Patent Application
20240309391
Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: 21,011 Byte (XML) file named “NCSU-39621-601” created on Jul. 12, 2022.
The present disclosure provides compositions, methods, and kits related to DNA transformation in bacteria. In particular, the present disclosure provides novel compositions and methods for enhancing DNA transformation by replicating a DNA methylation pattern used in a bacterial host strain. The compositions, methods, and systems described herein utilize cell-free transcription-translation mixtures and DNA methylation complexes to increase transformation efficiency and efficacy for any bacterial host.
The study and manipulation of bacteria is a central part of basic research, biotechnology, agriculture, and human health. In basic research, bacteria are found in virtually every habitable environment and exhibit wide ranging genetic architectures, metabolisms, and physiologics that are being widely studied. Many of these metabolic and physiological capabilities are also being exploited within industrial biotechnology, such as for chemical overproduction. Many naturally occurring bacteria associate with plants, creating opportunities to enhance agriculture through these bacteria. Finally, bacteria are closely associated with human health, whether as pathogens that cause disease or as members of the human microbiota that have been linked to health benefits as well as various metabolic, autoimmune, and neurological health conditions. Probiotic and commensal bacteria can also be engineered as non-invasive recorders of health status, as vaccines, or as cell-based therapies. Being able to genetically manipulate these bacteria is essential in all of these areas.
One of the key steps to genetic manipulation is transforming a bacterium with DNA. The transformed DNA can express genes-of-interest or be used to modulate gene expression or alter the genome of the bacterium. DNA can also be introduced into a bacterial cell through different means, such as electroporation, chemical competence, natural competence, phage delivery, conjugation, and nanoparticle delivery. Once DNA is in the cell though, it must circumvent a battery of defense systems meant to eliminate foreign DNA associated with mobile genetic elements such as phages. One of the most prominent defense systems in bacteria are restriction-modification (R-M) systems. These systems assess the methylation state of all DNA in the cell and cut DNA with the incorrect pattern. In general, a DNA methyltransferase (MTase) methylates specific DNA sequences at a particular location, while a restriction endonuclease (REase) cleaves at sequences either with or without a specific methylation. The exact configuration of these systems varies across the different types, such as Type I systems that rely on a specificity protein to direct the separate MTase and restriction REase to a particular site, while Type II systems encode specificity within the MTase and REase. These systems can be so efficient that an introduced DNA with the wrong pattern is cleaved immediately in numerous locations, resulting in massive drops in the apparent transformation efficiency. Because these patterns vary between strains, isolating plasmid DNA with the correct pattern is extremely difficult to create, particularly for any randomly selected bacterium.
Different methods of bypassing R-M systems to boost DNA transformation efficiency have been developed in bacteria to address this challenge. The most direct technique is to delete R-M genes responsible for sequence recognition and restriction. However, this technique is limited to strains with an adequate level of prior DNA transformation to permit homologous recombination. If DNA motifs recognized by R-M systems are well characterized, they can be systematically removed from transformed plasmids to bypass restriction. This approach is effective but requires extensive characterization for each new strain, and the required mutations to the plasmid could be problematic (e.g., within a resistance gene or the origin-of-replication). The most common method of bypassing R-M systems is to alter the methylation pattern of the plasmid prior to transformation to match the host's methylome. For some strains, this can be done in vitro if commercial MTases are available that methylate similarly to the host MTases, by incubating DNA with purified host MTases, or a cell lysate prepared from cells containing active host MTases. However, the most common approach is to methylate a plasmid in vivo by passaging it through an E. coli strain expressing some of the host MTases. While this approach successfully boosted transformation in several species, it is time intensive to set up for each new strain and often fails due to inefficient methylation or toxicity in s coil, especially when multiple MTases are required. For instance, many strains can contain upwards of 10 MTases, yet only two can be reasonably expressed in a given bacterium. Thus, there is a need for a widespread approach to rapidly boost DNA transformation across diverse bacteria.
One unexplored route involves cell-free transcription-translation systems (TXTL). These specially prepared lysates or reconstituted systems recapitulate transcription, translation, and some aspects of metabolism. As a result, adding DNA encoding genes-of-interest results in the production of active RNAs and proteins. Traditionally. TXTL was used for producing individual proteins considered cytotoxic, such as inhibitors of DNA replication or cell wall synthesis. More recently TXTL has been used for characterizing gene circuits, performing simple metabolic reactions, and for producing on-demand vaccines. However, they have not been harnessed as a means for actively modifying added components. Expressing MTases that create DNA modifications known to alter gene expression, in some cases proving cytotoxic, could also pose a major challenge for TXTL. Nonetheless, TXTL offers a unique opportunity to express one or even multiple MTases at a time without the need for protein purification or expression in E. coli. As disclosed further herein, it was surprisingly found that TXTL systems offered a robust platform for methylating added DNA, whether with one or multiple MTases, and the resulting methylated DNA significantly enhances transformation efficiency. In accordance with these findings, a high-throughput strategy for identifying MTases for a given host strain was developed. This approach represents a broadly applicable platform for enhancing transformation that overcomes challenges associated with current approaches.
Embodiments of the present disclosure include a composition for methylating target DNA for transformation into a host cell. In accordance with these embodiments, the composition includes a cell-free transcription-translation mixture and at least one expression construct encoding one or more components of a methylation complex. In some embodiments, the methylation complex replicates at least a portion of a methylation pattern used by the host cell on the target DNA.
In some embodiments, replicating the methylation pattern used by the host cell results in enhanced transformation efficiency. In some embodiments, replicating the methylation pattern used by the host cell bypasses the host cell's restriction modification (RM) system.
In some embodiments, the host cell is a bacterial cell. In some embodiments, the bacterial cell is selected from the group consisting of an E. coli cell, a Bifidobacterium cell, a Bacillus cell, a Campylobacter cell, a Clostridium cell, a Corynebacterium cell, a Cyanobacterium cell, a Fusobacterium cell, a Geobacillus cell, a Helicobacter cell, a Klebsiella cell, a Lactobacillus cell, a Mycobacterium cell, a Neisseria cell, a Paenibacillus cell, a Prevotella cell, a Pseudomonas cell, a Ralstonia cell, a Salmonella cell, a Serratia cell, a Shewanella cell, a Staphylococcus cell, a Streptococcus cell, a Vibrio cell, and a Yersinia cell, or any variants thereof.
In some embodiments, the cell free transcription-translation mixture is derived from bacterial cell lysate. In some embodiments, at least one component of the cell free transcription-translation mixture is purified.
In some embodiments, one or more components of the methylation complex comprises at least one of: (i) a methyltransferase, and a specificity protein associated with a Type I RM system in the host cell; (ii) a methyltransferase associated with a Type II RM system in the host cell; (iii) a methyltransferase associated with a Type III RM system in the host cell; and/or (iv) an orphan methyltransferase.
