockwave by delivering a controlled explosion to the surface of the cell suspension. The business end of the device is a plastic tube lined with explosive. One end, the end furthest away from the cells, is plugged with an electronically-controlled spark-plug. The spark ignites the explosive lining the tube and the explosion propagates along the tube as a wave of expanding gasses.

Shock wave radiation

It works just like a shotgun, only there are no pellets and the propellant lines the barrel rather than sitting in a cartridge. What holds for shotguns also holds for Chakravortty’s device: when such a wave of hot gasses mixed with combustion products reaches the end of a gun barrel, it creates a shock wave radiating out from the mouth of the barrel. It is this shock wave that the device deploys in transforming the bacteria. Remember, this isn’t a gene-gun. The DNA you want to get into the bacteria is added to the culture medium, not the device itself.

At this point I am reminded of a word of advice I was given years ago – don’t go fishing with a rifle. If you put the end of the barrel in water and fire, the fish will live, you won’t. That is because the shock wave leaving the barrel finds hard, incompressible water instead of the soft, squashy air it was expecting. Like a hyperactive child in church, it suddenly finds itself full of energy and nowhere to put it. So it vents its frustration by sending the shock wave back up the barrel, which then explodes under the strain.

Of course, there isn’t any danger of being blown apart by Chakravortty’s propylene barrel. All the same, if the bacteria are to be transformed you have to place the opening of the tube at just the right distance from the culture medium. And how do you do that without blowing the culture medium around the lab and creating a very unhealthy soda fountain? The trick is a tiny sheet of brass foil, just strong enough to withstand the shock and thin enough not to absorb too much of it. The foil lies on the surface of the culture medium, with just the right gap between it and the gun to ensure the best transfer of compressive energy to the liquid.

Does it work? As you might expect, Chakravortty’s paper presents some impressive results. They got about 1.5 x 10-5 transformants per bacterium using E. coli with a single shot from the gun. And just to be sure the bacteria weren’t just soaking up the DNA from the medium anyway, they checked that when you just place DNA in the medium and hold fire, there were no measurable transformants in the controls. The rate of transformation they got using shock waves was about the same value they got using electroporation and it was even higher than what they got with heat shock. This is not just because there is something quirky about E. coli, either. They got the same transformation rates with P. aeruginosa and S. typhimurium, with values similar to when they used electroporation for these species. In fact, they got glowing results, with plates full of bright red bacteria under fluorescent light after being bombed with mCherry, testifying to the success of the transformation.

So should you ditch your electroporator and build a bomb? To be sure, if Chakravortty’s claims are true, it looks as if shocking your bacteria has some clear advantages over zapping them. Firstly, the bacteria themselves seem to prefer it. After all, electroporation does some pretty horrible and irreversible things to membranes, and most cells just don’t ever get over it. Compared to the pretty high cell death rate they got using electroporation, Chakravortty claims they got almost no cell loss even up to 10 minutes after transformation. “But hang on,” some might say, “don’t people use micro shockwaves to kill bacteria?” Yes they do, but like so many things in life it is a matter of degree: Chakravortty’s device just doesn’t deliver enough energy to kill.

The second thing to consider about shockwave transformation is its ease: it is a lot quicker. Compared to the three hours or so it takes to set up an electroporation, the bug-bomber only takes about an hour to set up. The other handy thing is that unlike zapping, Chakravortty’s method doesn’t depend on the bacteria’s growth phase. And on top of all that, don’t forget the cost: an electroporator won’t leave you with much change out of €3,000, whereas Chakravortty’s group built their bomb for less than €100.

