Abstract:
A method and apparatus for electroporation includes placing a mixture of bacterial suspension and transforming DNA into an electroporation cuvette. The resulting sample is subjected through a current-limiting device to a complex 5 waveform including a burst of high-voltage radio-frequency current, which in some embodiments is superimposed on a biphasic high-voltage DC pulse, and in other embodiments on a high-voltage lower-frequency AC burst. The total waveform has at least an initial portion greater than eleven thousand volts per centimeter of electrode spacing, and a later portion in some embodiments is reduced to less than thirty percent 10 of magnitude of the initial portion. Transformed bacteria are selected by culture in selective medium in an embodiment. The high-voltage radio-frequency current is between 3 and 125 MHz, and in an embodiment is 24 MHz.

Description:
RELATED APPLICATIONS 
       [0001]    This application claims priority to U.S. patent application 60/568,756, filed May 6, 2004, entitled “USE OF INDUCED OSCILLATIONS TO ACHIEVE HIGH EFFICIENCY TRANSFORMATION OF DIFFICULT TO TRANSFORM BACTERIA” the disclosure of which is incorporated herein by reference. 
     
    
     US GOVERNMENT RIGHTS 
       [0002]    The United States Government has certain rights in this invention pursuant to contract Phase I SBIR DE-FG02-03ER83593 awarded by the Department of Energy. 
     
    
     FIELD OF THE INVENTION 
       [0003]    The present document relates to methods and apparatus for engineering genomes of microorganisms. In particular, the present document relates to transformation of bacteria by driving DNA segments across a bacterial cell membrane with an electric field. 
       BACKGROUND OF THE INVENTION 
     Engineering Bacterial Genomes 
       [0004]    Bacteria typically have genomes including one or a few chromosomal strands of Deoxyribose Nucleic Acid (DNA) having thousands of segments known as genes. Each gene includes a sequence of nucleotides that code for one or more peptides, or proteins, together with regulatory nucleotide sequences such as promoters, start codons, and stop codons. Bacteria may also incorporate shorter DNA segments such as plasmids and dormant bacteriophages, which may also contain genes and which may reside in the cytoplasm alone or be incorporated into the bacterial genome. 
         [0005]    Typical bacterial species encode proteins that have evolved according to the needs of the species in the environment it normally inhabits. These proteins typically include proteins for reproduction of the bacterial cell, for energy production, for producing fundamental building blocks of the cell like nucleotides, for producing motility structures like flagella, and for toxins that give that species a survival advantage over other species in the same environment. 
         [0006]    Bacteria have been engineered to produce proteins unnecessary for survival of that species, but of interest to humans. This has been done by isolating or creating a new segment of DNA encoding a desired protein and inserting the new segment into the bacterial genome; once inserted the new segment reproduces with the bacteria and, if properly designed and inserted, may produce the desired protein. The process of inserting the new DNA segment is known as transformation. 
         [0007]    Similarly, transformation can be used to disable selected genes normally present in the bacterial genome, or to increase production of preferred products. 
         [0008]    Alteration of bacterial genomes can be of use in adapting an organism to survive in a different environment, or to modify the organism&#39;s metabolic pathways to produce non-protein metabolic products of interest to humans. 
       Electroporation 
       [0009]    Electroporation is a term describing transport of hydrophilic molecules across a hydrophobic membrane via electrically formed pores (electropores). 
         [0010]    While some bacteria can be transformed simply by adding a solution of new DNA to culture media; most bacteria require further manipulation to transport the new DNA across the bacteria&#39;s cell wall and membrane into the interior of the bacterial cell. One such technique is Electroporation. 
         [0011]    DNA generally has a negative charge in aqueous solution; DNA is an acid and liberates hydrogen ion in solution at physiological pH. DNA therefore tends to move towards a positively charged electrode when an electric field is applied to a DNA solution, a phenomenon known as electrophoresis. 
