WO2023178544A1 - 表达元件组合、表达载体、宿主、应用及组装方法 - Google Patents

表达元件组合、表达载体、宿主、应用及组装方法 Download PDF

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WO2023178544A1
WO2023178544A1 PCT/CN2022/082446 CN2022082446W WO2023178544A1 WO 2023178544 A1 WO2023178544 A1 WO 2023178544A1 CN 2022082446 W CN2022082446 W CN 2022082446W WO 2023178544 A1 WO2023178544 A1 WO 2023178544A1
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assembly
fragments
genome
phage
limited
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French (fr)
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孔令欣
刘文峰
肖敏凤
程丽
陶皓然
李俊桦
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深圳华大生命科学研究院
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention relates to the field of genome fragment assembly, and in particular to expression element combinations, expression vectors, hosts, applications and assembly methods of phage genome fragments.
  • phages have strict host specificity and a very narrow bactericidal spectrum.
  • a phage can only kill one type of bacteria or one strain of a type of bacteria. This is mainly due to the phage adsorption protein and the host cell membrane. It is determined by the molecular structure of the surface receptor and its complementarity.
  • CRISPR-Cas system the natural genetic immune system
  • Jaschke et al. used PCR products and synthetic fragments to directly assemble the 5.4 kb phage X174 genome in yeast for the first time, and activated it in E. coli to successfully obtain phage plaques.
  • cell systems magazine reported that the natural homologous recombination system in Saccharomyces cerevisiae was used to successfully assemble 5-10 fragments ranging in size from 1.3-10.1kb (including the plasmid vector YAC 3.0kb).
  • the assembled phage genome length is 37 -Varies from 45kb.
  • Synthetic artificial phage currently faces a technical bottleneck in genome assembly.
  • in vitro assembly or in vivo assembly in yeast are mostly used.
  • the success rate of in vitro assembly of large genome fragments is extremely low; in vivo assembly technology in yeast has a long experimental cycle and a high technical threshold.
  • Gibson Assembly mainly relies on homologous recombination of homologous sequences at the ends of DNA. The assembly efficiency decreases with the increase of DNA fragments. This method has low efficiency in assembling phage genomes.
  • Golden Gate Assembly utilizes Type IIS restriction endonucleases to generate DNA fragments with short single-stranded overhangs and DNA ligase to join the fragments together.
  • the assembly relies on the target vector, which contains resistance selection markers for screening positive clones, but is prone to false positive problems; it still requires subsequent PCR and sequencing verification, which increases the experimental cycle and cost.
  • This method has a high technical threshold and low universality
  • the present invention provides expression element combinations, expression vectors, hosts, applications and assembly methods of genome fragments.
  • the present invention provides a combination of expression elements, including but not limited to:
  • the expression elements of the lambda Red homologous recombination system in the expression element combination include but are not limited to exo, bet and gam.
  • the present invention also provides an expression vector, including the combination of expression elements according to claim 1 or 2.
  • the expression vector includes, but is not limited to, pRARE plasmid and pKD46 plasmid.
  • the present invention also provides a host transformed or transfected with the expression vector.
  • the host is a prokaryotic or eukaryotic organism, including but not limited to bacteria, actinomycetes, rickettsiae, chlamydia, mycoplasma, cyanobacteria, archaea, yeast and molds one or more of them.
  • the bacteria include, but are not limited to, Gram-negative bacteria or Gram-positive bacteria;
  • the Gram-negative bacteria include but are not limited to Escherichia coli; more preferably, the Escherichia coli include but are not limited to Escherichia coli DH10B and Stellar;
  • the Gram-positive bacteria include but are not limited to cell wall-deficient L-type bacteria; more preferably, the cell wall-deficient L-type bacteria include but are not limited to L-type Listeria monocytogenes.
  • the fourth aspect of the present invention also provides the application of the expression element combination, the expression vector, or the host in genome fragment assembly.
  • the present invention also provides the expression element combination, the expression vector, or the host in molecular cloning, synthesis of artificial phage, phage therapy, preparation of products for phage therapy, detection of microorganisms, preparation of products for detection of microorganisms, Use of one or more medicaments for preventing and/or treating infections or diseases caused by microorganisms, or for preparing medicaments for preventing and/or treating infections or diseases caused by microorganisms.
  • the fragments include, but are not limited to:
  • the number of fragments includes but is not limited to:
  • the present invention also provides a method for assembling genome fragments, which assembles genome fragments based on the expression element combination, the expression vector, or the host.
  • the assembly includes, but is not limited to:
  • the assembly method includes, but is not limited to, the following steps:
  • Step A Obtain the fragments of the genome to be assembled
  • Step B Perform in vitro Gibson Assembly assembly
  • Step C Assemble in the host body
  • the assembly method may include step B, or may not include step B.
  • the fragments involved in the assembly method include, but are not limited to:
  • the reaction temperature of the in vitro Gibson Assembly includes but is not limited to 37°C to 50°C;
  • reaction temperature of the in vitro Gibson Assembly includes but is not limited to:
  • reaction time of the in vitro Gibson Assembly assembly in step B of the assembly method includes but is not limited to 15min to 105min;
  • reaction time of the in vitro Gibson Assembly includes but is not limited to:
  • the amount of the fragment is not less than 30 fmol
  • the dosage of the fragments includes but is not limited to:
  • the genome mentioned in the present invention refers to the complete set of genetic material of a cell or organism, or all nucleic acids carrying genetic information, including nucleic acids on all chromosomes.
  • the genome also includes nucleic acids on mitochondria.
  • the size of the genomes of various organisms varies greatly, with the smallest being only 5000bp (base pair), such as the simplest viruses, and the largest being 1011bp, such as some higher plants.
  • Genome size is not directly related to organism complexity. The positions of certain components in the genome are not static, and the genome size or number of genes is not absolutely fixed between different individuals of the same species. Changes in genome structure may even lead to changes in function.
  • the human genome consists of 3.2′109bp of DNA, including the nuclear chromosomal genome and the cytoplasmic mitochondrial genome. About half of the nuclear chromosome genome consists of different repetitive sequences, containing 25,000 to 35,000 genes. The genome controls the growth and development of organisms and life activities. In order to understand the nature of living things, we must first understand the genome, master all the nucleic acid sequences of the genome, analyze all the genetic information of the genome, and understand the organizational structure of genetic information and its expression in the organism.
