Patent Application
20230257790
This application was filed with a Sequence Listing XML in ST.26 XML format accordance with 37 C.F.R. § 1.831. The Sequence Listing XML file submitted in the USPTO Patent Center, 10 “013670-0012-WO01_sequence_listing_xml_18-JAN-2023.xml,” was created on Jan. 18, 2023, contains 22 sequences, has a file size of 45.6 Kbytes, and is incorporated by reference in its entirety into the specification.
Described herein are compositions and methods for recombination-based assembly of long dsDNA molecules. One embodiment described herein is a method for creating large recombinant plasmids in a competent host cell using a plurality of double stranded DNA fragments containing overlapping fragments using a recombinase and exonuclease.
Nucleic acid recombination lies at the core of molecular biology and biotechnology. The efficiency whereby recombinant nucleic acid technology is achieved can dictate the outcome of certain biotechnology implementations. The ability to perform DNA assembly or the ability to physically link multiple double stranded DNA (dsDNA) fragments together to generate longer dsDNA fragments is a key technology in synthetic biology.
What are needed are methods and compositions to overcome the existing challenges of current nucleic acid recombination technologies and reduce the complexity of workflows, increase DNA assembly efficiency and fidelity, while reducing overall assembly cost.
One embodiment described herein is a method for the assembly of a plurality of double stranded DNA (dsDNA) fragments into a covalently bound circular dsDNA molecule, the method comprising: (a) combining a plurality of distinct dsDNA fragments with a reaction mixture comprising an exonuclease and a recombinase to form a DNA reaction mixture; wherein each individual dsDNA fragment comprises one or more terminal single-stranded nucleotides that are complementary to terminal single-stranded nucleotides of an independent dsDNA fragment; (b) subjecting the DNA reaction mixture to a hybridization incubation to form a hybridized DNA reaction mixture; (c) subjecting the hybridized DNA reaction mixture to a deactivation incubation to form a deactivated DNA reaction mixture; (d) transforming the deactivated DNA reaction mixture into a competent host cell; and (e) incubating the transformed competent host cell under conditions sufficient to assemble and replicate one or more covalently bound circular dsDNA molecules comprising the plurality of distinct dsDNA fragments. In one aspect, the recombinase is selected from Uvsx from a bacteriophage, Rad51 or Dmc1 from a eukaryote, RadA from archaea, or RecA from E. coli. In another aspect, the recombinase is RecA from E. coli. In another aspect, the reaction mixture further comprises ATP. In another aspect, the exonuclease is T5 Exonuclease. In another aspect, the reaction mixture further comprises a DNA polymerase and a ligase. In another aspect, the competent host cell is an E. coli cell. In another aspect, the one or more terminal single-stranded nucleotides that are complementary overlap with terminal single-stranded nucleotides of the independent dsDNA fragment by about 10 nucleotides to about 120 nucleotides. In another aspect, the one or more terminal single-stranded nucleotides that are complementary overlap with terminal single-stranded nucleotides of the independent dsDNA fragment by about 20 nucleotides to about 60 nucleotides. In another aspect, the one or more terminal single-stranded nucleotides that are complementary overlap with terminal single-stranded nucleotides of the independent dsDNA fragment by about 20 nucleotides to about 35 nucleotides. In another aspect, the one or more terminal single-stranded nucleotides that are complementary overlap with terminal single-stranded nucleotides of the independent dsDNA fragment by about 25 nucleotides to about 30 nucleotides. In another aspect, the hybridization incubation comprises a hybridization temperature of about 25° C. to about 50° C. for about 5 minutes to about 120 minutes. In another aspect, the hybridization incubation comprises a hybridization temperature of about 35° C. to about 45° C. for about 10 minutes to about 20 minutes. In another aspect, the hybridization incubation comprises a hybridization temperature of about 42° C. for about 20 minutes. In another aspect, the deactivation incubation comprises a deactivation temperature of about 60° C. to about 70° C. for about 5 minutes to about 120 minutes. In another aspect, the deactivation incubation comprises a deactivation temperature of about 65° C. for about 20 minutes. In another aspect, the deactivation incubation comprises a deactivation temperature of less than about 5° C. for about 20 minutes. In another aspect, the reaction mixture further comprises one or more crowding agents, one