In some embodiments, one or more components of the methylation complex comprises at least one methyltransferase. In some embodiments, at least one methyltransferase is derived from the host cell.
In some embodiments, at least one methyltransferase is mutated. In some embodiments, the mutated methyltransferase comprises a mutation that enhances methylation of the target DNA. In some embodiments, the mutation occurs in the N-terminal domain of a Type IA methyltransferase. In some embodiments, the mutated methyltransferase comprises a mutation that enhances recognition ofunmethylated DNA.
In some embodiments, the composition further comprises S-Adenosyl methionine (SAM) as a methyl donor. In some embodiments, the composition further comprises a suitable methylation buffer. In some embodiments, the composition further comprises RNase A and/or Proteinase K.
Embodiments of the present disclosure also include a kit for methylating target DNA for transformation into a host cell. In accordance with these embodiments, the kit includes a cell-free transcription-translation mixture and at least one expression construct encoding one or more components of a methylation complex. In some embodiments, the methylation complex replicates at least a portion of a methylation pattern used by the host cell on the target DNA.
In some embodiments of the kit, one or more components of the methylation complex comprises at least one of: (i) a methyltransferase, and a specificity protein associated with a Type I RM system in the host cell; (ii) a methyltransferase associated with a Type II RM system in the host cell; (iii) a methyltransferase associated with a Type III RM system in the host cell; and/or (iv) an orphan methyltransferase.
In some embodiments of the kit, the one or more components of the methylation complex comprises a mutated methyltransferase, wherein the mutation enhances recognition ofunmethylated DNA.
In some embodiments, the kit further comprises one or more of: (i)S-Adenosyl methionine (SAM) as a methyl donor; (ii) a suitable methylation buffer; (iii) RNase A; and/or (iv) Proteinase K.
In some embodiments, the kit further comprises a set of barcoded plasmid DNA constructs for determining a methylation pattern in a host cell.
In some embodiments, the kit further comprises a lookup table comprising a list of methyltransferases associated with a host cell.
In some embodiments of the kit, the host cell is selected from the group consisting of an E. coli cell, a Bifidobacteria cell, a Bacillus cell, a Campylobacter cell, a Clostridium cell, a Helicobacter cell, a Mycobacterium cell, a Neisseria cell, a Pseudomonas cell, a Salmonella cell, a Staphylococcus cell, a Streptococcus cell, a Vibrio cell, and a Yersinia cell, or any variants thereof.
Embodiments of the present disclosure also include a method of methylating a target DNA for transforming into a host cell. In accordance with these embodiments, the method includes expressing one or more components of a methylation complex in a cell-free transcription-translation mixture comprising the target DNA. In some embodiments, the methylation complex replicates at least a portion of a methylation pattern used by the host cell on the target DNA.
In some embodiments of the method, replicating the methylation pattern used by the host cell bypasses the host cell's restriction modification (RM) system and enhances transformation efficiency.
In some embodiments of the method, the host cell is selected from the group consisting of an E. coli cell, a Bifidobacterium cell, a Bacillus cell, a Campylobacter cell, a Clostridium cell, a Corynebacterium cell, a Cyanobacterium cell, a Geobacillus cell, a Helicobacter cell, a Klebsiella cell, a Lactobacillus cell, a Mycobacterium cell, a Neisseria cell, a Paenibacillus cell, a Prevotella cell, a Pseudomonas cell, a Ralstonia cell, a Salmonella cell, a Serratia cell, a Shewanella cell, a Staphylococcus cell, a Streptococcus cell, a Vibrio cell, and a Yersinia cell, or any variants thereof.
In some embodiments of the method, one or more components of the methylation complex comprises at least one of: (i) a methyltransferase, and a specificity protein associated with a Type I RM system in the host cell; (ii) a methyltransferase associated with a Type II RM system in the host cell; (iii) a methyltransferase associated with a Type III RM system in the host cell; and/or (iv) an orphan methyltransferase.
In some embodiments of the method, one or more components of the methylation complex comprises a mutated methyltransferase, wherein the mutation enhances recognition of unmethylated DNA.
In some embodiments, the method further comprises isolating and/or purifying the methylated target DNA prior to transforming into the host cell.
In some embodiments, the method further comprises determining the one or more components of a methylation complex that are compatible with the host strain.
In some embodiments of the method, determining the one or more components of a methylation complex that are compatible with the host strain comprises at least one of: (i) consulting a lookup table comprising a list of methyltransferases associated with the host cell; (ii) use of a set of barcoded plasmid DNA constructs for determining the methylation pattern used in the host cell, and/or (iii) conducting bioinformatics analysis.
Embodiments of the present disclosure also include a method of identifying a methyltransferase compatible with a host cell. In accordance with these embodiments, the method includes: (i) generating a library of plasmid DNA constructs, wherein at least one plasmid in the library comprises a barcode associated with a candidate methyltransferase or set of methyltransferases; (ii) expressing each of the candidate methyltransferase or set of methyltransferases in a cell-free transcription-translation mixture comprising the associated barcoded plasmid construct from the library of DNA plasmid constructs, thereby methylating the associated barcoded plasmid construct; (iii) transforming the library of barcoded and methylated plasmid DNA constructs into a host cell; and (iv) determining the frequency of each of barcode and methylated plasmid DNA construct isolated from the host cell, thereby identifying the candidate methyltransferase or set of methyltransferases compatible with the host cell.
In some embodiments, the host cell is selected from the group consisting of an E. coli cell, a Bifidobacterium cell, a Bacillus cell, a Campylobacter cell, a Clostridium cell, a Corynebacterium cell, a Cyanobacterium cell, a Geobacillus cell, a Helicobacter cell, a Klebsiella cell, a Lactobacillus cell, a Mycobacterium cell, a Neisseria cell, a Paenibacillus cell, a Prevotella cell, a Pseudomonas cell, a Ralstonia cell, a Salmonella cell, a Serratia cell, a Shewanella cell, a Staphylococcus cell, a Streptococcus cell, a Vibrio cell, and a Yersinia cell, or any variants thereof.
In some embodiments, the methyltransferase comprises at least one of: (i) a methyltransferase, and a specificity protein associated with a Type I RM system in the host cell; (ii) a methyltransferase associated with a Type II RM system in the host cell; (iii) a methyltransferase associated with a Type III RM system in the host cell; and/or (iv) an orphan methyltransferase.
Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description.
The present disclosure provides compositions, methods, and kits related to DNA transformation in bacteria. In particular, the present disclosure provides novel compositions and methods for enhancing DNA transformation by replicating a DNA methylation pattern used in a bacterial host strain. The compositions, methods, and systems described herein utilize cell-free transcription-translation mixtures and DNA methylation complexes to increase transformation efficiency and efficacy for any bacterial host.