  • Genomes of prokaryotes The genomes of prokaryotes are relatively small and have low amounts of DNA. Much of the information in prokaryotic genomes is required to maintain basic functions of the cell, such as constructing and replicating DNA, producing new proteins, and obtaining and storing energy. In prokaryotic genomes, the density of gene distribution is very high. Most of the DNA molecules are used to encode proteins, and only a very small part is not transcribed. This is different from eukaryotic DNA molecules. Bacteria contain chromosomal and extrachromosomal plasmid DNA. The genomic DNA of E. coli is a single double-stranded circular DNA molecule, and some bacteria also contain small circular double-stranded DNA that exists in the cytoplasm. Extrachromosomal DNA may also contain genetic information and can replicate itself and pass genetic information to daughter cells.
  • Eukaryotic genome Eukaryotic organisms are much more complex than prokaryotes in all aspects, including rich internal membrane systems, complex genetic information expression systems, etc. In terms of genomes, the size of eukaryotic genomes is much larger than that of prokaryotes, with complex organization and high information content. In the entire DNA sequence, protein-coding regions account for only a small part, while non-coding sequences account for a large part. The eukaryotic gene structure is also far more complex and changeable than the prokaryotic gene structure. Eukaryotic cells contain organelles such as mitochondria or chloroplasts. These organelles also contain DNA, but unlike nuclear genomic DNA, they are usually small, circular, double-stranded DNA molecules.
  • eukaryotic genes are composed of protein-coding sequences and non-protein-coding sequences.
  • the coding sequences in genes are called exons, and the non-coding sequences in genes are called introns.
  • the exons encoding different regions of a certain protein are not continuously arranged together, but are often isolated by introns of different lengths, forming a mosaic arrangement of fragments. Therefore, eukaryotic Genes are sometimes called split genes. Different genes have very different numbers and sizes of introns.
  • the collagen gene is about 40kb long and has at least 40 introns, of which the short one is only 50bp and the long one can reach 2000bp.
  • introns Many eukaryotic genes have large and sometimes very large introns; for example, the gene involved in human cystic fibrosis contains 24 introns, which together exceed 1 Mb in length. And a few genes, such as histones, have no introns at all. The physiological function of introns is currently unclear.
  • exons code for discrete structural and functional units of the protein.
  • the central exon of the myoglobin and hemoglobin genes codes for a heme-binding region that can reversibly bind O2.
  • Other exons encode exclusively alpha-helical segments that allow the protein to be inserted into the cell membrane.
  • a domain of a protein may be encoded by a single exon.
  • One hypothesis is that during evolution, new proteins emerge due to the rearrangement of exons that encode discrete structural units, structural parts, and catalytic sites. Mixing of exons is a rapid and efficient way to generate new genes.
  • Splitting genes has another advantage, that is, the nascent RNAs spliced in different ways may produce a series of related proteins that vary in a basic characteristic according to a developmental program.
  • exon-intron junction region is a highly conserved and specific base sequence.
  • the exon-intron junction region refers to the interface between exons and introns, also known as boundary sequences.
  • the exon-intron junction region although short, is highly conserved. This conserved sequence is closely related to the splicing mechanism and is the signal sequence for RNA splicing.
  • a complete gene includes not only the coding region, but also specific sequences of varying lengths on both sides of the 5'-end and 3'-end. Although these sequences do not code for amino acids, they play an important role in the gene expression process. Therefore, strictly "gene", the molecular biological definition of this term is: all the nucleotide sequences necessary to produce a polypeptide chain or functional RNA.
  • RNA polymerase binds to a certain nucleic acid sequence in the 5'-end upstream region of the gene, and then begins transcription to synthesize an RNA copy of the gene.
  • This nucleic acid sequence is called a promoter. Promoters determine the direction, speed and accuracy of DNA transcription. RNA polymerase must accurately identify where gene transcription begins. RNA polymerase cannot look for any specific nucleotide when looking for the starting point of a gene, because single nucleotides occur very frequently in the cell's DNA molecules. However, a specific combination of nucleotides is not likely to occur randomly, and the greater the number of nucleotides involved, the less likely it is to occur.
  • n (1/4)n, where n is the number of nucleotides.
  • Prokaryotic RNA polymerase scans the entire DNA, looking for a specific nucleotide sequence of approximately 13 nucleotides in length that marks the start of a gene (one nucleotide is the transcription start site, and 6 nucleotides are located between 10 bases upstream of the start site and another 6 nucleotides 35 bases upstream of the start site), and the relative positions between these promoter sequences are fixed.
  • RNA polymerase Since the genomes of most prokaryotes are only a few million nucleotides, the chance that these promoter sequences come together is about 1 in 70 million, which is enough to allow RNA polymerase to reliably and uniquely identify the start position of the gene. .
  • the genomes of eukaryotes are several orders of magnitude larger than those of prokaryotes. Accordingly, eukaryotic RNA polymerases must recognize more complex and longer promoter sequences in order to reliably identify the start position of a gene.
  • a TATA box 30 bp upstream of the transcription start point there is a CAAT box 80 bp upstream, and a GCCACACCC or GGGCCGGG sequence near 80 bp to 110 bp upstream, which is called a GC box. (GC BOX).
  • a promoter is a DNA sequence with a relatively fixed position upstream of the transcription start point, including TATA box, CAAT box and GC box, generally within the range from the transcription start point to 150 bp upstream. These upstream conserved sequences are collectively called upstream promoter elements (UPE).
  • UEP upstream promoter elements
  • the TATA box has different functions from the other two UPEs.
  • the main function of the former is to initiate transcription accurately. If the TATA box is removed or mutated, the relative value of the decrease in the transcript product is not as obvious as that after mutation of the CAAT box or GC box, but it is found that the starting point of the obtained RNA product is not fixed.
  • the CAAT box and the GC box mainly control the initiation frequency of transcription and are basically not involved in the determination of the start site.
  • the CAAT box has the greatest impact on the frequency of transcription initiation.
  • the change of any base in this region will greatly affect the transcription intensity of the target gene, while the substitution of one or two bases in other sequences in the initiation region will not have much impact on the transcription intensity. Impact.
  • the insertion of nucleotides between the TATA box and the adjacent UPE also weakens transcription. Although the above three UPE sequences all have important functions, not every gene promoter region contains these three sequences.
  • the promoter is certainly important for transcription, but does the sequence upstream of the promoter have nothing to do with transcription?
  • the sequence upstream of the promoter have nothing to do with transcription?
  • Enhancers have several notable characteristics: (1) Enhancer sequences are long, up to hundreds of base pairs. Sometimes it is a repetitive sequence, which often contains a core sequence "(G)TGGA/TA/TA/T(G)". (2) The range of action is relatively long and can be thousands of bases away from the gene it acts on. (3) The effect has no directionality: the sequence has the same effect if it is reversed; (4) The position is not fixed: it can be upstream of the 5'-end of a gene, or downstream of the 3'-end, or even within the introns of genes. (5) Enhancers are generally tissue or cell specific. (6) The activity of enhancer is related to its spatial directionality in the DNA double helix structure.