or more chaperone agents, or a combination thereof. In another aspect, the one or more crowding agents comprises polyethylene glycol (PEG). In another aspect, the one or more chaperone agents comprises a diol or a polyol selected from substituted straight or branched alkylene glycols, pentaerythritol, sorbitol, diethylene glycol, dipropylene glycol, neopentyl glycol, propylene glycol and ethylene glycol ethers, 1,2-ethylene glycol, 1,2-PrD, 1,3-PrD, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, 2-methyl-1,3-propanediol, 2,2′-dimethylpropylene glycol, 1,3-butylethylpropanediol, methyl propanediol, methyl pentanediols, propylene glycol methyl ether, propylene glycol ethyl ether, propylene glycol butyl ether, diethylene glycol phenyl ether, propylene glycol phenol ether, propylene glycol methyl ether, tri-propylene glycol methyl ether, propylene glycol isobutyl ether, ethylene glycol methyl ether, or combinations thereof. In another aspect, the method further comprises isolating the covalently bound circular dsDNA molecules comprising the plurality of distinct dsDNA fragments from one or more competent host cells. In another aspect, the method further comprises sequencing the covalently bound circular DNA molecule comprising the plurality of distinct dsDNA fragments following isolation from the competent host cell.
Another embodiment described herein is a system for the assembly of a plurality of double stranded DNA (dsDNA) fragments into a covalently bound circular DNA molecule, the system comprising: (a) a plurality of distinct dsDNA fragments, wherein each individual dsDNA fragment comprises one or more terminal single-stranded nucleotides that are complementary to terminal single-stranded nucleotides of an independent dsDNA fragment from the plurality of distinct dsDNA fragments; (b) a reaction mixture comprising an exonuclease and a recombinase; and (c) a competent host cell. In another aspect, the recombinase is selected from Uvsx from a bacteriophage, Rad51 or Dmc1 from a eukaryote, RadA from archaea, or RecA from E. coli. In another aspect, the recombinase is RecA from E. coli. In another aspect, the reaction mixture further comprises ATP. In another aspect, the exonuclease is T5 Exonuclease. In another aspect, the reaction mixture further comprises a DNA polymerase and a ligase. In another aspect, the competent host cell is an E. coli cell. In another aspect, the reaction mixture further comprises one or more crowding agents, one or more chaperone agents, or a combination thereof.
Another embodiment described herein is a kit for the assembly of a plurality of double stranded DNA (dsDNA) fragments into a covalently bound circular DNA molecule, the kit comprising: (a) an exonuclease; (b) a recombinase; (c) one or more buffers, crowding agents, or chaperone agents, or ATP; (d) optionally, a competent host cell; and (e) optionally, instructions or directions for use.
Another embodiment described herein is the use of an exonuclease and a recombinase for the assembly of a plurality of double stranded DNA (dsDNA) fragments into a covalently bound circular DNA molecule, comprising: (a) combining a plurality of distinct dsDNA fragments with a reaction mixture comprising an exonuclease and a recombinase to form a DNA reaction mixture, wherein each individual dsDNA fragment comprises one or more terminal single-stranded nucleotides that are complementary to terminal single-stranded nucleotides of an independent dsDNA fragment from the plurality of distinct dsDNA fragments; (b) subjecting the DNA reaction mixture to a hybridization incubation to form a hybridized DNA reaction mixture; (c) subjecting the hybridized DNA reaction mixture to a deactivation incubation to form a deactivated DNA reaction mixture; (d) transforming the deactivated DNA reaction mixture into a competent host cell; and (e) incubating the transformed competent host cell under conditions sufficient to assemble a covalently bound circular DNA molecule comprising the plurality of distinct dsDNA fragments.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of biochemistry, molecular biology, immunology, microbiology, genetics, cell and tissue culture, and protein and nucleic acid chemistry described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.
As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” “vector,” “polypeptide,” and “protein” have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.
As used herein, the terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising.” The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.
As used herein, the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified.
As used herein, the term “or” can be conjunctive or disjunctive.
As used herein, the term “substantially” means to a great or significant extent, but not completely.