         [0012]    A desired new DNA plasmid is prepared in aqueous solution of low ionic strength, and added to bacterial cells suspended in an electroporation buffer. The mixture is typically kept on ice to prevent DNA degradation and to avoid overheating the bacteria during electroporation. Electroporation is performed by exposing the mixture of DNA solution and suspended bacteria to a high-intensity, brief, electric field. The intense field carries DNA molecules across the hydrophilic cell wall by electrophoresis. The intense field also carries DNA molecules through a temporary electropore in the hydrophobic cell membrane into some, but far from all, of the bacteria. 
         [0013]    The desired new DNA plasmid may include sequences homologous to portions of the bacterial chromosome at which they can insert into the bacterial chromosome. The new plasmid may alternatively include portions that code for integrases that incorporate portions of the plasmid into the bacterial chromosome. The new plasmid may be capable of surviving and replicating within the bacteria. 
         [0014]    The bacteria are then cultured under conditions favorable to growth of bacteria incorporating the desired new DNA. Typically a gene encoding for resistance to an antibiotic is included in the new DNA, and that same antibiotic is included in a post-electroporation culture media. Transformed bacteria are selected for because they have a survival advantage over untransformed bacteria in this media. 
         [0015]    Typically, electroporation is performed by placing the bacterial suspension and the transforming DNA between electrodes of a chilled electroporation cuvette and applying an electric pulse to the cuvette. A high, DC, or RF modulated DC voltage pulse is applied to the electrodes for a preset time typically up to several dozen milliseconds. 
       Difficult to Transform Bacteria. 
       [0016]    Some bacteria with complex intracellular morphology and/or complex cell development cycle are known as “difficult to electrotransform”. When transforming these bacteria, it is important to identify the right growth medium, specific growth stage of the culture, and some other biological conditions, such that the cells become as “electrocompetent” as possible before performing electroporation. 
         [0017]    When bacteria of some species are subjected to stressful conditions, they may form hardy endospores. Endospores are generally smaller than normal vegetative cells, and much more resistant to chemical, thermal, dessication, and other environmental hazards than normal vegetative cells. Electroporation may provide sufficient chemical, electrical, and thermal stress to trigger spore formation in some bacteria. As spores form, much of the cellular contents, often including the newly inserted and desired DNA plasmid if sporulation occurs immediately after electroporation, is excluded from the spore. Spore forming bacteria with complex lifecycles therefore are often difficult to transform. 
         [0018]    Some bacteria, such as  Acinetobacter  species, have intracellular granules or vesicles that can block inserted DNA plasmids from the nucleoid region of the cell where the genome and DNA replication enzymes are located. These bacteria can also be difficult to transform because only DNA inserted into the proper part of the cell is likely to be expressed, and because these granules and vesicles may block plasmids from reaching the proper part of the cell. 
         [0019]      Clostridium  is a genus of Gram-positive, spore-forming, anaerobic, bacteria, including several species that are difficult to transform, typically derive energy through fermentation and for which free oxygen is toxic. Example members of the genus include Clostridium perfringens, pathogenic in animals and man;  Clostridium botulinum , noted for production of potent toxins; as well as  Clostridium thermocellum , capable of fermenting cellulose at 60° C. 
         [0020]    Prior techniques for transformation of difficult-to-transform bacteria have included modifying the bacterial cell walls by growing the bacteria in media containing ingredients that damage developing cell walls, or by partially digesting the cell walls. The weakened cell walls then allow desired DNA plasmids to reach the cell membrane more rapidly, so that plasmids are more likely to pass through electropores into the cells. It has been found that weakening cell walls often adversely affect viability of the bacteria, viability is often so poor that electroporation yield remains unacceptably low. 
         [0021]    It is desirable to improve transformation yield of difficult-to-transform bacteria to expedite research performed with such bacteria. 
       Selection of Transformed Bacteria 
       [0022]    Once electroporation is performed, transformed bacteria may be selected by culturing the bacteria in a selective media. Selective medium contains at least one antibiotic for which a gene of resistance is included in the desired DNA plasmid. In such media, only transformed bacteria thrive. 