  • Silencers can inhibit gene expression or selectively express genes.
  • Single-copy genes and gene families Genes that appear only once in the genome are called single-copy genes. Single-copy genes are mostly genes encoding proteins and are generally more complex. These genes are often fragmented genes, that is, the coding regions are discontinuous. In eukaryotes, there are also transcription units similar to the prokaryotic operon structure. The primary transcripts transcribed from them can produce more than one protein through different splicing methods, that is, a DNA sequence can encode multiple proteins. 25% to 50% of the protein-coding genes in eukaryotic cells exist as a single gene in the genome, while the remaining protein-coding genes belong to gene families composed of two or more similar genes.
  • genes diverged from an ancestral gene forming a family of structurally and functionally related gene groups that are either clustered together or dispersed throughout the genome.
  • This group of genes that come from the same ancestral gene and encode similar but not identical proteins is called a gene family.
  • the proteins encoded by these genes are called protein families.
  • Gene families range from just a few genes to hundreds of genes.
  • the gene families corresponding to a-globin and b-globin are the most studied gene families.
  • Repeated sequences There are a large number of repeated sequences in the DNA of eukaryotic cells, especially for vertebrates. Repeated sequences can be roughly divided into three categories, namely low repeat sequences, moderate repeat sequences and highly repeat sequences.
  • the number of repeats of moderately repetitive sequences is between 10 and 105, accounting for 10% to 40% of the total DNA.
  • the moderately repetitive sequences in the mouse genome account for 20% and the Drosophila genome accounts for 15%.
  • the number of repeats of highly repetitive sequences is greater than 106, and the length of the basic repetitive sequences ranges from a few bases to several hundred bases or longer, accounting for 10% to 60% of the genome.
  • Highly repetitive sequences can be divided into: (1) satellite DNA (satellite DNA): the length of repeated sequences is 5 to 200bp. These sequences are gathered together and arranged in series, with a total length of up to 100mb (mega base). They are not highly polymorphic in the human population.
  • satellite DNA satellite DNA
  • Minisatellite DNA The length of repeated sequences ranges from 5 bp to dozens of bp, arranged in series, and the total length is between 0.5 and 30 kb. This type of repetitive sequence is highly polymorphic in the human population.
  • Microsatellite DNA The length of the repeated sequence is between 2 and 6 bp, and it is also highly polymorphic.
  • Gene mutation refers to a mutation in the DNA sequence of a gene. What is currently most understood about molecular evolution is point mutation, or a single base mutation in a nucleic acid sequence. Single base mutation refers to the change of one base to another base, or the insertion of a new base in the sequence, or the deletion of an original base. The insertion or deletion of bases in the coding region will cause the reading frame of the gene to be frameshifted, causing the original codons to be scrambled, resulting in abnormal gene transcription.
  • Gene recombination and gene movement are the driving force of biological evolution and have important biological significance.
  • the so-called genetic recombination refers to the combination of DNA sequences derived from two or more different parents into a new genetic DNA sequence.
  • Gene recombination is a common phenomenon in the biological world.
  • Homologous recombination is the exchange between any two genetic DNA sequences that share a homologous sequence.
  • the condition for homologous recombination to occur is the existence of homologous sequences between two DNA molecules, regardless of the actual base sequence order. As long as they are similar, and the longer the similar segments, the more frequently recombination will occur. If the homologous sequence is too short, recombination will be difficult to occur.
  • Homologous recombination is also an important mechanism for DNA damage repair.
  • a damaged strand needs to be repaired and the other complementary strand cannot be used as a repair template, another homologous sequence must be used to repair the damaged strand using the homologous recombination repair mechanism.
  • Another type of recombination is site-specific recombination, which occurs in site-specific short sequence regions. Precise cleavage reactions and ligation reactions occur during recombination, and DNA is neither lost nor synthesized.
  • There are also a type of mobile gene sequences in the genome that can be moved from one part of the chromosome to a new location on the same chromosome or another chromosome.
  • genes that can move are called mobile genes.
  • Mobile genes are generally moved in the form of transposons. Recombination and movement can produce new sequences and functions, or change the functions of original sequences.
  • Chromosomes may undergo aberrations, causing changes in the genetic material. Chromosomal aberrations include chromosome loss, translocation, displacement, etc. These changes are actually changes in DNA molecules, such as partial loss of chromosomes, which actually means the loss of part of the DNA molecule. Mutation is the basic condition for evolution. People have gradually understood the molecular mechanism of evolution, and through comparative sequence analysis of related molecules, the evolutionary relationship between species can be obtained. On the other hand, similarities and differences between related molecules provide important information about molecular structure and function. Molecular evolution also involves selection and inheritance. Inheritance requires that genes from parents can be passed on to offspring, while selectivity suggests that beneficial mutations will be retained.
  • Human genome The coding region of the human genome is less than 3% of all DNA sequences, and the number of genes is 25,000 to 35,000. Repeated sequences account for a large part of the human genome, about 50%. These repeated sequences may have certain functions, but they may not have any function.
  • the repetitive sequences of the human genome can be divided into four types: (1) short interspersed elements (SINEs); (2) long interspersed elements (LINEs); (3) long terminal repeat elements (LTR); (4) DNA transposons ( transposons).
  • the remaining DNA sequences of the genome include promoters, transcriptional regulatory sequences, and other sequences of unknown properties.
  • the genomes described in the invention include, but are not limited to, bacteriophage genomes.
  • Bacteriophage is a type of virus that infects bacteria (including actinomycetes, fungi and prokaryotes). It is a non-cellular organism and has similar characteristics to other viruses. It is mainly composed of protein and nucleic acid (DNA or RNA). It is widely distributed in nature and can be found in soil, air, water or living organisms.
  • DNA or RNA protein and nucleic acid
  • phage genomes vary in size, ranging from 3,435 bp for Cytococcum L5 phage to 316,674 bp for Pseudomonas 201phi2-1 phage.
  • most phages with larger genomes are myotail phages (with contractile tails), and the long-tailed phage with the largest genome is Bacillus phage SPB c2 (134,416 bp).
  • Phage populations have significant genetic diversity. Generally speaking, the genomes of phages that do not share a common host rarely have sequence similarity, even if the host bacteria are related to a certain extent. For example, between the 4 Streptomyces phage genomes and the 50 Mycobacteriophage genomes, although the host mycobacteria and Streptomyces both belong to the genus Actinobacteria, there is no obvious similarity in their nucleotide sequences.