As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In one aspect, the term “about” refers to any values, including both integers and fractional components that are within a variation of up to ±10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value. As used herein, the symbol “−j” means “about” or “approximately.”
All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1, 0.2, 0.3, 0.4 . . . 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to ±10% of any value within the range or within 3 or more standard deviations, including the end points.
As used herein, the terms “control,” or “reference” are used herein interchangeably. A “reference” or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. “Control” also refers to control experiments or control cells.
As used herein, the terms “inhibit,” “inhibition,” or “inhibiting” refer to the reduction or suppression of a given biological process, condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.
As used herein, the phrase “room temperature,” “RT,” or “ambient temperature” indicates a temperature of about 20-27° C.; about 25° C.±10%; or −25° C., at standard atmospheric pressure.
As used herein, the term “additive” refers to one or more components added to the buffers described herein to enhance hybridization and improve overall specificity.
As used herein, the term “nucleic acid” may refer to DNA, RNA, dsDNA, dsRNA, ssDNA, ssRNA, or hybrids of DNA/RNA complexes or sequences obtained from any source, containing target and non-target sequences. For example, a nucleic acid sample can be obtained from artificial sources or by chemical synthesis, or by enzymatic synthesis, or from viruses, prokaryotic cells including microbes, or eukaryotic cells. Biological samples may be vertebrate, including human or excluding humans, invertebrates, plants, microbes, viruses, mycoplasma, fungi, or Archaea.
As used herein, the terms “base pair” or “bp” refer to the interaction of one or more nucleotides in a single stranded nucleic acid molecule with one or more nucleotides in a complementary single stranded nucleic acid molecule via hydrogen bonding to form a double stranded nucleic acid molecule (e.g., double stranded DNA). The base pairs may be Watson-Crick, Hoogsteen, or other noncanonical base paring interactions. Typically, the base pairs are Watson-Crick base pairs, e.g., A-T (or A-U) with two hydrogen bonds, and C-G, with three hydrogen bonds.
As used herein, “complementary” refers to the ability of one nucleic acid to form base pairs with another nucleic acid. Typically, one strand runs 5′→3′ and pairs with complementary nucleotides in a second strand in the 3′→5′ direction. For example, 5′-GAATC-3′ is complementary to 5′-GATTC-3′ (i.e., 3′-CTTAG-5′) and can form a double stranded nucleic acid where each nucleotide forms a Watson-Crick base pair with its respective complement on the other strand.
As used herein, the terms “overlap” or “overlapping” refer to double stranded nucleic acids that have non-base-paired nucleotides at one or both of the 5′- or 3′-termini (e.g., “overhangs”) that are capable of base-paring with complementary non-base paired nucleotides at one or both of the 5′- or 3′-termini of another double stranded sequence (e.g., “complementary overhangs”). See
As an illustrative example, a plurality of distinct dsDNA fragments could contain four dsDNA fragments, F1, F2, F3, and F4, each top strand oriented 5′-3′. To assemble a long dsDNA, fragment F1 would have a 3′-terminus that would overlap the 5′-terminus of F2. F2 would have a 5′-terminus that would overlap the 3′-terminus of F1 and a 3′-terminus that would overlap the 5′-terminus of F3. Likewise, F3 would have a 5′-terminus that would overlap the 3′-terminus of F2 and a 3′-terminus that would overlap the 5′-terminus of F4. F4 would have a 5′-terminus that would overlap the terminus of F3.
As used herein, the terms “long nucleic acid” or “large nucleic acids” refer to a nucleic acid that is greater than 100 nucleotides (or “base pairs” for double stranded nucleic acids). This includes single stranded nucleic acids, double stranded nucleic acids, plasmids, vectors, or other constructs known in the art. Long nucleic acids can be 100 to 20,000 nucleotides or greater, including all integers and subranges within the specified range. For example, long nucleic acids can include 100-1000, 100-5000, 500-2000, 1000-5000 nucleotides, or other subranges within 100-20,000 nucleotides.