As described further herein, DNA transformation represents an ongoing and substantial challenge for those working with non-model bacteria. This step is critical for performing genetic manipulations, yet remains one of the most significant challenges for many bacteria. This is part due to the fact that the various ways to improve transformation developed for one bacterium do not necessarily transfer to even related strains. In many cases, the principal barrier is posed by restriction-modification systems that adorn host DNA with a specific methylation pattern and actively cleave DNA with a different pattern. Prior work has shown that mimicking a host's methylation pattern can radically boost DNA transformation. To date, creating this pattern has required expressing DNA methyltransferases (MTases) in E. coli or purifying the MTases to for in vitro reactions. However, only one or two MTases can be generally expressed in E. coli and many are cytotoxic, while MTase purification can be a laborious and time-consuming process. Additionally, these approaches have principally relied on Type II MTases encoded by a single protein, while other types require expressing multiple proteins. As a result, these approaches remain significant challenges and only partially replicate the methylation pattern.
To address these issues, the IMPRINT (Imitating Methylation Patterns Rapidly IN TXTL) DNA transformation platform was developed. This method involves expressing MTases in a cell-free transcription-translation (TXTL) reaction mixture, and combining the expressed MTases in a methylation reaction with target plasmid DNA. The plasmid DNA can then be isolated and transformed into the target bacterium. The process that includes obtaining the MTase constructs and then the purified target plasmid DNA takes significantly less time than methods currently used, which can take days to weeks. In addition, TXTL reaction mixtures can be developed and used for all types of MTases and can be combined into a single methylation reaction. The time/costs savings and the ability to fully replicate a bacterium's methylation pattern represents a disruptive capability that would benefit bacterial researchers in biomedical, agricultural, and environmental sciences, as well as for strain engineering for cell-based therapies and industrial biotechnology (see, e.g.,
Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.
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. In case of conflict, the present document, including definitions, will control. Preferred 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 present disclosure. The phrase “in one embodiment” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. 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.
For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
“Correlated to” as used herein refers to compared to.
As used herein, the term “nucleic acid molecule” refers to any nucleic acid containing molecule, including but not limited to, DNA or RNA. The term encompasses sequences that include any of the known base analogs of DNA and RNA including, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethvl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacctic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.
The term “gene” refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences for the production of a polypeptide, precursor, or RNA (e.g., rRNA, tRNA, sRNA, microRNA, lincRNA). The polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, immunogenicity, etc.) of the full-length or fragment are retained. The term also encompasses the coding region of a structural gene and the sequences located adjacent to the coding region on both the 5′ and 3′ ends for a distance of about 1 kb or more on either end such that the gene corresponds to the length of the full-length mRNA. Sequences located 5′ of the coding region and present on the mRNA are referred to as 5′ non-translated sequences. Sequences located 3′ or downstream of the coding region and present on the mRNA are referred to as 3′ non-translated sequences. The term “gene” encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed “introns” or “intervening regions” or “intervening sequences.” Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.
As used herein, the term “heterologous gene” refers to a gene that is not in its natural environment. For example, a heterologous gene includes a gene from one species introduced into another species. A heterologous gene also includes a gene native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to non-native regulatory sequences, etc.). Heterologous genes are distinguished from endogenous genes in that the heterologous gene sequences are typically joined to DNA sequences that are not found naturally associated with the gene sequences in the chromosome or are associated with portions of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally expressed).
As used herein, the term “oligonucleotide,” refers to a short length of single-stranded polynucleotide chain. Oligonucleotides are typically less than about 300 residues long (e.g., between 15 and 100), however, as used herein, the term is also intended to encompass longer polynucleotide chains. Oligonucleotides are often referred to by their length. For example, a 24-residue oligonucleotide is referred to as a “24-mer.” Oligonucleotides can form secondary and tertiary structures by self-hybridizing or by hybridizing to other polynucleotides. Such structures can include, but are not limited to, duplexes, hairpins, cruciforms, bends, and triplexes.
The term “homology” and “homologous” refers to a degree of identity. There may be partial homology or complete homology. A partially homologous sequence is one that is less than 100% identical to another sequence.
As used herein, the terms “complementary” or “complementarity” are used in reference to polynucleotides (e.g., a sequence of nucleotides such as an oligonucleotide or a target nucleic acid) related by the base-pairing rules. For example, for the sequence “5′-A-G-T-3′” is complementary to the sequence “3′-T-C-A-5′.” Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids. Either term may also be used in reference to individual nucleotides, especially within the context of polynucleotides. For example, a particular nucleotide within an oligonucleotide may be noted for its complementarity, or lack thereof, to a nucleotide within another nucleic acid strand, in contrast or comparison to the complementarity between the rest of the oligonucleotide and the nucleic acid strand.
In some contexts, the term “complementarity” and related terms (e.g., “complementary,” “complement”) refers to the nucleotides of a nucleic acid sequence that can bind to another nucleic acid sequence through hydrogen bonds, e.g., nucleotides that are capable of base pairing, e.g., by Watson-Crick base pairing or other base pairing. Nucleotides that can form base pairs, e.g., that are complementary to one another, are the pairs; cytosine and guanine, thymine and adenine, adenine and uracil, and guanine and uracil. The percentage complementarity need not be calculated over the entire length of a nucleic acid sequence. The percentage of complementarity may be limited to a specific region of which the nucleic acid sequences that are base-paired, e.g., starting from a first base-paired nucleotide and ending at a last base-paired nucleotide. The complement of a nucleic acid sequence as used herein refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5′ end of one sequence is paired with the 3′ end of the other, is in “antiparallel association.” Certain bases not commonly found in natural nucleic acids may be included in the nucleic acids of the present invention and include, for example, inosine and 7-deazaguanine. Complementarity need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs.
Thus, in some embodiments, “complementary” refers to a first nucleobase sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complement of a second nucleobase sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more nucleobases, or that the two sequences hybridize under stringent hybridization conditions. “Fully complementary” means each nucleobase of a first nucleic acid is capable of pairing with each nucleobase at a corresponding position in a second nucleic acid. For example, in certain embodiments, an oligonucleotide wherein each nucleobase has complementarity to a nucleic acid has a nucleobase sequence that is identical to the complement of the nucleic acid over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more nucleobases.
As used herein, a “double-stranded nucleic acid” may be a portion of a nucleic acid, a region of a longer nucleic acid, or an entire nucleic acid. A “double-stranded nucleic acid” may be, e.g., without limitation, a double-stranded DNA, a double-stranded RNA, a double-stranded DNA/RNA hybrid, etc. A single-stranded nucleic acid having secondary structure (e.g., base-paired secondary structure) and/or higher order structure comprises a “double-stranded nucleic acid”. For example, triplex structures are considered to be “double-stranded.” In some embodiments, any base-paired nucleic acid is a “double-stranded nucleic acid.”
