# US 2014/0193916 A1 · Electrotransformation of Clostridium pasteurianum > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0193916 A1 - Document ID: US20140193916A1 - Status: Published application - Kind: A1 - Application number: 14/150,764 - Filed: 2014-01-08 - Published: 2014-07-10 - Earliest claimed: 2013-01-08 - Assignees: Algaeneers Inc. - Inventors: Michael Evan Pyne; Murray Moo Young; Duane Andrew Chung; Chih-Hsiung Perry Chou - Web page: https://patentgrep.com/patent/US20140193916A1 ## CPC - H04W 72/04: Wireless resource allocation - C12N 1/20: Bacteria; Culture media therefor - C12N 15/74: Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora - C12N 15/87: Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation - H04W 74/0816: with collision avoidance - H04W 84/18: Self-organising networks, e.g. ad-hoc networks or sensor networks - H04W: WIRELESS COMMUNICATION NETWORKS (broadcast communication H04H; communication systems using wireless links for non-selective communication, e.g. wireless extensions H04M1/72) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract By this invention, for the first time, a method for high-efficiency genetic transformation of the anaerobic bacterium Clostridium pasteurianum is provided. Clostridium pasteurianum is a bacterium of substantial industrial importance, due to its selectivity and high productivity of the biofuel and biochemical n-butanol, and its ability to grow on a wide variety of inexpensive substrates. Notable among the substrates that it can utilize as a sole source of carbon and energy is glycerine, which is produced in increasing quantities globally as a by-product of biodiesel processing. The industrial exploitation of Clostridium pasteurianum has previously been impeded by the lack of genetic engineering tools for this bacterium. This invention provides such tools for the first time. Included in the invention is a means for protecting newly introduced DNA from degradation by a restriction enzyme within C. pasteurianum. Then, a detailed protocol is given, which enables high-efficiency transformation of C. pasteurianum via a series of treatments and electroporation conditions which successfully negotiate the resistant cell wall of C. pasteurianum. Finally, the invention discloses selection markers and vector components, which round out the tools required to successfully perform genetic engineering in C. pasteurianum for the first time. ## Claims ### Claim 1 A method for introducing recombinant DNA constructs into one or more bacterium. ### Claim 2 (depends on 1) The method of claim 1 wherein said bacterium is a gram-positive bacteria. ### Claim 3 (depends on 1) The method of claim 1 wherein said bacterium belongs to the genus Clostridia. ### Claim 4 (depends on 1) The method of claim 1 wherein said bacterium is Clostridium pasteurianum. ### Claim 5 (depends on 1) The method of claim 1 wherein said method involves the delivery of one or more electrical pulses to said bacterium. ### Claim 6 (depends on 1) The method of claim 1 wherein said method involves the use of methylation to block the activity of a restriction enzyme within said bacterium. ### Claim 7 (depends on 1) The method of claim 1 wherein said method involves the use of a methylase which methylates a cytosine residue within the deoxyribonucleotide sequence 5′-cytosine-guanine-cytosine-guanine-3′. ### Claim 8 (depends on 1) The method of claim 1 wherein said method involves the use of a cell-wall weakening agent. ### Claim 9 (depends on 8) The method of claim 8 wherein said cell-wall weakening agent is selected from the group consisting of glycine and DL-threonine. ### Claim 10 (depends on 9) The method of claim 9 wherein said method involves the use of an osmoprotectant agent. ### Claim 11 (depends on 10) The method of claim 10 wherein said osmoprotectant is selected from the group consisting of sucrose, lactose, sorbitol, and mannitol. ### Claim 12 (depends on 9) The method of claim 9 where said method involves the use of ethanol. ### Claim 13 (depends on 4) The method of claim 4 where said recombinant DNA construct contains an origin of replication selected from the group consisting of pCB102 from Clostridium butyricum, pCD6 from Clostridium difficile, and pIM13 from Bacillus subtilis. ### Claim 14 (depends on 4) The method of claim 4 wherein said recombinant DNA construct contains a DNA sequence which encodes an enzyme that confers resistance to an antibiotic selected from the group consisting of thiamphenicol, clarithromycin, and erythromycin. ### Claim 15 (depends on 4) The method of claim 4 wherein said method comprises: (i) the delivery of one or more electrical pulses to said bacterium. (ii) the use of one or more cell-wall weakening agents selected from the group comprising glycine and DL-threonine. (iii) the use of one or more osmoprotectants selected from the group consisting of sucrose, lactose, mannitol, and sorbitol. ### Claim 16 A bacterial cell which contains one or more recombinant DNA constructs. ### Claim 17 (depends on 16) The bacterial cell of claim 16 wherein said bacterial cell is from a gram-positive bacteria. ### Claim 18 (depends on 16) The bacterial cell of claim 16 wherein said bacterial cell is from a bacteria that is a member of the genus Clostridium. ### Claim 19 (depends on 16) The bacteria cell of claim 16 wherein said bacterial cell is from Clostridium pasteurianum. ### Claim 20 (depends on 16) The bacterial cell of claim 16 wherein said one or more recombinant DNA constructs were introduced into said bacterial cell by a method involving the delivery of one or more electrical pulses to said bacterium. ### Claim 21 (depends on 16) The bacterial cell of claim 16 wherein said one or more recombinant DNA constructs were introduced into said bacterial cell by a method involving the use of methylation to block the activity of a restriction enzyme within said bacterium. ### Claim 22 (depends on 16) The bacterial cell of claim 16 wherein said one or more recombinant DNA constructs were introduced into said bacterial cell by a method involving the use of a methylase which methylates a cytosine residue within the deoxyribonucleotide sequence 5′-cytosine-guanine-cytosine-guanine-3′. ### Claim 23 (depends on 16) The bacterial cell of claim 16 wherein said one or more recombinant DNA constructs were introduced into said bacterial cell by a method involving the use of a cell-wall weakening agent. ### Claim 24 (depends on 23) The bacterial cell of claim 23 wherein said cell-wall weakening agent is selected from the group consisting of glycine and DL-threonine. ### Claim 25 (depends on 24) The bacterial cell of claim 24 wherein said method involves the use of an osmoprotectant agent. ### Claim 26 (depends on 25) The bacterial cell of claim 25 wherein said osmoprotectant is selected from the group consisting of sucrose, lactose, sorbitol, and mannitol. ### Claim 27 (depends on 24) The bacterial cell of claim 24 where said method involves the use of ethanol. ### Claim 28 (depends on 19) The bacterial cell of claim 19 where said one or more recombinant DNA constructs contain an origin of replication selected from the group consisting of pCB102 from Clostridium butyricum, pCD6 from Clostridium difficile, and pIM13 from Bacillus subtilis. ### Claim 29 (depends on 19) The bacterial cell of claim 19 wherein said one or more recombinant DNA constructs contain a DNA sequence which encodes an enzyme that confers resistance to an antibiotic selected from the group consisting of thiamphenicol, clarithromycin, and erythromycin. ### Claim 30 (depends on 19) The bacterial cell of claim 19 wherein said one or more recombinant DNA constructs are introduced into said bacterial cell by a method comprising: (i) the delivery of one or more electrical pulses to said bacterium. (ii) the use of one or more cell-wall weakening agents selected from the group comprising glycine and DL-threonine. (iii) the use of one or more osmoprotectants selected from the group consisting of sucrose, lactose, mannitol, and sorbitol.