  • phages sharing a common host with related sequences For example, 30 phage genomes isolated from Pseudomonas (33), Staphylococcus (48), and Mycobacterium tuberculosis (50) possess related sequences. But new research finds that phages sharing the same host may also have little or no sequence similarity. For example, Escherichia coli K-12 is a common host of lambda phage and T4 phage, but the two phages have completely different lifestyles, and the sequence similarity between the two phages is also minimal.
  • Another study used the Gepand program to analyze the relationship between 30 mycobacterial phages and 50 Pseudomonas phage nucleoplasmic acid sequences isolated from a common bacterial host, and several points were revealed through comparison.
  • Phage genomics research has made tremendous progress in the past few years. Although there are still no definite answers to the study of phage diversity and evolutionary pathways, the understanding of these issues has become increasingly clear, and the foundation for related research has been laid. In the next few years, with the development of sequencing technology, more new phage sequence information will be obtained. In addition, the application of phage preparations to control bacterial infections has shown certain therapeutic prospects. The widespread drug resistance of pathogenic bacteria also urgently requires new ways to solve this threat. Therefore, phage therapy or combined antibiotic therapy may become a very valuable approach. It is worth noting that in order to ensure the safety and controllability of phage therapy, more detailed and clear experimental evidence is needed on the biological characteristics of phages and the interaction between specific phages and host bacteria.
  • the fragment assembly method of the phage genome provided by the present invention will provide important technical support for the research on the sequence and function of the phage, especially the research on the therapeutic function.
  • the assembly in step C also includes an activation step
  • the activation includes but is not limited to: inoculating the host into a medium containing Ca 2+ or Mg 2+ and culturing it at 37°C for no less than 6 hours.
  • the evaluation criteria for the success of the assembly method include but are not limited to: the occurrence of expected plaques or the complete plate phenomenon, the phage genome assembly is successful.
  • the present invention also provides a method for assembling a genome, including a method for assembling the above-mentioned genome fragments.
  • the genome includes, but is not limited to, a bacteriophage genome.
  • the present invention also provides a method for synthesizing artificial phage, including a method for assembling the above-mentioned genome fragments.
  • the present invention also provides a method for assembling DNA multimolecules, including the method for assembling the above-mentioned genome fragments.
  • the present invention also provides a method for DNA cloning, including the assembly method of the above-mentioned genome fragments.
  • the successfully assembled phage genome has the same activity as natural phage, overcoming the current technical problem that the phage genome assembled in yeast is difficult to activate;
  • the genome assembly achieved by this invention does not rely on the target vector, and can achieve one-step assembly and activation of the phage genome, avoiding the tedious screening steps after in vitro Golden Gate Assembly and yeast in vivo assembly, and greatly reducing the experimental cycle. and cost;
  • This invention is one of the key steps in the synthesis of artificial phages.
  • This invention can be combined with synthetic biology technologies such as the construction of phage element libraries, site-directed mutations, and de novo synthesis to synthesize artificial phages with the potential to clinically treat pathogenic bacteria;
  • the genome assembly that can be achieved by this invention is not limited to phage genomes, but can be extended to the assembly of other genomes.
  • the present invention can realize the direct assembly of phage genome fragments obtained by PCR or de novo synthesis in E. coli, thus laying a technical foundation for the synthesis of artificial phages with the potential to clinically treat pathogenic bacteria. It can be used to synthesize artificial phages, improve phage therapy, and treat infections caused by bacteria, especially clinical multi-drug-resistant bacteria.
  • the present invention has a short experimental period, good universality and high success rate in phage genome assembly, is conducive to promoting the synthesis of artificial phages, improving phage therapy, and has good market prospects.
  • Figure 1 shows the phage genome fragment assembly flow chart provided by the invention
  • Figure 2 shows an example of the assembly results of 4 to 5 ⁇ 10kb genome fragments using PH-Kp7215-A and PH-Kp5295 of the present invention (full clear); wherein, A represents the combination of E. coli in vivo assembly and in vitro Gibson Assembly assistance; B represents The in vivo assembly of E. coli is combined with the assistance of Gibson Assembly in vitro; C shows the direct assembly of the genome in E. coli;
  • Figure 3 shows an example of negative control, the host bacteria Kp7215 is plated (overgrown with host bacteria);
  • Figure 4 shows a comparative example: only an example of the assembly results of 5 ⁇ 10kb genome fragments of PH-Kp5295 by Gibson Assembly in vitro (overgrown with host bacteria);
  • Figure 5 shows a negative control example of phage PH-Kp5295 genome fragment assembly. One fragment is randomly selected (overgrown with host bacteria);
  • Figure 6 shows a comparative example: the gel image of the assembly results of 5 ⁇ 10kb genome fragments of PH-Kp5295 in vitro Gibson Assembly; the maker in the picture is 1kb extent; A- is the genome fragment without Gibson Assembly; A1 is the genome fragment Gibson Assembly 15min result , gDNA1 is the result of gDNA Gibson Assembly 15min, that is, genome circularization, which serves as a control for the assembly effect; A1-5, gDNA1-5 are respectively the genome fragments and gDNA Gibson Assembly reaction 15min, 30min, 45min, 60min, 75min; you can see It can be seen that there is a significant difference between the fragment assembly results and the size of the complete genome.
  • the complete genome is about 50kb, while the Gibson assembly product is about 20-30Kb, and there are unassembled DNA fragments;
  • Figure 7 shows a comparative example: in vitro Gibson Assembly's PH-Kp9495-AB1 assembly result gel image of 10 2kb ⁇ 7kb genome fragments; the illustration is the same as Figure 6, except for the 1kb extent, the first two markers used are 1kb DNA Ladder and DL15000DNA marker; the reaction time of genome fragments and gDNA Gibson Assembly is 45min, 60min, 75min, 90min, 105min; it can be seen that there is a significant difference between the results of fragment assembly and the size of the complete genome.