The term “isolated” when used in relation to a nucleic acid, as in “an isolated oligonucleotide” or “isolated polynucleotide” refers to a nucleic acid sequence that is identified and separated from at least one component or contaminant with which it is ordinarily associated in its natural source. Isolated nucleic acid is such present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids as nucleic acids such as DNA and RNA found in the state they exist in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs that encode a multitude of proteins. However, isolated nucleic acid encoding a given protein includes, by way of example, such nucleic acid in cells ordinarily expressing the given protein where the nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid, oligonucleotide, or polynucleotide may be present in single-stranded or double-stranded form. When an isolated nucleic acid, oligonucleotide or polynucleotide is to be utilized to express a protein, the oligonucleotide or polynucleotide will contain at a minimum the sense or coding strand (i.e., the oligonucleotide or polynucleotide may be single-stranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide or polynucleotide may be double-stranded).
As used herein, the term “purified” or “to purify” refers to the removal of components (e.g., contaminants) from a sample. For example, antibodies are purified by removal of contaminating non-immunoglobulin proteins; they are also purified by the removal of immunoglobulin that does not bind to the target molecule. The removal of non-immunoglobulin proteins and/or the removal of immunoglobulins that do not bind to the target molecule results in an increase in the percent of target-reactive immunoglobulins in the sample. In another example, recombinant polypeptides are expressed in bacterial host cells and the polypeptides are purified by the removal of host cell proteins; the percent of recombinant polypeptides is thereby increased in the sample.
Preferred 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 present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
As described further herein, embodiments of the present disclosure represent a novel way of increasing DNA transformation efficiency and efficacy in recalcitrant bacteria by quickly assessing and overcoming the DNA restriction barrier posed by host restriction-modification (R-M) systems. The compositions and methods provided in the present disclosure involve producing host methyltransferases using cell-free transcription-translation (TXTL) machinery from E. coli lysate and/or various purified TXTL components, and imbuing shuttle plasmids (e.g., target DNA) with the produced methyltransferases to rapidly methylate DNA before transforming it into the bacterial host. These compositions and method represent a stark contrast to the current state-of-the art methods for overcoming the R-M restriction barrier that require producing the desired methyltransferases inside an E. coli host, which is not only time intensive to set up, it also often results in a failed transformation due to induced cellular toxicity by the expressed heterologous methyltransferases.
The compositions and methods of the present disclosure are able to replicate rapidly the methylation patterns of a given bacterial host strain using a TXTL reaction mixture that involves three basic steps and takes approximately 18 hours to complete. In some embodiments, a first 12-14 hour TXTL reaction is set up to produce the desired methyltransferases (method of identifying a compatible methyltransferase are described further below). Then, a methylation reaction is conducted by combining the plasmid of interest with methylation buffer and a small amount of the TXTL mix containing the methyltransferases. A methyl donor (e.g., S-Adenosyl methionine or SAM) can be added to boost methylation, although it is not explicitly required because it is often times already present in the TXTL mixture. Finally, the target DNA plasmid is purified from the TXTL and methylation components using Proteinase K. RNase A, and column purification to yield clean, methylated DNA that is suitable for transformation into the bacterial host.
As described further herein, this method was demonstrated by expressing E. coli MG1655 Dam and Dcm methyltransferases, and validating complete methylation of a target plasmid with restriction digestion by methyl-sensitive restriction enzymes DpnI and PspGI. The method was then used to assess and quickly overcome the R-M barrier to DNA transformation in Salmonella enterica LT2, which involved expressing a Type III methyltransferase and Type I methyltransferase with the associated specificity protein from this strain. It was found that the Type I R-M methyltransferase does not efficiently utilize unmethylated DNA as a substrate, and improved methylation was achieved by mutating two amino acids in this methyltransferase that are conserved in the E. coli Type I R-M methyltransferase EcoK and have been found to promote methylation of unmethylated substrates. The method was then used to increase DNA transformation in the probiotic bacterium Bifidobacterium breve UCC2003 by expressing three well-characterized Type II R-M methyltransferases. As would be recognized by one of skill in the art based on the present disclosure, this method can be applied to any bacterial strains, including those of Bifidobacteria, that are currently difficult or virtually impossible to successfully transform with conventional methods/compositions.
Additionally, a high-throughput approach was developed to quickly determine the minimal methylation pattern for efficient DNA transformation in diverse Bifidobacteria strains by barcoding an E. coli-Bifidobacteria shuttle plasmid and sequencing a pool of plasmids from a single transformation containing every combination of methyltransferases. Overall, this approach provides a customized platform for rapidly improving and/or making feasible DNA transformation in any bacterium harnessing R-M defense systems.
In accordance with these embodiments, the present disclosure provides a composition for methylating target DNA for transformation into a host cell. In some embodiments, the composition includes a cell-free transcription-translation mixture and at least one expression construct encoding one or more components of a methylation complex. In some embodiments, the methylation complex replicates at least a portion of a methylation pattern used by the host cell on the target DNA. As demonstrated herein, replicating at least a portion of the methylation pattern used by a host cell on any given DNA plasmid results in enhanced transformation efficiency and efficacy as the methylated target DNA plasmid bypasses the host cell's restriction modification (RM) system.
As would be readily recognized by one of ordinary skill in the art based on the present disclosure, the IMPRINT methodology described herein can be applied to any bacterial host cell, and is particularly applicable for use in bacterial strains that are difficult to transform (e.g., the methylation patterns and/or methyltransferases in that host strain have not been characterized). For example, bacterial host strains that may be used with the IMPRINT methods of the present disclosure include, but are not limited to E. coli, Bifidobacterium, Bacillus, Campylobacter, Clostridium, Corynebacterium, Cyanobacterium, Geobacillus, Helicobacter, Klebsiella, Laclobacillus, Mycobacterium, Neisseria, Paenibacillus, Prevotella, Pseudomonas, Ralsionia, Salmonella, Serratia, Shewanella, Staphylococcus, a Streptococcus, Vibrio, and Yersinia, or any variants thereof.
In some embodiments, a composition for methylating target DNA for transformation into a host cell includes a cell-free transcription-translation (TXTL) reaction mixture that is derived from bacterial lysate, including any of the bacterial strains listed above. In some embodiments, at least one component of the cell free transcription-translation mixture is purified. That is, the TXTL mixtures of the present disclosure can include any combination of lysate and purified components, as long as the mixture (with or without a purified component) is able to facilitate the expression of a desired transcript into a functional protein. In other embodiments, such as those involving competent bacterial host strains, the cell free transcription-translation mixture is added directly to a host cell culture without purification.
