Almost everyone in the BioBricks realm seems to use a standard method for modifying their organisms: chemical transformation. Yet there is another method which is very promising.
In chemical transformation [3], some standard bacteria is grown, purified, mixed with some chemicals which cut open the bacteria, the new DNA plasmid is added to create some modified bacteria, the new DNA plasmid flows through the cut into the bacteria, everything is mixed with some more chemicals, allowed to heal & grow, and purified. (My naive translation of the process)
Note all the chemicals used? Chemicals can be expensive. And the amount of modified bacteria which results from this process is pretty low.
There’s an alternative method used more for yeast than bacteria: voltage-based transformation, electroporation [1]. With electroporation, some standard bacteria is grown, mixed with some simple chemicals, the new DNA plasmid is added, everything is given a quick high voltage zap (like lightening) which cuts open the bacteria, the new DNA plasmid flows through the cut into the bacteria, the bacteria is allowed to heal & grow, and purified. (Again, my naive translation of the process)
This eliminates some chemicals, although the process still requires some specialized equipment which can be troublesome (and expensive) — the voltage can be as high as 5 kV at 20 mA. (As high as the internal components of a CRT television, which, if accidentally touched, can be easily fatal.)
There’s another method though, that I haven’t seen mentioned: sonic transformation, sonoporation [2]. In sonic transformation, some standard bacteria is grown, some chemicals are added, optionally producing small bubbles, the new DNA plasmid is added, everything is given a loud blast of ultrasound (for example, at 40 kHz) which cuts open the bacteria, the new DNA plasmid flows through the cut into the bacteria, the bacteria is allowed to heal & grow, and purified.
In the research quoted below, sonoporation has shown to be much more effective at modifying bacteria than either chemical transformation or electroporation; plus, this is done without the expensive chemicals necessary for chemical transformation and without high voltage equipment necessary for electroporation.
From [2]:
We have employed a standard low frequency 40 kHz ultrasound bath to successfully transfer plasmid pBBR1MCS2 into Pseudomonas putida UWC1, Escherichia coli DH5{alpha} and Pseudomonas fluorescens SBW25 with high efficiency. Under optimal conditions: ultrasound exposure time of 10 s, 50 mM CaCl2, temperature of 22°C, plasmid concentration of 0.8 ng/µl, P. putida UWC1 cell concentration of 2.5 x 109 CFU (colony forming unit)/ml and reaction volume of 500 µl, the efficiency of ultrasound DNA delivery (UDD) was 9.8 ± 2.3 x 10–6 transformants per cell, which was nine times more efficient than conjugation, and even four times greater than electroporation. We have also transferred pBBR1MCS2 into E. coli DH5{alpha} and P. fluorescens SBW25 with efficiencies of 1.16 ± 0.13 x 10–6 and 4.33 ± 0.78 x 10–6 transformants per cell, respectively.
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We propose a mechanism of ultrasound-mediated plasmid transfer of bacteria in which plasmid transfer is enhanced in the presence of CaCl2 : Plasmid DNA and bacteria are initially well-mixed in an aqueous solution. The addition of CaCl2 causes changes in the conformation of plasmid DNA or cellular membrane structures that promote transformation. As low frequency 40 kHz ultrasound is applied to the solution the transmitted energy causes temporary porosity in the cell membrane, which enables the plasmids to enter through the pores. When the ultrasound is switched off the cell membrane repairs itself and the transformed cell retains the plasmid DNA. Bacteria acquire new functions, such as growing on a selective medium, after taking up plasmid DNA.
Of course, the basic procedure for all of the methods should be obvious from the comparison: grow some standard bacteria, find a way to cut the cells open (carefully, without killing them; see the wikipedia link, “cell disruption”), the new DNA plasmid will enter through the cut, let the bacteria heal & grow, then purify. See, genetic engineering isn’t so difficult.
Ultrasound is used with microbubble agents for sonoporation. Of course, various experiments using ultrasound with and without microbubble agents have been performed, showing that microbubbles enhance plasmid uptake [9]:
The effect of ultrasound alone has been studied and has been shown to increase cell permeability on its own, without the addition of microbubbles. However, microbubbles in the presence of ultrasound with high acoustic pressure has an additional effect in increasing cell permeability. First, microbubbles, by acting as cavitation nuclei, can lower the threshold for cavitation. Stride and Saffari made an analysis of the conditions in the shell of the microbubble under influence of ultrasound and concluded that extremely high shell stresses and ‘bubble like behaviour’, including cavitation may be expected. In body tissue or blood, cavitation sets fluid in motion and creates small shock waves that give rise to microstreaming along the endothelial cell. Destruction of microbubbles may cause high-energy microstreams, or microjets, that will cause shear stress on the membrane of an endothelial cell and increase its permeability.