As used herein, the terms “distinct” or “different” refer to double stranded DNA molecules or “fragments” that have different nucleotide sequences. For example, “a plurality of distinct dsDNA fragments” refers to multiple dsDNA molecules each having a particular sequence. In the plurality mixture, there may be multiple copies of each distinct dsDNA molecule, collectively forming a plurality of distinct dsDNA fragments. As used herein, the distinct dsDNA fragments may have sequences at one or both of the 5′- or 3′-termini that are capable of base-paring with complementary nucleotides at one or both of the 5′- or 3′-termini of another “independent” distinct dsDNA fragment (once the complementary regions are converted to single strands). As used herein, the term “independent” refers to a distinct dsDNA fragment that has a different dsDNA sequence as compared to another distinct dsDNA fragment.
Described herein are methods and compositions for improved DNA assembly of long double stranded nucleic acid molecules using a combination of in vitro alignment and in vivo methods. The methods are improved relative to the current state of the art.
Described herein are methods and compositions for a method of creating large recombinant plasmids in a competent host cell using a plurality of fragments of DNA containing sequences that overlap with adjacent fragments. In one aspect, the methods and compositions are useful for the assembly of two or more double-stranded DNA fragments using an exonuclease capable of creating single stranded overhangs from double stranded DNA and a recombinase to catalyze and stabilize complementary base pairing of the single-stranded (ssDNA) ends. This enables multi-fragmented dsDNA assemblies without the need for ligase or polymerase. While these assemblies are not covalently linked, the stabilization of the overlapping regions by the recombinase allows for transformation into E. coli followed by ligation and replication by the cellular machinery. In one aspect, the method comprises using a recombinase to stabilize the complementary regions to permit transformation into a cell and subsequent replication of the DNA to create a dsDNA replicate.
Additionally described herein are methods and compositions for assembly of double-stranded (dsDNA) fragments with a recombinase protein. In one aspect, the methods and compositions are useful for the assembly of two or more double-stranded DNA fragments using RecA to catalyze complementary base pairing of the single stranded DNA (ssDNA) ends in an ATP-dependent manner, enabling multi-fragmented dsDNA assemblies when performed in the presence of an exonuclease and optionally a polymerase and ligase.
One embodiment described herein is a reaction mixture of a plurality of distinct dsDNA fragments containing sequences that overlap with adjacent fragments by at least 10 base pairs, an exonuclease that creates single stranded overhangs of DNA, and a recombinase that facilitates the hybridization and stabilization of the newly created single strands into a competent cell host. Following the hybridization incubation at a temperature between 25° C. and 50° C. for 5 to 120 minutes and a subsequent deactivation incubation at 65° C. for 5 to 120 minutes, the mixture is transformed into a competent host cell. Following transformation and subsequent incubation a covalently bound circular molecule of DNA containing all the overlapping fragments is made. In one aspect, the reaction mixture optionally contains a crowding agent and/or a chaperone. In another aspect, the covalently bound circular DNA molecule containing the overlapping fragments is then isolated from the transformed cell. Exemplary methods include plasmid isolations or PCR amplification directly using the cell as the target nucleic acid. In another aspect, the sequences of the isolated fragments are verified by Sanger Sequencing or Next Generation Sequencing (NGS). Another aspect described herein, is a method for carrying out the reaction mixture, transforming cells, isolating the DNA, and sequencing the isolated DNA.
One embodiment described herein is a reaction mixture of a plurality of distinct dsDNA fragments containing sequences that overlap with adjacent fragments by 10 to 120 base pairs, an exonuclease that creates single stranded overhangs of DNA, and a recombinase that facilitates the hybridization of the newly created single strands, a polymerase that fills in gaps of single stranded DNA after the recombinase facilitated annealing, and a ligase that covalently bonds the fragments of DNA after fill in. Following the initial incubation at a constant temperature, the mixture is transformed into a competent host cell. Following transformation, a covalently bound circular molecule of DNA containing all the overlapping fragments is made. In another aspect, the reaction mixture further optionally contains a crowding agent, including but not limited to a polyethylene glycol (PEG). Another aspect described herein, are methods for performing the reaction mixture, transforming cells, isolating the DNA, and sequencing the isolated DNA.