As described further herein, the use of a transcription-translation mixture offers a unique method to methylate target DNA that is significantly easier, faster, and more scalable than existing approaches that rely on purified MTases or expressing MTases in F co/i. TXTL systems have been used to produce different types of proteins, although the proteins themselves represent the endpoint of the reaction, or these proteins or their products are purified for downstream applications. However, producing MTases using TXTL systems has not been explored for methylating DNA, and in particular, has not been explored for the purpose of enhancing DNA transformation. Therefore, prior to the results described in the present disclosure, the applicability of TXTL systems for DNA methylation applications was not known, including whether MTases could be sufficiently produced using TXTL systems to allow for methylation of target plasmid DNA. As described further herein, embodiments of the present disclosure demonstrate the utility of this approach.
In some embodiments, a composition for methylating target DNA for transformation into a host cell includes at least one expression construct encoding one or more components of a methylation complex. In some embodiments, the one or more components of the methylation complex includes at least one of a methyltransferase, and a specificity protein associated with a Type I RM system in the host cell. In some embodiments, the one or more components of the methylation complex includes a methyltransferase associated with a Type II RM system in the host cell. In some embodiments, the one or more components of the methylation complex includes a methyltransferase associated with a Type III RM system in the host cell. In some embodiments, the one or more components of the methylation complex includes an orphan methyltransferase. An orphan methyltransferase (e.g., Dam methylase) is a methyltransferase that is not part of a restriction-modification system but operates independently to regulate gene expression, mismatch repair, and bacterial replication amongst many other functions. In some embodiments, the one or more components of the methylation complex includes an orphan methyltransferase that is associated with a host cell or that is obtained from a different bacterium but produces the desired methylation pattern. In some embodiments, the one or more components of the methylation complex includes at least one methyltransferase. In some embodiments, the at least one methyltransferase is derived from the host cell.
As described further herein, existing approaches for overcoming R-M systems to facilitate DNA transformation in a bacterial host cell have generally been limited to Type II MTases, single proteins that methylate without other proteins, and usually only one or two of these MTases are used at a time. However, results of the present disclosure demonstrate the use of MTases from all types, and these be combined to methylate DNA with many MTases at one time (i.e., multiplexed). This facilitates the implementation of more complicated DNA methylation patterns that in turn greatly enhance transformation efficiency and efficacy compared to current approaches.
In some embodiments, methods and compositions of the present disclosure include using IMPRINT in a multiplexed platform (e.g., using multiple MTases). As described further herein, the identification of certain mutations in MTases can facilitate the use of multiple MTases in a given methylation reaction. In some embodiments, at least one methyltransferase is mutated. In some embodiments, the mutated methyltransferase comprises a mutation that enhances methylation of the target DNA. In some embodiments, the mutation occurs in the N-terminal domain of a Type IA methyltransferase. In some embodiments, the mutated methyltransferase comprises a mutation that enhances recognition ofunmethylated DNA.
In some embodiments, a composition for methylating target DNA for transformation into a host cell includes various other components. For example, in some embodiments, the composition comprises S-Adenosyl methionine (SAM) as a methyl donor. In some embodiments, the composition comprises a suitable methylation buffer. In some embodiments, the composition further RNase A and/or Proteinase K.
As would be recognized by one of ordinary skill in the art based on the present disclosure, IMPRINT can be used to enhance DNA transformation efficacy and/or efficiency in bacterial host strains that are cultured in isolation. However. IMPRINT compositions and methods can also be used with various other suitable delivery vehicles and/or devices to implement IMPRINT in other settings where genetic engineering can be advantageous, such as in situ microbiome editing, which includes but is not limited to, targeting bacteria in the environment, in the soil of agricultural fields, in human microbiomes, and the like (see, e.g.,
Additionally, IMPRINT can be used to enhance transformation with any DNA transformation methods, including conjugation (e.g., DNA transfer between bacteria through formation of a pilus) and phagemid delivery (e.g., delivery of a plasmid packaged by a bacteriophage particle). For example, as described further herein, bacteria can be engineered to express an MTase or a combination of MTases to facilitate the methylation of a plasmid DNA that can then be transferred via conjugation to another bacteria. IMPRINT is also compatible with any in vitro methylation reaction performed using an MTase or a combination of MTases. Furthermore, IMPRINT can be used with circular DNA as well as linear DNA. For example, circular and linear DNA can be used to express an MTase or a combination of MTases to perform IMPRINT, and circular and linear DNA can be methylated using IMPRINT to improve transformation efficiency.
In accordance with these embodiments, the present disclosure also includes a method of methylating a target DNA for transforming into a host cell. In some embodiments, the method includes expressing one or more components of a methylation complex in a cell-free transcription-translation mixture comprising the target DNA. In some embodiments, the methylation complex replicates at least a portion of a methylation pattern used by the host cell on the target DNA. In some embodiments of the method, replicating the methylation pattern used by the host cell bypasses the host cell's restriction modification (RM) system and enhances transformation efficiency.
In some embodiments of the method, the host cell includes, but is not limited to, E. coli, Bifidobacterium, Bacillus, Campvlobacter, Clostridium, Corynebacterium, Cyanobacterium, Geobacillus, Helicobacter, Klebsiella, Lactobacillus, Mycobacterium, Neisseria, Paenmbacillus, Prevotella, Pseudomonas, Ralstonia, Sallmonella, Serratia, Shewanella, Staphylococcus, a Streptococcus, Vibrio, and Yersinia, or any variants thereof.
In some embodiments of the method, one or more components of the methylation complex includes (i) a methyltransferase, and a specificity protein associated with a Type I RM system in the host cell; (ii) a methyltransferase associated with a Type II RM system in the host cell; (iii) a methyltransferase associated with a Type III RM system in the host cell; and/or (iv) an orphan methyltransferase. In some embodiments of the method, one or more components of the methylation complex comprises a mutated methyltransferase, wherein the mutation enhances recognition ofunmethylated DNA.
In some embodiments, the method further comprises isolating and/or purifying the methylated target DNA prior to transforming into the host cell. As would be recognized by one of ordinary skill in the art based on the present disclosure, methods for purifying a target DNA or plasmid prior to transformation into a bacterial host cell can include the use of a conventional DNA purification column, and/or treatment with RNase and/or Proteinase K.
Embodiments of the present disclosure also include a system or kit for methylating target DNA for transformation into a host cell. In accordance with these embodiments, the kit includes a cell-free transcription-translation (TXTL) mixture and at least one expression construct encoding one or more components of a methylation complex. In some embodiments, the methylation complex replicates at least a portion of a methylation pattern used by the host cell on the target DNA.