Simple construction

But before you rush out to your local supplier, we need to think of the down sides. The first, obviously, is that your favourite supplier doesn’t stock it. You’ll have to make it yourself. That being said, the construction should not be a challenge to a standard departmental workshop. Apart from cutting a chamber assembly to specifications that will hold the foil in the right position and deliver the explosive gasses, there isn’t much to it. Most, if not all, of the materials can be easily bought from standard suppliers and there would be no shortage of graduate students keen to play with the explosions. And if you were wondering where to get the explosive from, try a supplier of mining charges. I’m serious. Chakravortty’s group bought the tubes ready made from Dyno Nobel, a Swedish company, who sells the tubes for detonating your way into a coal face. Now, that would raise eyebrows in your purchasing department. In all, Chakravortty’s paper contains enough detail for a capable engineer to design and build a device in a few hours.

But even when you have built your device, your next job would be optimising the setup. Here again, Chakravortty’s group has blazed a trail. They tried varying the length of the polymer tube and found that the longer you made it, the more bacteria were transformed. Presumably, a longer tube means a bigger shock wave. They tried it up to 40 cm – perhaps any longer than that and you get a bacterial soda-fountain. The thickness of the foil and what it is made of, don’t seem to matter much, though. They tested brass and copper foils from 100 to 180 μm and found little difference in performance. They also looked at the effect of hitting them with one, two or up to five shots and it seems that one hit is as effective as five.

As for the composition of the medium and its effects on transformation efficiency, it looks like the usual rules apply. For instance, just like in any other transformation experiment, you have to get the glycerol concentration right. Chakravortty found 10% was best, while pushing it up to 40% almost wiped out the effect. In the same vein, the calcium concentration and plasmid concentration both had an inverted U-shaped effect: the best calcium concentration was around 200 mM (600 mM blocks transformation) and the best plasmid concentration was around 3ng/μl. No surpris­es­ there.

So just how does it work? The honest answer would be that we don’t really know, while Chakravortty suggests it might have something to do with a process called cavitation. Not being at all sure what cavitation is, I typed “cavitation” and “shockwave” into a well-known internet search engine. Scrolling down past the links to a well-known provider of computer animation and invitations to sign up with a dentist, I eventually found my answer.

Micro shockwaves cause the formation, followed by the sudden implosion, of tiny bubbles in the medium. As the alternating pressure of the shock wave passes through the medium, the low pressure half of the wave causes minute bubbles of vapour to form. When the high pressure half arrives, the bubbles collapse so rapidly that two things happen. First, they create their own localised secondary shock waves. Second, the wall of one side of the bubble collapses inwards, propelling liquid through the centre of the bubble and creating a microjet of liquid. Perhaps, Chakravortty surmises, either of these, or a combination of them, momentarily disintegrates a miniscule area of membrane, allowing the DNA message to enter the cell. Not unlike electroporation, really, only perhaps the cavitations restrict their damage more locally than pulses of voltage would do.

Sonoporation

Indeed, it was the discovery that cavitation is the means, by which ultrasound delivers DNA into cells that fired the growth of “sonoporation” over the last few years as an emerging DNA therapy. One of the spin-offs from that was the idea of including ultrasound contrast agents and it would be interesting to know if adding these would improve the performance of the Chakravortty device.

So, using micro shockwaves is nothing new. We have noted above that they have been used to kill bacteria but they are also regularly used in removing kidney stones, cleaning labware and even for tenderising meat. What is more, several reports are in the literature, describing the technique working for eukaryotic cells. Lauer et al. reported that one can deliver DNA into cells using a lithotripter (Gene Therapy 1997, 4, 710-15). Off to our favourite internet search engine again – apparently, it’s the device they use to smash kidney stones using ultrasound. Bit of an overkill for bacteria, though, and how many labs have a lithotripter under the bench? Chakravortty is right in saying that their device is the first of its kind for bacteria; furthermore, it’s cheaper than a lithotripter!

But given the time and trouble it would inevitably take to design and build the device, one wonders whether there might be other ways to deliver shockwaves to cells. Indeed, Fechheimer et al. reported that they got 20% transfection of mammalian cells using a standard laboratory sonicator (PNAS, 1987, 84(23), 8463-7). Perhaps bacteria cell walls are tougher than mammalian membranes. After all, Chakravortty had to use a mining explosive to get into their bacteria.





Last Changed: 10.11.2012




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