Another embodiment described herein is a mixture of a plurality of distinct dsDNA fragments containing sequences that overlap with adjacent fragments by 10 to 120 base pairs, an exonuclease that creates single stranded overhangs of DNA, and a recombinase that facilitates the hybridization of the newly created single strands, a polymerase that fills in gaps of single stranded DNA after a recombinase facilitated annealing, and a ligase that covalently bonds the fragments of DNA after fill in. Following the initial incubation at a constant temperature, the mixture is transformed into a competent host cell. Following transformation, a covalently bound circular molecule of DNA containing all the overlapping fragments is made. In one aspect the mixture optionally contains a chaperone, including but not limited to, a diol or polyol. Examples of diols or polyols include optionally substituted straight or branched alkylene glycols, pentaerythritol, sorbitol, diethylene glycol, dipropylene glycol, neopentyl glycol, such as propylene glycol and ethylene glycol ethers, such as 1,2-ethylene glycol, 1,2-PrD, 1,3-PrD, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, 2-methyl-1,3-propanediol, 2,2′-dimethylpropylene glycol, 1,3-butylethylpropanediol, methyl propanediol, methyl pentanediols, propylene glycol methyl ether, propylene glycol ethyl ether, propylene glycol butyl ether, diethylene glycol phenyl ether, propylene glycol phenol ether, propylene glycol methyl ether, tri-propylene glycol methyl ether, propylene glycol isobutyl ether, ethylene glycol methyl ether, or mixtures thereof. Another aspect described herein, is a method for carrying out the reaction mixture, transforming cells, isolating the DNA, and sequencing the isolated DNA.
In one embodiment described herein the recombinase proteins comprise one or more of Uvsx from bacteriophages, Rad51 and Dmc1 From eukaryotes, RadA from archaea or RecA from E. coli. In another aspect, the recombinase proteins are ATP dependent. In another aspect, the recombinase is RecA or recA orthologs that contain DNA binding domains and promote DNA recombination.
In one embodiment described herein, the exonuclease is thermostable. In another aspect, the exonuclease is a 5′ to 3′ exonuclease or 3′ to 5′ exonuclease. In another aspect, the exonuclease is a 3′ to 5′ exonuclease. In yet another aspect, the exonuclease is T5 exonuclease.
In another embodiment described herein, the dsDNA nucleic acid fragments have overlapping ends. That is the 5′ and 3′ ends of adjoining fragments are complementary to each other. In one aspect the overlapping ends are 5 to 100 base pairs (bp), including all integers within and the endpoints of the specified range. In another aspect the overlapping ends are 5 bp, 10 bp, 15 bp, 20 bp, 25 bp, 30 bp, 35 bp, 40 bp, 45 bp, 50 bp, 55 bp, 60 bp, 65 bp, 70 bp, 75 bp, 80 bp, 85 bp, 90 bp, 95 bp, or 100 bp. In another aspect the overlapping ends are between 10 bp and 60 bp. In another aspect the overlapping ends are 20 bp, 25 bp, 30 bp, 35 bp, 40 bp, 45 bp, or 50 bp.
In one embodiment, the dsDNA and reaction incubation time ranges from 10 minutes to 120 minutes, including all integers within and the endpoints of the specified range. In another aspect the deactivation incubation time ranges from 10 minutes to 120 minutes, including all integers within and the endpoints of the specified range. In another aspect, the hybridization conditions comprise an incubation temperature ranging from about 25° C. to about 50° C., including all integers within and the endpoints of the specified range. In another aspect, the deactivation conditions comprise an incubation temperature ranging from about 60° C. to about 70° C., including all integers within and the endpoints of the specified range. In another aspect, the deactivation conditions comprise an incubation temperature of about less than 5° C.