In some embodiments of the kit, one or more components of the methylation complex includes (i) a methyltransferase, and a specificity protein associated with a Type I RM system in the host cell; (ii) a methyltransferase associated with a Type II RM system in the host cell; (iii) a methyltransferase associated with a Type III RM system in the host cell; and/or (iv) an orphan methyltransferase.
In some embodiments of the kit, the one or more components of the methylation complex comprises a mutated methyltransferase, wherein the mutation enhances recognition ofunmethylated DNA. In some embodiments, the kit comprises one or more of: (i)S-Adenosyl methionine (SAM) as a methyl donor; (ii) a suitable methylation buffer; (iii) RNase A; and/or (iv) Proteinase K.
As described further herein, a kit for methylating target DNA for transformation into a host cell can include a set of barcoded plasmid DNA constructs for determining a methylation pattern in a host cell (see, e.g.,
As described further herein, embodiments of the present disclosure include methods of methylating a target DNA for transforming into a host cell, including expressing one or more components of a methylation complex in a cell-free transcription-translation mixture comprising the target DNA. In accordance with these embodiments, the present disclosure includes methods for determining whether one or more components of a methylation complex are compatible with a particular host strain. In some embodiments of the method, determining whether one or more components of a methylation complex are compatible with the host strain includes consulting a lookup table comprising a list of methyltransferases associated with the host cell. In some embodiments of the method, determining whether one or more components of a methylation complex are compatible with the host strain includes the use of a set of barcoded plasmid DNA constructs for determining the methylation pattern used in the host cell (see, e.g.,
In some embodiments, the method can be used to identify a methyltransferase that is compatible with a given host strain, such that it can be expressed as part of the IMPRINT methodology described herein, and used to enhance DNA transformation in that host cell. In some embodiments, one or more aspects of a method for identifying a suitable methyltransferase in a particular host strain can be performed by a user carrying out IMPRINT. In other embodiments, one or more aspects of a method for identifying a suitable methyltransferase in a particular host strain can be performed by an off-site scientific professional that supports a user that will carry out IMPRINT.
For example, a user that wishes to implement IMPRINT can provide answers to the questions in the workflow provided in
In accordance with these embodiments, a user can consult an off-site scientific professional for support in identifying a suitable methyltransferase for a desired bacterial host strain (see, e.g.,
Embodiments of the present disclosure also include a method of identifying a methyltransferase compatible with a host cell. In accordance with these embodiments, the method includes: (i) generating a library of plasmid DNA constructs, wherein at least one plasmid in the library comprises a barcode associated with a candidate methyltransferase or set of methyltransferases; (ii) expressing each of the candidate methyltransferase or set of methyltransferases in a cell-free transcription-translation mixture comprising the associated barcoded plasmid construct from the library of DNA plasmid constructs, thereby methylating the associated barcoded plasmid construct; (iii) transforming the library of barcoded and methylated plasmid DNA constructs into a host cell; and (iv) determining the frequency of each of barcode and methylated plasmid DNA construct isolated from the host cell, thereby identifying the candidate methyltransferase or set of methyltransferases compatible with the host cell.
In some embodiments, the host cell is selected from the group consisting of an E. coli cell, a Bifidobacterium cell, a Bacillus cell, a Campylobacter cell, a Clostridium cell, a Corynebacterium cell, a Cyanobacterium cell, a Geobacillus cell, a Helicobacter cell, a Klebsiella cell, a Lactobacillus cell, a Mycobacterium cell, a Neisseria cell, a Paenibacillus cell, a Prevotella cell, a Pseudomonas cell, a Ralstonia cell, a Salmonella cell, a Serratia cell, a Shewanella cell, a Staphylococcus cell, a Streptococcus cell, a Vibrio cell, and a Yersinia cell, or any variants thereof. In some embodiments, the methyltransferase comprises at least one of: (i) a methyltransferase and a specificity protein associated with a Type I RM system; (ii) using a set of barcoded plasmid DNA constructs associated with a methyltransferase or a combination of methyltransferases for determining a methylation pattern and identifying a corresponding methyltransferase or combination of methyltransferases that is compatible with host cell; (iii) a methyltransferase associated with a Type III RM system; and/or (iv) an orphan methyltransferase that produces the same methylation pattern.
Standard growth conditions. E. coli and S. enterica propagation was performed in LB medium (10 g/L NaCl, 5 g/L yeast extract, 10 g/L tryptone) while being shaken at 250 rpm at 37° C., aside from E. coli KL740 which was grown at 30° C. Plasmids were maintained at the following antibiotic concentrations; ampicillin (50 μg/mL), chloramphenicol (34 μg/mL).
Bifidobacteria were routinely grown in MRS liquid broth (BD CN #288130) supplemented with 0.05% L-cysteine and MRS agar (BD CN #288210) or Reinforced Clostridial Agar (RCA, Thermo CN #CM0151B) and incubated at 37° C. in an anaerobic chamber. Tetracycline was used to maintain plasmids at a concentration of 20 μg/mL.
Plasmid generation. Plasmid pJV170 was used to readily clone new methyltransferases under P70a expression. Cloning was performed by amplifying plasmid pJV170 and host genomic DNA with primers that include homology tails, then by performing Gibson assembly according to manufacturer's protocols (NEB CN #E261IS). The assembly mix was transformed into E. coli KL740 by electroporation, and colonies were screened using colony PCR and Sanger sequencing to determine if the clone was correct.
When necessary, small insertions or mutations were inserted into plasmids using Q5 site-directed mutagenesis (NEB CN #E0554S) according to manufacturer's protocols and transforming chemically competent NEB 10-beta cells (NEB CN #C3019), where colony PCR and Sanger sequencing were again used to determine if the clone was correct.
To clone a library of barcoded E. coli-Bifidobacteria shuttle vectors, Q5 mutagenesis was performed according to manufacturer's protocols where four random nucleotides were added to the 5′ end of one of the primers. The Q5 mutagenesis mix was transformed into NEB 10-beta chemically competent cells, and individual barcodes were isolated using colony PCR and Sanger sequencing to determine the barcode sequence of each clone.
In vitro methylation of plasmid DNA. To quickly methylate DNA in vitro using IMPRINT, TXTL reactions were first set up by incubating TXTL master mix (Arbor Biosciences CN #507024) with each methyltransferase plasmid harboring the methyltransferase gene(s) to be expressed at 29° C. for 12-16 hours. The shuttle plasmid was first propagated in methyltransferase-deficient E. coli EC135 to yield an un-methylated shuttle plasmid. Then, a methylation reaction was set up (50 μL) by adding a total of 1 μL of the TXTL reaction(s) to the shuttle vector along with 1× dam methyltransferase buffer (NEB CN #M0222) and 640 μM AdoMet (NEB CN #B9003), and incubating at 37° C. for 2 hours. Following methylation, the reaction was first treated with 100 μg/mL Proteinase K and incubated at 50° C. for 30 minutes then with 100 μg/mL RNase A and incubated at 37° C. for 1 hour to remove impurities from the TXTL mix. Finally, the shuttle plasmid was purified using column purification (Zymo Research CN #D4013) according to manufacturer protocols.