  • the complete genome is about 50kb, while the Gibson assembly product is the largest No more than 15kb, there are a large number of unassembled DNA fragments;
  • Figure 8 shows an example of assembly results using Palz-36 ⁇ 15kb genome fragments of the present invention (full clear and with plaques);
  • A shows the in vivo assembly of E. coli combined with the assistance of Gibson Assembly in vitro;
  • B shows the in vivo assembly of E. coli Combined with the assistance of Gibson Assembly in vitro;
  • C shows the direct assembly of the genome in E. coli;
  • Figure 9 shows a comparative example: only an example of assembly results of Palz-36 ⁇ 15kb genome fragments of Gibson Assembly in vitro (overgrown with host bacteria);
  • Figure 10 shows an example of negative control, the host strain Pao1 is plated (covered with host bacteria);
  • Figure 11 shows a negative control example of Palz-3 phage genome fragment assembly. Five fragments were randomly selected for in vivo assembly in Escherichia coli (overgrown with host bacteria);
  • Figure 12 shows an example of the assembly results of 10 2kb ⁇ 7kb PH-Kp9495-AB1 genome fragments using the invention (full clear and with plaques); among them, A shows the direct assembly of the genome in E. coli; B shows the assembly and assembly in E. coli In vitro Gibson Assembly-assisted combination; C shows the in vivo assembly of E. coli and in vitro Gibson Assembly-assisted combination;
  • Figure 13 shows a comparative example: only an example of the assembly results of 10 2kb ⁇ 7kb PH-Kp9495-AB1 genome fragments from Gibson Assembly in vitro (overgrown with host bacteria);
  • Figure 14 shows a negative control example of phage PH-Kp9495-AB1 genome fragment assembly. Six fragments were randomly selected for assembly in Escherichia coli (overgrown with host bacteria);
  • Figure 15 shows an example of negative control, the host bacteria Kp9495 is plated (covered with host bacteria);
  • Figure 16 shows an example of the results of cloning a 1kb fragment using the psgRNA N20 GFP plasmid of the present invention
  • A shows the combination of in vivo assembly of E. coli and the assistance of Gibson Assembly in vitro
  • B shows the direct assembly of the plasmid fragment in E. coli;
  • Figure 17 shows an example of a negative control, with plasmid fragments and vectors used as negative controls respectively;
  • Figure 18 shows an example of the assembly results of 12 2.7-5kb, 13 2.2-5kb, 14 2.6-4.7kb, and 15 2.5-4.5kb PH-Kp9495-AB1 genome fragments using the invention (full clear); among them, A B shows the assembly of 12 2.7-5kb PH-Kp9495-AB1 genome fragments using the combination of in vivo assembly of Escherichia coli and the assistance of Gibson Assembly in vitro. Genome fragments; C shows the assembly of 14 PH-Kp9495-AB1 genome fragments of 2.6 to 4.7kb using the combination of in vivo assembly of E.
  • D shows the assembly of 15 PH-Kp9495-AB1 genome fragments of 2.5 to 4.5 kb using the combination of in vivo assembly of E. coli and the assistance of Gibson Assembly in vitro kb PH-Kp9495-AB1 genomic fragment
  • Figure 19 Comparative example: A, B, C, and D are 12 2.7-5kb, 13 2.2-5kb, 14 2.6-4.7kb and 15 2.5-4.5kb PH-Kp9495-AB1 genomes of only in vitro Gibson Assembly Example of fragment assembly results (overgrown with host bacteria);
  • Figure 20 shows an example of a negative control. 15 2.5-4.5 kb continuous and complete PH-Kp9495-AB1 genome fragments were activated with stellar/pRARE without a homologous recombination system (overgrown host bacteria).
  • the present invention discloses the combination of expression elements of genome fragments, expression vectors, hosts, applications and assembly methods. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters for implementation. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention.
  • the methods and applications of the present invention have been described through preferred embodiments. Relevant persons can obviously make modifications or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of this invention.
  • Preparation of phage genome fragments to be assembled First, split the phage genome into multiple fragments by designing primers. There should be at least 40-100bp homologous regions at the ends of adjacent genome fragments that can be spliced together. If the number of assembled fragments is large, , is larger, the length of the homology arm can be appropriately increased, while ensuring that the absolute value of the secondary structure free energy of the homology region and its downstream 20 bp region at 50°C is small (the secondary structure free energy is small, theoretically The assembly effect is better, predicted by NUPACK); prepare the phage genome fragments to be assembled through PCR and gel recovery;
  • in vitro assembly of E. coli and phage activation The efficiency of in vitro assembly of large DNA fragments is very low. In the final reaction mixture, only a very small amount of all the fragments may be assembled into a complete genome, and most of them are unassembled fragments or 2-4 splices. intermediate product. The efficiency of in vivo assembly of large DNA fragments is much higher than that of in vitro assembly, and a key factor affecting the in vivo assembly efficiency is the probability that all fragments enter the same competent cell. Therefore, in order to further improve the assembly efficiency and increase the amount of complete assembly products, consider combining in vitro assembly and in vivo assembly. First, in vitro assembly ensures that a large number of large fragments can be spliced together.
  • the laboratory of our research group has E. coli DH10B containing the pRARE plasmid expressing rare codons. This strain is often used to activate phage genomes, and the pKD46 plasmid containing the lambda Red homologous recombination system.
  • the Lambda Red homologous recombination system has the characteristics of short homologous sequence (40-60bp) and high recombination efficiency. It is composed of three genes of lambda phage exo, bet and gam, encoding three proteins of Exo, Beta and Gam respectively (see the schematic diagram of the principle) Figure Lambda Red homologous recombination system).
  • Exo protein is an exonuclease, a cyclic trimer molecule with a hollow channel in the middle. One end of the channel can accommodate double-stranded DNA molecules, and the other end can only accommodate single-stranded DNA. Exo protein can bind to the end of double-stranded DNA and degrade DNA from the 5' end to the 3' end of the DNA double strand, causing the DNA molecule to form a 3' sticky end; Beta protein is an annealing protein that plays a role in Red homologous recombination.
  • Beta protein is a 16kD polypeptide molecule that can bind to RecBCD exonuclease, inhibit its boundary effect on foreign DNA, and prevent foreign DNA from being degraded by the host after entering the cell.
  • the two plasmids pRARE and pKD46 can coexist in E. coli DH10B.
  • the pKD46 plasmid expressing the homologous recombination system is transferred to DH10B/pRARE E. coli, which is commonly used for phage genome activation, thus constructing an intermediate that can achieve assembly and activation in one step. Host E. coli.
  • the present invention aims to realize the direct assembly of phage genome fragments obtained by PCR or de novo synthesis in E. coli.
  • This goal can be achieved by two technical routes: 1. Direct assembly of phage genome in E. coli; 2. An assembly method that combines the in vivo assembly of E. coli with the assistance of in vitro Gibson Assembly. That is, after completing the in vitro Gibson Assembly assembly reaction, the mixture obtained after the reaction is directly transferred to the pKD46 plasmid containing the lambda Red homologous recombination system. E. coli DH10B competent cells are used for in vivo assembly of E. coli. This E.