One embodiment described herein is a method for the assembly of a plurality of double stranded DNA (dsDNA) fragments into a covalently bound circular dsDNA molecule, the method comprising: (a) combining a plurality of distinct dsDNA fragments with a reaction mixture comprising an exonuclease and a recombinase to form a DNA reaction mixture; wherein each individual dsDNA fragment comprises one or more terminal single-stranded nucleotides that are complementary to terminal single-stranded nucleotides of an independent dsDNA fragment; (b) subjecting the DNA reaction mixture to a hybridization incubation to form a hybridized DNA reaction mixture; (c) subjecting the hybridized DNA reaction mixture to a deactivation incubation to form a deactivated DNA reaction mixture; (d) transforming the deactivated DNA reaction mixture into a competent host cell; and (e) incubating the transformed competent host cell under conditions sufficient to assemble and replicate one or more covalently bound circular dsDNA molecules comprising the plurality of distinct dsDNA fragments. In one aspect, the recombinase is selected from Uvsx from a bacteriophage, Rad51 or Dmc1 from a eukaryote, RadA from archaea, or RecA from E. coli. In another aspect, the recombinase is RecA from E. coli. In another aspect, the reaction mixture further comprises ATP. In another aspect, the exonuclease is T5 Exonuclease. In another aspect, the reaction mixture further comprises a DNA polymerase and a ligase. In another aspect, the competent host cell is an E. coli cell. In another aspect, the one or more terminal single-stranded nucleotides that are complementary overlap with terminal single-stranded nucleotides of the independent dsDNA fragment by about 10 nucleotides to about 120 nucleotides. In another aspect, the one or more terminal single-stranded nucleotides that are complementary overlap with terminal single-stranded nucleotides of the independent dsDNA fragment by about 20 nucleotides to about 60 nucleotides. In another aspect, the one or more terminal single-stranded nucleotides that are complementary overlap with terminal single-stranded nucleotides of the independent dsDNA fragment by about 20 nucleotides to about 35 nucleotides. In another aspect, the one or more terminal single-stranded nucleotides that are complementary overlap with terminal single-stranded nucleotides of the independent dsDNA fragment by about 25 nucleotides to about 30 nucleotides. In another aspect, the hybridization incubation comprises a hybridization temperature of about 25° C. to about 50° C. for about 5 minutes to about 120 minutes. In another aspect, the hybridization incubation comprises a hybridization temperature of about 35° C. to about 45° C. for about 10 minutes to about 20 minutes. In another aspect, the hybridization incubation comprises a hybridization temperature of about 42° C. for about 20 minutes. In another aspect, the deactivation incubation comprises a deactivation temperature of about 60° C. to about 70° C. for about 5 minutes to about 120 minutes. In another aspect, the deactivation incubation comprises a deactivation temperature of about 65° C. for about 20 minutes. In another aspect, the deactivation incubation comprises a deactivation temperature of less than about 5° C. for about 20 minutes. In another aspect, the reaction mixture further comprises one or more crowding agents, one or more chaperone agents, or a combination thereof. In another aspect, the one or more crowding agents comprises polyethylene glycol (PEG). In another aspect, the one or more chaperone agents comprises a diol or a polyol selected from substituted straight or branched alkylene glycols, pentaerythritol, sorbitol, diethylene glycol, dipropylene glycol, neopentyl glycol, propylene glycol and ethylene glycol ethers, 1,2-ethylene glycol, 1,2-PrD, 1,3-PrD, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, 2-methyl-1,3-propanediol, 2,2′-dimethylpropylene glycol, 1,3-butylethylpropanediol, methyl propanediol, methyl pentanediols, propylene glycol methyl ether, propylene glycol ethyl ether, propylene glycol butyl ether, diethylene glycol phenyl ether, propylene glycol phenol ether, propylene glycol methyl ether, tri-propylene glycol methyl ether, propylene glycol isobutyl ether, ethylene glycol methyl ether, or combinations thereof. In another aspect, the method further comprises isolating the covalently bound circular dsDNA molecules comprising the plurality of distinct dsDNA fragments from one or more competent host cells. In another aspect, the method further comprises sequencing the covalently bound circular DNA molecule comprising the plurality of distinct dsDNA fragments following isolation from the competent host cell.