Electroporation. Electroporation of S. enterica was performed as follows: an overnight culture was diluted 1:20 in fresh LB media and grown until the OD600=0.6. Then, cells were harvested by centrifugation at 5,000 rpm and 4° C., before being washed twice with 25 mL of ice-cold 10% glycerol. Harvested cells were then suspended in 0.5 mL ice-cold 10% glycerol. 0.1 mL of cell suspension was added to 1-mm electroporation cuvettes with 50-100 ng of plasmid DNA, and the cells were electroporated at 1.8 kV, 200Ω resistance, and 25 μF. 0.9 mL of pre-warmed LB broth was then added to the electroporated cells, and a recovery was set up for 1 hour at 37° C. before cells were plated on LB agar supplemented with chloramphenicol. Colonies were counted after overnight incubation at 37° C.
To transform B. breve and B. longum by electroporation, cells were first made electrocompetent by adapting a previously published protocol50. In short, 4 mL of an overnight culture was added to 50 mL of MRS supplemented with 1% glucose and 0.05% L-cysteine, and cells were grown at 37° C. in an anaerobic chamber until the OD600 reached 0.6 (B. breve) or 1.0 (B. longum). Then, cells were harvested by centrifugation at 4,500 rpm and 4° C., and washed twice with 25 mL of ice-cold wash buffer (0.5 M sucrose, 1 mM ammonium citrate, pH 6.0) then once more with 1 mL of ice-cold wash buffer before resuspending the cells in 0.375 mL ice-cold wash buffer. To perform electroporation, 90 μL of the cell suspension was added to 1-mm electroporation cuvettes with 200-500 ng of plasmid DNA, and electroporation was performed at 2.0 kV, 200Ω resistance, and 25 μF. For B. longum, the cells were incubated with the plasmid DNA on ice for at least 5 minutes before performing electroporation. After electroporation, 0.9 mL of pre-warmed Reinforced Clostridial Medium (RCM) was added to the cells and a recovery was set up for 3 hours at 37° C. in an anaerobic chamber. Finally, cells were plated on Reinforced Clostridial Agar (RCA) supplemented with tetracycline, and colonies were counted after 2-3 days of incubation at 37° C.
HT-IMPRINT. To determine the optimal methylation pattern required for transformation of different Bifidobacteria strains, 4-nt barcodes were cloned into the E. coli-Bifidobacterium shuttle plasmid. Then, 2-3 barcodes were assigned to each combination of methyltransferases for a given strain. IMPRINT reactions were performed to methylate the barcoded shuttle plasmids with each combination of the methyltransferases present in the strain. The purified, barcoded shuttle vectors from the IMPRINT reactions were then pooled together and transformed into the Bifidobacteria strain, and cells were both plated on RCA supplemented with tetracycline and diluted in MRS broth supplemented with 0.05% L-cysteine and tetracycline. After 2-3 days, the transformed plasmids were prepped from the back-diluted cultures, or from the grown colonies by first adding 1 mL PBS to the agar plates to resuspend colonies. In both cases, the plasmid was used as a template to amplify a segment of the plasmid harboring the barcode. The PCR amplicon was submitted for Amplicon-EZ Sequencing performed by Genewiz (genewiz.com/en/Public/Senvices/Next-Generation-Sequencing/Amplicon-Sequencing-Services/Amplicon-EZ). The number of reads mapping to each barcode was counted within each.fastq.gz file using the command zgrep-c “CTGCNNNN” *.fastq.gz for pJV420_U barcodes or zgrep-c “GCTTNNNN” *.fastq.gz for pJV420_D barcodes. From there, the median barcode count (if using three barcodes per sample) or average barcode count (if using two barcodes per sample) was calculated for each methylation sample in the transformed cells and in the pool control. The ratio of each sample in the transformed cells compared to the pool control was compared to the ratio of total reads in the transformed cells relative to the pool control to calculate the extent of barcode enrichment or depletion.
Statistical analyses. A student's t-test was utilized to determine statistical significance in DNA transformation from different MTases. In all cases, the t-test was a two-sample, equal variance dataset with a two-tailed distribution.
It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure, which is defined solely by the appended claims and their equivalents.
All publications and patents mentioned in the above specification are herein incorporated by reference as if expressly set forth herein. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and may be made without departing from the spirit and scope thereof.
It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present disclosure described herein are readily applicable and appreciable, and may be made using suitable equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are merely intended only to illustrate some aspects and embodiments of the disclosure, and should not be viewed as limiting to the scope of the disclosure. The disclosures of all journal references, U.S. patents, and publications referred to herein are hereby incorporated by reference in their entireties.
The present disclosure has multiple aspects, illustrated by the following non-limiting examples.
While physical barriers such as the cell wall can interfere with transformation, arguably the most common barrier is posed by restriction-modification (R-M) systems. These systems are one of a growing set of defenses but represent the most prevalent across bacteria. To confer immunity. R-M systems rely on DNA methyltransferases (MTases) that methylate genomic DNA with a unique methylation pattern using S-adenosyl methionine (SAM) as a methyl donor as well as restriction endonucleases (REases) that cleave DNA with foreign patterns. Four types have been defined based on the need for a specificity protein to guide the MTase and REase (Type I), the MTase and REase acting independently (Type II), the MTase and REase forming a complex capable of both methylation and restriction (Type III), and the REase cleaving methylated DNA (Type IV). Transformed DNA possessing the wrong methylation pattern undergoes extensive cleavage, resulting in a massive drop in the number of transformed colonies. As a further challenge, bacteria often possess not one but multiple R-M systems conferring unique methylation patterns that can vary even between strains.