         [0023]    Alternatively, electroporated bacteria may be cultured into colonies on an agar plate and bacterial products blotted onto a membrane. The membrane can then be stained with fluorescent antibodies to proteins encoded on the desired DNA plasmid. Colonies expressing those proteins will then have associated fluorescent marks on the membrane, thereby allowing identification of colonies that express those proteins. 
         [0000]    Modified  Clostridium thermocellum  May Help Produce Biofuels 
         [0024]    Most plants produce sugars by photosynthesis in abundance. Typical plants process most of the sugars they produce into cellulose, forming much of the cell wall and supporting fiber of angiosperms. Only a small proportion of sugars become starches in seed. 
         [0025]    There are many uses for the seed of corn, wheat, oats, or other grains, both for food, animal feed, and for fermentation into alcohols. Many organisms, including humans, produce amylase enzymes capable of hydrolyzing starch. 
         [0026]    Mammals, yeast, and other eukaryotic organisms lack enzymes for hydrolyzing cellulose; sugars linked with beta-glucoside bonds in cellulose are not well used and often become waste. Grass, wood, agricultural residues (including cornstalks, wheat and oat straw, and manure), and municipal solid waste (paper) have high cellulose content. 
         [0027]      Clostridium thermocellum  has the ability to hydrolyze cellulose, it ferments the resulting sugars into a mixture of alcohols and organic acids, including acetic acid. 
         [0028]    It has been proposed that a strain of  Clostridium thermocellum  suitable for use in industrial production of ethanol from cellulose can be more easily engineered if this organism&#39;s resistance to transformation by electroporation can be overcome since multiple, substantial, modifications of its genome are required. In particular, it is desirable to decrease organic acid production and increase both ethanol production and ethanol tolerance. 
       SUMMARY 
       [0029]    A method of electroporation includes placing a mixture of bacterial suspension and transforming DNA into an electroporation cuvette. The resulting sample is subjected through a current-limiting resistor to a complex waveform including a burst of high-voltage radio-frequency current, which in some embodiments is superimposed on a biphasic high-voltage DC pulse. The total waveform has at least an initial portion greater than eleven thousand volts per centimeter of electrode spacing. In some embodiments the waveform in a later portion is reduced to between ten and thirty percent of the magnitude of the initial portion. Transformed bacteria are selected by culture in selective medium. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0030]      FIG. 1  is a block diagram of a prior-art electroporation apparatus. 
           [0031]      FIG. 2  illustrates the principle of electroporation. 
           [0032]      FIG. 3  is a block diagram of the present electroporation apparatus. 
           [0033]      FIG. 4  is an illustration of DC and AC components of the high-voltage burst. 
           [0034]      FIG. 5  is an illustration of DC and AC components of an alternative high-voltage burst. 
           [0035]      FIG. 6  is an abbreviated flow chart of a method of bacterial transformation. 
           [0036]      FIG. 7  is an illustration of DC and AC components of an alternative high-voltage burst. 
           [0037]      FIG. 8  is an illustration of a composite-AC high-voltage burst. 
       
    
    
     DETAILED DESCRIPTION OF THE EMBODIMENTS 
       [0038]      FIG. 1  illustrates prior-art electroporation apparatus similar to that described in Tyurin M. V., Padda R., Ke-xue Huang, Wardwell S., Caprette D., Bennett G. N. (2000):  Electrotransformation of Clostridium acetobutylicum ATCC  824  Using High - Voltage Radio Frequency Modulated Square Pulses //Journal of Applied Microbiology. 88(2): 220-227 (hereinafter Tyurin, 2000), the text of which is hereby incorporated by reference. A suspension of cultured bacteria in a solution  102  of desired-sequence DNA is placed in an electroporation cuvette  104 . Care is taken to ensure that the solution  102  is as free of salt as possible, nonionic solutes are used to provide appropriate osmolarity. The cuvette  104  has a first electrode  106  and a second electrode  108  spaced both exposed to the solution  102  but electrically insulated by a polypropylene microcentrifuge tube  110  from a surrounding ice block  112 . When working with anaerobic bacteria, the entire cuvette  104  and ice-block  112  are placed in an oxygen-free glovebox  114 . The first  106  and second  108  electrodes are connected to the output of a high-voltage electrical function generator  120 , or operating under control of pulse timing circuits  122 . 