Another embodiment described herein is a system for the assembly of a plurality of double stranded DNA (dsDNA) fragments into a covalently bound circular DNA molecule, the system comprising: (a) a plurality of distinct dsDNA fragments, wherein each individual dsDNA fragment comprises one or more terminal single-stranded nucleotides that are complementary to terminal single-stranded nucleotides of an independent dsDNA fragment from the plurality of distinct dsDNA fragments; (b) a reaction mixture comprising an exonuclease and a recombinase; and (c) a competent host cell. In another aspect, the recombinase is selected from Uvsx from a bacteriophage, Rad51 or Dmc1 from a eukaryote, RadA from archaea, or RecA from E. coli. In another aspect, the recombinase is RecA from E. coli. In another aspect, the reaction mixture further comprises ATP. In another aspect, the exonuclease is T5 Exonuclease. In another aspect, the reaction mixture further comprises a DNA polymerase and a ligase. In another aspect, the competent host cell is an E. coli cell. In another aspect, the reaction mixture further comprises one or more crowding agents, one or more chaperone agents, or a combination thereof.
Another embodiment described herein is a kit for the assembly of a plurality of double stranded DNA (dsDNA) fragments into a covalently bound circular DNA molecule, the kit comprising: (a) an exonuclease; (b) a recombinase; (c) one or more buffers, crowding agents, or chaperone agents, or ATP; (d) optionally, a competent host cell; and (e) optionally, instructions or directions for use.
Another embodiment described herein is the use of an exonuclease and a recombinase for the assembly of a plurality of double stranded DNA (dsDNA) fragments into a covalently bound circular DNA molecule, comprising: (a) combining a plurality of distinct dsDNA fragments with a reaction mixture comprising an exonuclease and a recombinase to form a DNA reaction mixture, wherein each individual dsDNA fragment comprises one or more terminal single-stranded nucleotides that are complementary to terminal single-stranded nucleotides of an independent dsDNA fragment from the plurality of distinct dsDNA fragments; (b) subjecting the DNA reaction mixture to a hybridization incubation to form a hybridized DNA reaction mixture; (c) subjecting the hybridized DNA reaction mixture to a deactivation incubation to form a deactivated DNA reaction mixture; (d) transforming the deactivated DNA reaction mixture into a competent host cell; and (e) incubating the transformed competent host cell under conditions sufficient to assemble a covalently bound circular DNA molecule comprising the plurality of distinct dsDNA fragments.
It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The exemplary compositions and formulations described herein may omit any component, substitute any component disclosed herein, or include any component disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the other components in the formulation are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof.
Various embodiments and aspects of the inventions described herein are summarized by the following clauses:
This example demonstrates dsDNA assembly using a reaction mix comprising an exonuclease, polymerase, and ligase.
A plurality of distinct dsDNA fragments with overlapping homologous ends were designed. See Table 1. 35 to 70 fmol of dsDNA fragments were added to a 20 μL reaction mix comprising 100 mM Tris·HCl, 10 mM MgCl2, 10 mM DTT, 1 M D-Sorbitol, 0.8 mM dNTPs, 0.004 U/μL T5 Exonuclease, 0.025 U/μL Polymerase, 0.2675 U/μL Ligase, 43 ng/μL. The plurality of distinct dsDNA fragments and reaction mix were incubated at 50° C. for 20 minutes, followed by incubation at 65° C. for 20 minutes. Following incubation, the reaction was transferred into competent E. coli DH5a cells and transformed via chemical transformation.
In this experiment, five linear double stranded DNA fragments were combined as described above. When correctly assembled, the fragments created a pUC-based plasmid that expressed green fluorescent protein (GFP). Evidence of correct assembly is shown by resistance to ampicillin and the fluorescent phenotype of the bacterial colonies when grown on LB agar plates (1% peptone, 0.5% yeast extract, 0.5% NaCl, 1.2% agar; 100 μg/mL ampicillin).
The transformation steps comprised transferring 2 μL of the incubated reaction mix to 15 μL of competent cells followed by transformation using heat shock at 42° C. for 30 seconds. Post transformation, 125 μL of SOC medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, and 20 mM glucose) was added to the heat shock cells followed by a 37° C. incubation for 1 hour. After the 37° C. incubation, the transformed cell and SOC media mixture was plated onto LB agar plates and incubated at 37° C. for 16 hours. Following the plate incubation, the total cells were counted as well as counting cells expressing fluorescent protein.
This example demonstrates dsDNA assembly using a reaction mix comprising recombinase, exonuclease, polymerase, and ligase.