  • coli also contains a pRARE plasmid expressing rare codons, which facilitates the activation of the phage genome in the intermediate host E. coli.
  • the transformed competent cells were incubated in LB medium containing Ca 2+ for 6 hours. After the incubation, they were taken out and vortexed with 5% chloroform to disrupt the intermediate host E. coli to release the activated phage particles.
  • the present invention introduces the implementation of this method by taking the genome assembly of Klebsiella pneumoniae phage as an example, and can also be applied to the assembly of other phage genomes, such as Pseudomonas aeruginosa, Acinetobacter baumannii, etc.
  • phage genomes whose hosts are Gram-negative bacteria.
  • phages whose hosts are Gram-positive bacteria currently reported efficient activation intermediate hosts include cell wall-deficient L-type bacteria such as L-type Listeria.
  • the subsequent phage genome assembly can not only be limited to the assembly in E. coli. After transforming the bacterium, if it is transferred to the homologous recombination system, it can be tried to assemble in the bacterium. It is also expected to achieve transformation, assembly and activation. Completed in one step.
  • the present invention can be used to transform and synthesize artificial phages, and these phages can be used for further improved phage therapy.
  • the present invention achieves the effect of completing assembly and activation in one step.
  • This method has a short experimental period, good universality and high success rate.
  • This invention realizes the direct assembly of large fragments of phage genome in E. coli for the first time.
  • This technology can realize the transformation, assembly and activation of phage genome in one step, greatly shortening the experimental cycle and improving the efficiency of transforming artificial phage.
  • This invention is also applicable to the assembly of large fragments (also applicable to small fragments) in other situations, and can be used in routine molecular cloning experiments.
  • Example 1 Klebsiella pneumoniae (Kp) phage PH-Kp9495-AB1, PH-Kp5295, PH-Kp7215-A genome, assembled from 4-5 to 10kb fragments.
  • Reaction mix used for PCR Hot Start High-Fidelity 2X Master Mix.
  • E. coli strains strains DH10B/pRARE; DH10B/pRARE/pKD46 preserved by the research team
  • Each phage assembly experimental set includes three types:
  • the amount of fragments used in the three different experimental groups was all 60 fmol/fragment.
  • Positive control The extracted phage genomic DNA is activated with the intermediate host DH10B/pRARE and the intermediate host DH10B/pRARE/pKD46. Theoretically, the results of the positive control should appear as plaque or clear results.
  • Randomly selected genome fragments (such as the 1st fragment of PH-Kp5295; the 4th fragment of PH-Kp9495-AB1);
  • Competent DH10B/pRARE/pKD46 perform the same experimental operations as the experimental group, but do not transform the fragments or genomes, to eliminate the interference of chloroform absorbed after adding chloroform to disrupt the cells on the experimental results.
  • the negative control results should be filled with the natural host bacteria of the phage.
  • A. Streak and isolate monoclonal colonies Take the preserved bacteria in glycerol and streak using the "three-line method". Among them, the DH10B/pRARE/pKD46 strain needs to be cultured at 30°C. Use a solid LB plate with ampicillin and chloramphenicol dual antibiotics. Monoclonal colonies of appropriate size can be grown in about 2 days; the DH10B/pRARE strain needs to be cultured at 37°C. , use a solid LB plate with chloramphenicol antibiotics and culture it for about 1 day to grow a monoclonal colony of appropriate size.
  • B. Preparation of overnight bacteria Pick the single clone colonies obtained above and put them into liquid LB medium.
  • the LB liquid medium used for DH10B/pRARE/pKD46 strain needs to add a final concentration of 10mM arabinose to induce Lambda Red homologous recombination.
  • two antibiotics, ampicillin and chloramphenicol must be contained and cultured at 30°C; the LB liquid medium used for the DH10B/pRARE strain must contain the antibiotic chloramphenicol and cultured at 37°C.
  • Preparation of logarithmic bacteria Calculate the volume of logarithmic bacteria to be prepared based on the 100X amount of competent cells required for the experiment.
  • the DH10B/pRARE strain was cultured at 37°C until the OD600 was between 0.5-0.55; the DH10B/pRARE/pKD46 strain was cultured at 30°C until the OD600 was between 0.5-0.55; the liquid LB medium used for both strains was the same as the previous step.
  • Competent state preparation "Calcium chloride method" prepares the above two different E. coli competent states, both of which are 200 ⁇ L 100X.
  • Experimental group 1 60 fmol/fragment, 15 ⁇ L ER Mix, reaction at 42°C for 90 minutes. Transfer the reaction mixture to the competent cells of Escherichia coli DH10B/pRARE, that is, add the mixture to the competent cells, mix gently, then incubate on ice for 30 minutes, heat shock at 42°C for 90 seconds, then incubate on ice for 3 minutes, add 1 ml containing LB medium with 5mM Ca 2+ , cultured at 37°C, 220rpm.
  • Experimental group 2 60 fmol/fragment, 15 ⁇ L ER Mix, reaction at 42°C for 90 minutes. Transfer the reaction mixture to the competent cells of Escherichia coli DH10B/pRARE/pKD46, that is, add the mixture to the competent cells, mix gently, then incubate on ice for 30 minutes, heat shock at 42°C for 90 seconds, then incubate on ice for 3 minutes, add 1 ml of LB medium containing 5mM Ca 2+ , cultured at 37°C, 220rpm.
  • Experimental group 3 Transfer 60 fmol/fragment directly into E. coli DH10B/pRARE/pKD46 competent cells, that is, add the fragment mixture to the competent cells, mix gently, then incubate on ice for 30 minutes, heat shock at 42°C for 90 seconds, and then ice again Bath for 3 minutes, add 1 ml of LB medium containing 5mM Ca 2+ , and culture on a shaker at 37°C and 220rpm.
  • Negative control 1 Randomly select a genome fragment, transfer 60 fmol directly into E. coli DH10B/pRARE/pKD46 competent cells, that is, add the fragment mixture to the competent cells, mix gently, then incubate on ice for 30 minutes, and heat accurately at 42°C. 90s, then incubate on ice for 3min, add 1ml of LB medium containing 5mM Ca 2+ , and culture on a shaking table at 37°C and 220rpm.