         [0039]    Reported apparatus includes high-voltage electrical function generators  120  having direct current DC output, and high-voltage electrical function generators having a one-hundred kilohertz AC burst superimposed on a DC pulse. In particular, Tyurin 2000 in his  FIG. 3  taught that better results were obtained with one-hundred kilohertz AC superimposed on a DC pulse than with higher frequencies in the range one hundred fifty to three hundred fifty kilohertz. 
         [0040]    When the prior apparatus of  FIG. 1  is used, the operator triggers the pulse timing circuits  122 , and the pulse generator provides either a square pulse as indicated  124 , an exponentially decaying pulse, or a superposition of a high-voltage DC pulse with one hundred kilohertz AC of amplitude one-tenth to one-third the magnitude of the DC pulse between the first  106  and second  108  electrodes of the cuvette. While DC pulses are standard in the art, Tyurin 2000 disclosed transformation of Clostridium species using a pulse having an AC signal of 100 kHz superimposed on a DC pulse. High-voltage pulses above one kilovolt are typically used. 
         [0041]    The high-voltage electric field applied between the electrodes  106 ,  108 , causes current to flow through the solution  102  of desired DNA plasmids  202  ( FIG. 2 ) with suspended bacteria  204 . DNA plasmids  202  are typically circular of DNA with a replication initiation site. The current, which readily flows through the saline intracellular fluid, burns minute holes in the bacterial cell walls  206 , and electrophoretically shifts DNA molecules in the solution, through the cell walls, and some molecules may shift through the minute holes. After the high-voltage current pulse, some desired DNA molecules  210  end up inside transformed  212  bacteria, where the desired DNA molecules  210  may be expressed and further incorporate into the bacterial genome. 
         [0042]    In the present electroporation apparatus as illustrated in  FIG. 3 , a suspension of cultured bacteria in a solution of desired-sequence DNA is placed in an electroporation cuvette  302 . The desired-sequence DNA is preferably in the form of a plasmid. The cuvette has first  304  and second  306  polished parallel-plate stainless-steel electrodes spaced approximately two millimeters (distance D) apart in a two-milliliter polypropylene disposable centrifuge tube  307 , and both the electrodes and cuvette contents are electrically insulated from, but thermally coupled to, a surrounding ice block  308 . In doing so, part of the tube  307  is placed within a cavity in the ice block  308 . When working with anaerobic bacteria such as  Clostridium thermocellum , the cuvette  302  and icewater bath  308  are kept in an oxygen-free glovebox  310 . The cuvette&#39;s electrodes  304 ,  306  are connected to a high-voltage burst generator  312 , controlled by a pulse generator  314 . 
         [0043]    When the present electroporation apparatus is operated, an operator triggers the pulse generator  314  to fire the high-voltage burst generator  312 . The high-voltage burst generator then provides a high-voltage burst  316  through a current limiting resistor  318 . 