Figure 1, From [9]. Destruction of microbubbles by ultrasound resulting in increased membrane permeability by shear stress, temperature rise and activation of reactive oxygen species. Drug delivery by microbubbles by: a: transient holes induced by shear stress; b: increase in membrane fluidity; c: endocytosis of microbubbles; d: fusion of the microbubble membrane with the cell membrane.
This increase in permeability is probably due to transient holes in the plasma membrane and possibly the nuclear membrane. A second proposed mechanism, the generation of reactive oxygen species in endothelial cells under influence of ultrasound, was investigated by Basta et al. A significant, time-dependent increase in intracellular radical production after exposure to ultrasound was demonstrated. As the use of microbubbles together with ultrasound lowers the threshold for cavitation, this could possibly result in an increased production of free radicals, which are associated with cell killing in vitro and, as a consequence, may be also involved in enhancement of permeability of endothelial cell layers. A third interesting aspect is the rise in temperature in tissue following the application of high pressure ultrasound. Bubble collapse following high energetic ultrasound can create high velocity jet streams that may cause a local, transient increase in temperature. As a rise in temperature influences the fluidity of phospholipid bilayer membranes, cell membrane permeability could possibly be changed directly as a consequence of the increased bilayer fluidity. Fourth, endocytosis or phagocytosis, active membrane transport mechanisms, may also be involved in the uptake of the bubble, bubble fragments or material entrapped in microbubbles.
Several different microbubble agents are available today :
- Optison
- Albunex
- Levovist
- BR1
- BR14
- … others?
Publications have compared the results of a few different agents [5]:
It is believed that the main mechanism is that microbubbles act to focus ultrasound energy by lowering the threshold for ultrasound bioeffects.
Li and colleagues studied three microbubbles, all of which have been developed for patient use, with different shell and gas contents. They used a physiotherapy ultrasound system and worked with plasmid DNA encoding a nontherapeutic reporter gene (a gene that encodes an easily detectable protein marker). They first studied the effects on cells in suspension. They found that Optison (albumin shell with a perfluorocarbon gas; Nycomed-Amersham, Oslo, Norway) behaved differently than the air-based agents Albunex (Molecular Biosystems, San Diego, Calif, distributed by Mallinckrodt, St Louis, Mo) and Levovist (Schering, Berlin, Germany). While Optison was much more effective at killing cells than were the other agents, when the concentration was reduced to correct for this, it was eight times better at transfection (ie, uptake of DNA into muscle fibers with consequent expression of the reporter gene). They then injected microbubbles with DNA into the quadriceps muscle of mice and applied ultrasound. With Optison, a marked increase was seen in the efficiency of transfection, while no difference was seen by using the other microbubbles or ultrasound alone.
Optison (PDF datasheet) is an ultrasound contrast agent (approved for human use), manufactured by GE Healthcare, intended for use in patients with suboptimal echocardiograms to opacify the left ventricle and to improve the delineation of the left ventricular endocardial borders. It contains Octafluoropropane, C3F8, arranged as a microsphere with diameter of 3.0-4.5µm. Datasheet Prescribing information for Optison.
Currently, the mechanism for sonoporation is not well understood. Ultrasound can also be used at low power levels for effects: [7]
The ultrasonic wave was generated by an arbitrary waveform generator (Agilent 33220A; Agilent Technology, Palo Alto, CA) and amplified (150A100B; EMV Benelux, Nieuwkoop, The Netherlands). Peak-to-peak acoustic pressure generated at the region of interest (ROI) was MI 0.1 or MI 0.5 [note: MI, mechanical index], with a duty cycle of 0.2% and pulse repetition frequency of 20 Hz.
Fig. 1. Experimental design. A: schematic drawing of the ultrasound setting on the microscope used for online measurements. B: schematic overview of experiments with 5-(6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA). C: schematic overview of experiments with fluo-4.
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The presence of ultrasound-exposed microbubbles in close proximity to cells has an effect on the permeability of cells. This permeability change is a result of a number of as yet undefined physical, chemical, and mechanical forces acting on microdomains of the cell membrane. Because of the difficulty of investigating ultrarapid alterations in cell membrane phospholipid bilayer organization, specific mechanisms of pore formation have not yet been definitely proven. Microbubble oscillation, cavitation, collapse, and the resulting shear forces are all suggested to play a role in the increase in permeability of the cell. We demonstrated increased permeability of the cell membrane by revealing Ca2+ influxes at both MI 0.1 and MI 0.5 in the presence of microbubbles, already with pulsed ultrasound with a duty cycle of 0.2% and pulse repetition frequency of 20 Hz. This is ultrasound with low energy compared with ultrasound used by other groups (6, 13, 20). This is an important finding, because it indicates that ultrasound exposed microbubbles can indeed provoke alterations in cell membrane permeability, which may have several consequences for intracellular ion homeostasis.
Hopefully, someone in need of a small lab project and BioBricks on their hands, has some time to test an ultrasound protocol for BioBricks.
Fascinating! Rather, I am also working on molecular aspects associated with sound exposure to biological systems.
Cheers!
Tarun