  • Negative control 2 Randomly select a tube of DH10B/pRARE/pKD46 competent and do not convert any fragments or genomes. Other operations remain consistent with the experimental group. That is, ice bath for 30 minutes, heat shock at 42°C for 90 seconds, then ice bath for 3 minutes, add 1 ml of LB medium containing 5mM Ca 2+ , and culture on a 37°C, 220rpm shaker
  • 200 ⁇ L of supernatant and 300 ⁇ L of the natural host bacteria of the corresponding phage in the logarithmic phase are mixed and incubated at 37°C for 4-5 hours. After that, spread a double-layer agar plate. Add 3 ml of soft agar containing 5mM Mg 2+ to the incubated mixture, vortex thoroughly and spread it on a solid LB plate containing 5mM Mg 2+ . Place it on the solid LB plate after solidification. Incubate at 37°C for at least 4-6 hours to observe the assembly results.
  • Example 2 Pseudomonas aeruginosa phage Palz-3 genome, assembled from 6 to 15 kb fragments.
  • E. coli strains strains DH10B/pRARE; DH10B/pRARE/pKD46 preserved by the research team
  • Each phage assembly experimental set includes three types:
  • the amount of fragments used in the three different experimental groups was all 60 fmol/fragment.
  • Positive control The extracted phage genomic DNA is activated with the intermediate host DH10B/pRARE and the intermediate host DH10B/pRARE/pKD46. Theoretically, the results of the positive control should appear as plaque or clear results.
  • Competent DH10B/pRARE/pKD46 perform the same experiment as the experimental group, but do not transform the fragments or genomes to eliminate the interference of chloroform absorbed after adding chloroform to disrupt the cells on the experimental results.
  • the negative control results should be filled with the natural host bacteria of the phage.
  • A. Streak and isolate monoclonal colonies Take the preserved bacteria in glycerol and streak using the "three-line method". Among them, the DH10B/pRARE/pKD46 strain needs to be cultured at 30°C. Use a solid LB plate with ampicillin and chloramphenicol dual antibiotics. Monoclonal colonies of appropriate size can grow in about 2 days; the DH10B/pRARE strain needs to be cultured at 37°C. , using a solid LB plate with chloramphenicol antibiotic, monoclonal colonies of appropriate size can grow in about 1 day.
  • Preparation of logarithmic bacteria Calculate the volume of logarithmic bacteria to be prepared based on the 100X amount of competent cells required for the experiment.
  • the DH10B/pRARE strain was cultured at 37°C until the OD600 was between 0.5-0.55; the DH10B/pRARE/pKD46 strain was cultured at 30°C until the OD600 was between 0.5-0.55; the liquid LB medium used for both strains was the same as the previous step.
  • Competent state preparation "Calcium chloride method" prepares the above two different E. coli competent states, both of which are 200 ⁇ L 100X.
  • Experimental group 1 60 fmol/fragment, 15 ⁇ L ER Mix, react at 42°C for 90 minutes. Transfer the reaction mixture to the competent cells of Escherichia coli DH10B/pRARE, that is, add the mixture to the competent cells, mix gently, then incubate on ice for 30 minutes, heat shock at 42°C for 90 seconds, then incubate on ice for 3 minutes, add 1 ml containing LB medium with 5mM Ca2+, cultured at 37°C, 220rpm.
  • Experimental group 2 60 fmol/fragment, 15 ⁇ L ER Mix, react at 42°C for 90 minutes. Transfer the reaction mixture to the competent cells of Escherichia coli DH10B/pRARE/pKD46, that is, add the mixture to the competent cells, mix gently, then incubate on ice for 30 minutes, heat shock at 42°C for 90 seconds, then incubate on ice for 3 minutes, add 1ml LB medium containing 5mM Ca2+, culture at 37°C, 220rpm.
  • Experimental group 3 Transfer 60 fmol/fragment directly into E. coli DH10B/pRARE/pKD46 competent cells, that is, add the fragment mixture to the competent cells, mix gently, then incubate on ice for 30 minutes, heat shock at 42°C for 90 seconds, and then ice again Bath for 3 minutes, add 1 ml of LB medium containing 5mM Ca2+, and culture on a shaker at 37°C and 220rpm.
  • Negative control 1 Randomly select a genome fragment, transfer 60 fmol directly into E. coli DH10B/pRARE/pKD46 competent cells, that is, add the fragment mixture to the competent cells, mix gently, then incubate on ice for 30 minutes, and heat accurately at 42°C. 90s, then incubate on ice for 3min, add 1ml of LB medium containing 5mM Ca2+, and culture on a shaking table at 37°C and 220rpm.
  • Negative control 2 Randomly select a tube of DH10B/pRARE/pKD46 competent and do not convert any fragments or genomes. Other operations remain consistent with the experimental group. That is, ice bath for 30 minutes, heat shock at 42°C for 90 seconds, then ice bath for 3 minutes, add 1 ml of LB medium containing 5mM Ca 2+ , and culture on a shaking table at 37°C and 220 rpm.
  • the assembly method of directly assembling the genome of Escherichia coli in vivo and combining the in vivo assembly of Escherichia coli with the assistance of Gibson Assembly in vitro resulted in plaques or clear plates in all three parallel experiments.
  • three parallel experiments of pure in vitro Gibson Assembly were unsuccessful, indicating that the stability of the results of the present invention is superior to that of traditional Gibson Assembly, and it can achieve up to six 15kb phage genomes more stably. Successful assembly of large fragments.
  • E. coli strains strains DH10B/pRARE; DH10B/pRARE/pKD46 preserved by the research team
  • Each phage assembly experimental set includes three types:
  • the amount of fragments used in the three different experimental groups was all 60 fmol/fragment.
  • Positive control The extracted phage genomic DNA is activated with the intermediate host DH10B/pRARE and the intermediate host DH10B/pRARE/pKD46. Theoretically, the results of the positive control should appear as plaque or clear results.
  • Competent DH10B/pRARE/pKD46 perform the same experimental operations as the experimental group, but do not transform the fragments or genomes, to eliminate the interference of chloroform absorbed after adding chloroform to disrupt the cells on the experimental results.
  • the negative control results should be filled with the natural host bacteria of the phage.
  • A. Streak and isolate monoclonal colonies Take the preserved bacteria in glycerol and streak using the "three-line method". Among them, the DH10B/pRARE/pKD46 strain needs to be cultured at 30°C. Use a solid LB plate with ampicillin and chloramphenicol dual antibiotics. Monoclonal colonies of appropriate size can grow in about 2 days; the DH10B/pRARE strain needs to be cultured at 37°C. , using a solid LB plate with chloramphenicol antibiotic, monoclonal colonies of appropriate size can grow in about 1 day.
  • Preparation of logarithmic bacteria Calculate the volume of logarithmic bacteria to be prepared based on the 100X amount of competent cells required for the experiment.