Recreating these patterns or mutating the short recognition sites have proven to be an effective way to restore transformation, in some cases radically boosting transformation. However, existing approaches have been heavily constrained in their ability to fully reproduce these patterns in a rapid and comprehensive means (
To tackle this challenge, experiments were conducted to evaluate TXTL as a distinct means to overcome DNA restriction. TXTL recapitulates transcription and translation in a lysate or solution of purified components, allowing the functional expression of RNA and protein in minutes to hours without the need for cell culturing or protein purification. A TXTL-based pipeline was developed in which DNA MTases identified in a host bacterium (e.g., through bioinformatics or listed on the REBASE database) are expressed and combined with DNA in a methylation reaction, and the resulting DNA is purified and transformed into the host. DNA lacking any methylation (e.g., PCR product, plasmid DNA extracted from a MTase-free strain) serves as the starting point to immediately circumvent Type IV R-M systems that cleave methylated DNA. The resulting pipeline was termed IMPRINT (Imitating Methylation Patterns Rapidly IN TXTL) (
IMPRINT was initially prototyped by expressing MTases from E. coli. Standard laboratory strains of E. coli (e.g. MG1655) possess two orphan DNA MTases, Dam (M.EcoKdam) and Dcm (M.EcoKdcm), which respectively methylate adenine in GATC and the second cytosine in CC(A/T)GG. To assess methylation, the restriction enzyme DpnI was used, which cuts GATC when methylated by Dam, and PspGI, which cuts CC(A/T)GG unless it is methylated by Dcm (
The next major question was whether DNA methylated through IMPRINT could boost transformation. The LT2 strain of the gram-negative pathogen Salmonella enterica was used, which is related to E. coli but restricts E. coli DNA through its set of well-characterized R-M systems. As listed on REBASE, the associated MTases include two orphan MTases Dan (M.SenLT2dam) and Dem (M.SenLT2dcm) homologous to those in E. coli, another orphan MTase (M.SenLT2IV), a Type I MTase requiring a specificity protein (MS.SenLT2II), and a Type III MTase (M.SenLT2I) (
It was observed that including any of the orphan MTases with the Types I and III MTases reduced transformation (
By applying different MTase combinations using IMPRINT, it was determined that the Type I and III MTases contributed to enhanced DNA transformation in S. enterica LT2, while the orphan MTases played no role in enhanced transformation. For bacteria with a large number of R-M systems or much more laborious and intensive transformation procedures, testing the MTases individually or in combinations would be a long and involved process. Therefore, experiments were conducted to create a high-throughput version of IMPRINT that tests different combinations of MTases in a single transformation (
To apply HT-IMPRINT and move beyond bacteria related to E. coli, Bifidobacteria was used. These gram-positive bacteria are common constituents of the human digestive tract, and many species are used as probiotics. These bacteria are also strict anaerobes with more involved transformation procedures, possess a wide variety of R-M systems, and have proven difficult to transform. Initial experiments were conducted using one strain, Bifidobacterium breve UCC2003, which harbors three characterized Type II R-M systems listed on REBASE and previously shown to interfere with DNA transformation (
Experiments were also conducted to apply HT-IMPRINT to Bifidobacterium longum ATCC 15707. This strain contains two R-M systems listed on REBASE, where the Type II system encodes two different MTases (M1.Blo1217ORF1038, M2.Blo1217ORF1038) and the Type I system encodes an MTase with two different specificity proteins (MS1S2.Blo1217ORF1481) (
HT-IMPRINT revealed the most consequential MTase combinations for different Bifidobacteria. In some cases, the methylation pattern for one strain would boost transformation in other strains, easing the process of transforming diverse bacteria. Experiments were then conducted to evaluate how the best MTase combination identified via IMPRINT in one strain impacts transformation in other strains. The best MTase combinations for S. enterica LT2, B. breve UCC2003, and B. longum ATCC 15707 were selected and these were applied to the Bifidobacterium-E. coli shuttle plasmid JV420 that can be propagated in all strains (
Taken together, the results of the present disclosure demonstrate that IMPRINT offers an effective means to recreate the pattern of DNA methylation in a host bacterium using TXTL, thereby boosting transformation of the methylated DNA into the host. Furthermore, applying HT-IMPRINT allowed the identification of the best MTase combination in a single transformation, simplifying subsequent transformation efforts. By avoiding cell culturing and protein purification required by traditional approaches, IMPRINT greatly accelerates the time from obtaining constructs for each MTase to achieving improved transformation. The results of the present disclosure further showed that methylation could be enhanced with mutations to Type I MTases that relieve the preference for hemimethylated substrates.
The final product of IMPRINT is purified DNA that can be transformed through different means, including electroporation, chemical transformation, and natural transformation, as well as packaged into nanoparticles. While IMPRINT utilizes naked DNA that is not readily compatible with cell-based delivery approaches such as conjugation or phage delivery, HT-IMPRINT identified a minimal set of important MTases to express in the donor/packaging strain. Outside of R-M systems, novel antiphage defense systems continue to be discovered throughout bacteria that could also impact DNA transformation. Systems that rely on DNA methylation (e.g., BREX, DISARM) or other forms of DNA chemical modification can be incorporated into IMPRINT to allow transformed DNA to circumvent an even wider assortment of bacterial defenses. Regardless of the specific delivery mode or type of MTase, increased transformation by IMPRINT provides for efficient genetic manipulation and harnesses the rich diversity of bacteria for sustainable chemical production, enhancing food production, and applying cell-based biosensors and therapeutics. IMPRINT is also useful for studying the role of R-M systems in host defense and gene regulation, creating opportunities to explore the functional roles played by DNA MTases in diverse bacteria.
Sequences. The various embodiments of the present disclosure described herein may include one or more of the sequences referenced below, which can be found in the corresponding sequence listing.
M.EcoKI (
EDDKKLVQAVFHNVSTfITEPKQITALVSNMDSLDSLDWYNGAH (SEQ ID NO: 1)
M.EcoKI_L85Q (
EDDKKQVQAVFHNVSTTITEPKQITALVSNMDSLDSLDWYNGAH (SEQ ID NO: 2)
M.EcoKI_LI 13R (
EDDKKLVQAVFHNVSTTITEPKQITALVSNMDSLDSRDWYNGAH (SEQ ID NO: 3)
M.SenLT2II (
SDEKKLVQAVFHNVSTTIEQPKQITELVSYMDALDSRDWYNGNH (SEQ ID NO: 4)
M.SenLT2II_L85Q (
EDEKKQVQAVFHNVSTTIEQPKQITELVSYMDSLDSRDWYNGNH (SEQ ID NO: 5)
M.SenLT2I1_L113R (
EDEKKLVQAVFHNVSTTIEQPKQITELVSYMDSRDSRDWYNGNH (SEQ ID NO: 6)
Primer sequences used in the various embodiments of the present disclosure are provided in Table 1 below.
Barcode sequences used in the various embodiments of the present disclosure are provided in Table 2 below.
Barcode sequences used in the various embodiments of the present disclosure are provided in Table 3 below.
Plasmid constructs used in the various embodiments of the present disclosure are provided in Table 4 below.
This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63/222,020 filed Jul. 15, 2021, which is incorporated herein by reference in its entirety for all purposes.
This invention was made with government support under grant number MCB1452902 awarded by the National Science Foundation. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US22/73690 | 7/13/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63222020 | Jul 2021 | US |