         [0044]    The high-voltage burst  316  can be described as illustrated in  FIG. 4  as the superposition of a high-voltage Initial Spike of Direct Current (DC) pulse having a DC component amplitude  402  with a superimposed sinusoidal high radio-frequency Alternating Current (AC) peak-to-peak amplitude  404  such that the Initial Spike Total Amplitude  406  is in the range of from 12 to 25 kilovolts per centimeter of electrode spacing D. The electroporation pulse has an initial spike pulsewidth  408  between approximately five and twenty percent, and in an embodiment is about ten percent of the total pulsewidth  410  of initial spike and plateau; the total pulsewidth  410  ranges from three to twenty-five milliseconds, and is preferably from eight to ten milliseconds. The plateau therefore is greater in length than half of the total pulsewidth  410 . The plateau continues the radio-frequency alternating current at approximately the same peak-to-peak amplitude  404  as during the initial spike, and has a plateau DC component  412  that is between ten and thirty percent of the initial spike DC component, and has magnitude greater than half of the AC peak-to-peak amplitude. The total magnitude  414  of the plateau of the voltage burst is the sum of the DC component  412  plus half the AC peak-to-peak amplitude  404 . The sinusoidal AC component has a frequency of between 3 and 125 MHz, being preferably within twenty percent of 24 MHz, and has peak-to-peak magnitude greater than six and a half percent of the initial spike total amplitude  406 . The high-voltage burst may be of either polarity, since relative voltages between the electrodes  304 ,  306  induce current flow in the electroporation cell. The composite of AC and DC components is known herein as a high-voltage burst. 
         [0045]    Since in the embodiment of  FIG. 4 , the AC component begins with the initial spike, the AC component overlaps the initial spike. 
         [0046]    It is believed that the initial spike serves to create pores in the bacterial cell membrane and start discharge through the cuvette, while the plateau portion serves to electrophorese the desired DNA plasmid through cell wall and through the pores into cells. It is also believed that the AC component causes current paths through the bacterial sample to vary, such that cell damage is spread out and not focused on any one portion of the cell. 
         [0047]    Pulses approximating that described in  FIG. 4 , such as those with a rapid exponential decay from the initial spike DC component to the plateau level, are expected to produce similar results. 
         [0048]    It is expected that with certain bacteria, especially difficult to transform members of genus  Clostridium , the present electroporator using the waveform of  FIG. 4  will give substantially better yields of transformed bacteria than achieved by other workers and with other apparatus. It was also found that a greater percentage of bacteria appeared largely intact after electroporation than with conventional electroporation. 
         [0049]    The present electroporator is also expected to be successful with bacterial strains from species of  Thermoanaerobacterium  and  Thermoanaerbacter , and other bacteria having characteristics resembling those of  Clostridium  species. It is also expected that the present electroporator will be successful with  Acinetobacter.    
         [0050]    Experiments where the AC component was lacking show significantly reduced transformation efficiency. 
         [0051]    An alternative high-voltage burst is illustrated in  FIG. 5  as the superposition of a high-voltage Initial Spike of Direct Current (DC) pulse having a DC component amplitude  502  in the range of from 12 to 24 kilovolts per centimeter of electrode spacing D, or 2400 to 4800 volts for electrodes spaced two millimeters apart. The electroporation pulse has an initial spike pulsewidth  504  approximately ten percent of the total pulsewidth  506  of initial spike and plateau; the total pulsewidth  506  ranges from three to twenty-five milliseconds, and is preferably from eight to ten milliseconds. The plateau has superimposed a sinusoidal radio-frequency alternating current (AC)  508  component, and has a plateau DC component  510  that is between ten and thirty percent of the initial spike DC component, and has magnitude greater than half of the AC peak-to-peak amplitude  508 . The total magnitude  512  of the voltage burst is the sum of the DC component  510  plus half the AC peak-to-peak amplitude  508 . The AC component has a frequency of between 3 and 125 MHz, being preferably about 24 MHz, and has a peak-to-peak magnitude greater than six and a half percent of the peak amplitude of the initial spike  502 . 
         [0052]    In the embodiment of  FIG. 5 , the AC component begins as the initial spike ends. 
         [0053]    In summary, the process of generating transformed bacteria begins by purifying a culture of bacteria  602  to remove most contaminating salts from the culture media. A solution of desired DNA plasmids is prepared  604 , this is combined  606  with the purified bacteria to produce a suspension of bacteria in a solution of DNA plasmids. The bacteria may be suspended in salt-free solvent and DNA solution added, or the bacteria may be suspended directly in the DNA solution. 