A plurality of distinct dsDNA fragments with overlapping homologous ends were designed. See Table 1 above. 35 to 70 fmol of dsDNA fragments were added to a 20 μL reaction mix comprising 100 mM Tris·HCl, 10 mM MgCl2, 10 mM DTT, 1 M D-Sorbitol, 0.8 mM dNTPs, 0.004 U/μL T5 Exonuclease, 0.025 U/μL Polymerase, 0.2675 U/μL Ligase, 43 ng/μL RecA, and 430 mM ATP. The plurality of distinct dsDNA fragments and reaction mix were incubated at 50° C. for 20 minutes, followed by incubation at 65° C. for 20 minutes. Following incubation, the reaction was transferred into competent E. coli DH5a cells via chemical transformation.
Five linear double stranded fragments of DNA were combined as described in Example 1; See Table 1 above. When correctly assembled, the fragments created a pUC-based plasmid that expressed green fluorescent protein (GFP). Evidence of correct assembly is shown by the resistance to ampicillin and the fluorescent phenotype of the bacterial colonies when grown on LB agar plates. The transformation steps comprised transferring 2 μL of the incubated reaction mix to 15 μL of competent cells followed by transformation using heat shock at 42° C. for 30 seconds. Post transformation, 125 μL of SOC media was added to the heat shock cells followed by a 37° C. incubation for 1 hour. After the 37° C. incubation, the transformed cell and SOC media mixture was plated onto LB agar plates and incubated at 37° C. for 16 hours. Following the plate incubation, the total cells were counted as well as counting cells expressing fluorescent protein.
This example demonstrates dsDNA assembly techniques utilizing a reaction mix containing exonuclease and a recombinase. The reaction mix in this example does not contain ligase or polymerase.
A plurality of distinct dsDNA fragments with overlapping homologous ends were designed. See Table 1 above. 35-70 fmol of dsDNA fragments were added to a 20 μL reaction isothermal assembly reaction mix comprising 100 mM Tris·HCl, 10 mM MgCl2, 10 mM DTT, 1 M D-Sorbitol, 0.8 mM dNTPs, 0.004 U/μL T5 Exonuclease, 43 ng/μL RecA, and 430 mM ATP. The plurality of distinct dsDNA fragments and isothermal assembly reaction mix was incubated in a thermocycler at 50° C. for 20 minutes followed by incubation at 65° C. for 20 minutes. Following incubation, the reaction mix was transferred into DH5a E. coli cells via chemical competent transformation as described in Example 2.
This example demonstrates that the hybridization between ssDNA ends and interaction with the recombinase are non-covalent and can be disrupted by heat denaturation.
Reactions were prepared as described in Example 3. The reactions were incubated in a thermocycler at 50° C. for 20 min, 65° C. for 20 min, with and without a 2 min 95° C. heat denaturation step. In the absence of the 2 min 95° C. heat denaturation step, the 65° C. incubation was extended by 2 min. Following incubation, the reaction mixtures were transferred into competent DH5a E. coli cells via chemical transformation as described in Example 2.
This example demonstrates the efficiency of the assembly recombinase and exonuclease assembly method under different hybridization incubation temperatures.
Reaction mix compositions were setup as previously described in Example 3 (reaction mixes contain recombinase and exonuclease with no polymerase or ligase) The plurality of distinct dsDNA fragments and reaction mix were incubated in a thermocycler at 25° C., 37° C., 42° C., or 50° C. for 20 min, followed by incubation at 65° C. for 20 min. Following incubation, the samples were transformed as described in Example 2.
This example demonstrates the effect of altered overhang length on the dsDNA fragments. The dsDNA fragments were designed such that the overhangs on the 3′- and 5′-ends of adjacent dsDNA fragments contained either 15 bp, 20 bp, 25 bp, 30 bp, or 35 bp of complementarity.
Reaction mix compositions were setup as previously described in Example 3. The plurality of distinct dsDNA fragments and reaction mix were incubated as previously described in Example 2 and transformed as previously described in Example 1.
This application claims priority to U.S. Provisional Patent Application No. 63/304,792 filed on Jan. 31, 2022, which is incorporated by reference herein in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63304792 | Jan 2022 | US |