         [0054]    The suspension of bacteria in DNA solution is placed  608  in the electroporation cuvette  302  such that the electrodes  304 ,  306  make contact with the solution. A burst as described above with reference to  FIG. 4  or  FIG. 5  is applied  610  to the suspension. Surviving bacteria are cultured  612  and selected  614  to provide a culture of transformed bacteria. 
         [0055]    The culturing  612  and selection  614  are in a first embodiment performed by including an antibiotic resistance gene in the DNA plasmids, and including the related antibiotic in culture media in which surviving bacteria are cultured  612 . 
         [0056]    In an alternate embodiment selection  614  is performed by blotting. 
         [0057]      FIG. 7  illustrates an alternative burst that has been successfully used to transform  Clostridium thermocellum  bacteria. The burst is essentially the superposition of a square DC pulse represented as DC component  702  having a pulsewidth  704  of between three and twenty-five milliseconds, in an embodiment the pulsewidth is between eight and twelve milliseconds. The DC component  702  is superimposed on an AC component  706  having peak-to-peak magnitude greater than six and a half percent of the magnitude of the DC component  702 , and in an embodiment a frequency of twenty-four megahertz. A total magnitude  710  of the voltage burst is the sum of the DC component  702  plus half the AC peak-to-peak amplitude  706 . The AC component has a frequency of between 3 and 125 MHz, being preferably within twenty percent of 24 MHz. The burst of  FIG. 7  has total magnitude  710  in the range of between eleven kilovolts and twenty-four kilovolts per centimeter of electrode spacing in the electroporation cuvette. 
         [0058]    It was found that, with difficult to transform members of genus  Clostridium , the present electroporator using the waveform of  FIG. 7  gave better yields of transformed bacteria than achieved by other workers and with other apparatus. It was also found that a greater percentage of bacteria appeared largely intact after electroporation than with conventional electroporation. 
         [0059]    In an alternative embodiment, illustrated in  FIG. 8 , a burst having a first and a second superimposed AC signals with combined zero-to-peak magnitude  802  reaching between eleven kilovolts and twenty-four kilovolts per centimeter of spacing between electrodes in the electroporation cuvette. The first AC signal has a frequency of between twenty and five hundred kilohertz, and preferably about one hundred kilohertz, the second AC signal has a frequency of between three and one hundred twenty-five, and preferably about twenty-four, megahertz. The second AC signal has a magnitude between six and thirty percent of the magnitude of the first AC signal. The burst has a width  804  of between three and twenty-five milliseconds, in an embodiment the width is between eight and twelve milliseconds. 
         [0060]    In yet another alternative embodiment, illustrated in  FIG. 9 , a damped AC burst having a center frequency of between three and one hundred twenty-five, and preferably about twenty-four, megahertz. The damped burst has an initial zero-to-peak magnitude  902  reaching between eleven kilovolts and twenty-four kilovolts per centimeter of spacing between electrodes in the electroporation cuvette. The burst tapers in amplitude to a level approximately ten percent of the initial magnitude over a width  904  of between three and twenty-five milliseconds, in an embodiment the width is between eight and twelve milliseconds. 
         [0061]    It is anticipated that the current limiting resistor  318  may be replaced by other forms of current limiting devices appropriate for the waveform applied. In particular, it is expected that a reactive component such as an inductor, or a reactive network incorporating one or more inductors, resistors, and capacitors, are particularly suitable for use as a current limiting device with waveforms incorporating the AC components herein described. In some embodiments, current sensing with current limiting through active feedback may also be used. 
         [0062]    With the use of substantial AC components in the transforming pulse as herein described, it is anticipated that some embodiments may embed the current limiting device into an RF or pulse transformer that serves to increase voltage between the burst generator and the cuvette. 
         [0063]    While the invention has been particularly shown and described with reference to a preferred embodiment thereof, it will be understood by those skilled in the art that various other changes in the form and details may be made without departing from the spirit and scope of the invention. It is to be understood that various changes may be made in adapting the invention to different embodiments without departing from the broader inventive concepts disclosed herein and comprehended by the claims that follow.