  • the DH10B/pRARE strain was cultured at 37°C until the OD600 was between 0.5-0.55; the DH10B/pRARE/pKD46 strain was cultured at 30°C until the OD600 was between 0.5-0.55; the liquid LB medium used for both strains was the same as the previous step.
  • Competent state preparation "Calcium chloride method" prepares the above two different E. coli competent states, both of which are 200 ⁇ L 100X.
  • Experimental group 1 60 fmol/fragment, 15 ⁇ L ER Mix, reaction at 42°C for 105 min. Transfer the reaction mixture to the competent cells of Escherichia coli DH10B/pRARE, that is, add the mixture to the competent cells, mix gently, then incubate on ice for 30 minutes, heat shock at 42°C for 90 seconds, then incubate on ice for 3 minutes, add 1 ml containing LB medium with 5mM Ca 2+ , cultured at 37°C, 220rpm.
  • Experimental group 2 60 fmol/fragment, 15 ⁇ L ER Mix, reaction at 42°C for 105 min. Transfer the reaction mixture to the competent cells of Escherichia coli DH10B/pRARE/pKD46, that is, add the mixture to the competent cells, mix gently, then incubate on ice for 30 minutes, heat shock at 42°C for 90 seconds, then incubate on ice for 3 minutes, add 1 ml of LB medium containing 5mM Ca 2+ , cultured at 37°C, 220rpm.
  • Experimental group 3 Transfer 60 fmol/fragment directly into E. coli DH10B/pRARE/pKD46 competent cells, that is, add the fragment mixture to the competent cells, mix gently, then incubate on ice for 30 minutes, heat shock at 42°C for 90 seconds, and then ice again Bath for 3 minutes, add 1 ml of LB medium containing 5mM Ca 2+ , and culture on a shaker at 37°C and 220rpm.
  • Negative control 1 Randomly select a genome fragment, transfer 60 fmol directly into E. coli DH10B/pRARE/pKD46 competent cells, that is, add the fragment mixture to the competent cells, mix gently, then incubate on ice for 30 minutes, and heat accurately at 42°C. 90s, then incubate on ice for 3min, add 1ml of LB medium containing 5mM Ca 2+ , and culture on a shaking table at 37°C and 220rpm.
  • Negative control 2 Randomly select a tube of DH10B/pRARE/pKD46 competent and do not convert any fragments or genomes. Other operations remain consistent with the experimental group. That is, ice bath for 30 minutes, heat shock at 42°C for 90 seconds, then ice bath for 3 minutes, add 1 ml of LB medium containing 5mM Ca 2+ , and culture on a shaking table at 37°C and 220 rpm.
  • 200 ⁇ L of supernatant and 300 ⁇ L of the natural host bacteria of the corresponding phage in the logarithmic phase are mixed and incubated at 37°C for 4-5 hours. After that, spread a double-layer agar plate. Add 3 ml of soft agar containing 5mM Mg 2+ to the incubated mixture, vortex thoroughly and spread it on a solid LB plate containing 5mM Mg 2+ . Place it on the solid LB plate after solidification. Incubate at 37°C for at least 4-6 hours to observe the assembly results.
  • E. coli strain strain DH10B/pRARE/pKD46 preserved by the research team
  • the plasmid cloning experimental group includes 2 types:
  • the amount of fragments used in the two different experimental groups was both 30 fmol/fragment.
  • Positive control The extracted psgRNA N20 GFP plasmid should theoretically contain dense E. coli monoclonal colonies in the positive control results.
  • Negative control Plasmid vector and fragment are used as negative controls respectively; theoretically, there should be no or less E. coli single clone colonies in the negative control results.
  • A. Streak and isolate monoclonal colonies Take the preserved bacteria in glycerol and streak using the "three-line method". Among them, the DH10B/pRARE/pKD46 strain needs to be cultured at 30°C, using a solid LB plate with ampicillin and chloramphenicol as dual antibiotics. Monoclonal colonies of appropriate size can grow in about 2 days.
  • Competent state preparation Prepare the above E. coli competent state by "calcium chloride method", the competent state is 200 ⁇ L 100X.
  • Experimental group 1 30 fmol/fragment, 15 ⁇ L ER Mix, react at 50°C for 15 minutes. Transfer the reaction mixture to the competent cells of Escherichia coli DH10B/pRARE/pKD46, that is, add the mixture to the competent cells, mix gently, then incubate on ice for 30 minutes, heat shock at 42°C for 90 seconds, then incubate on ice for 3 minutes, add 1ml LB medium, culture at 37°C, 220rpm for 1 hour. Then, centrifuge the culture solution at 14,000 rpm for 3 minutes, absorb and discard the supernatant to 800 ml of culture medium, use the remaining culture medium to mix the bacterial pellet, and then coat the solid LB plate.
  • Experimental group 2 30 fmol/fragment was directly transferred to Escherichia coli DH10B/pRARE/pKD46 competent cells, that is, the fragment mixture was added to the competent cells, mixed gently, then incubated on ice for 30 minutes, heat shock at 42°C for 90 seconds, and then incubated on ice. 3min, add 1ml LB medium, and incubate for 1h on a shaking table at 37°C and 220rpm. Then, centrifuge the culture solution at 14,000 rpm for 3 minutes, absorb and discard the supernatant to 800 ml of culture medium, use the remaining culture medium to mix the bacterial pellet, and then coat the solid LB plate.
  • Negative control plasmid fragment or vector, transfer 30 fmol directly into E. coli DH10B/pRARE/pKD46 competent cells, that is, add the fragment mixture to the competent cells, mix gently, then incubate on ice for 30 minutes, heat shock at 42°C for 90 seconds. Then ice bath for 3 minutes, add 1ml LB medium, incubate for 1 hour at 37°C, 220rpm shaker, centrifuge the culture medium at 14000rpm for 3 minutes, absorb and discard the supernatant ⁇ 800 ⁇ L medium, use the remaining medium to mix the bacterial sediment Post-coat solid LB plates.
  • both the direct assembly of the genome in E. coli and the combination of in vivo assembly in E. coli and the assistance of Gibson Assembly in vitro can realize the connection of vectors and fragments to construct complete plasmids, indicating that the present invention is also suitable for conventional molecular cloning experiments.
  • E. coli strains Stellar/pRARE strains preserved by the research team; stellar/pRARE/pKD46
  • Each phage assembly experimental set includes three types:
  • the amount of fragments used in the three different experimental groups was all 30 fmol/fragment.
  • Positive control The extracted phage genomic DNA is activated with the intermediate host stellar/pRARE and the intermediate host stellar/pRARE/pKD46. Theoretically, the results of the positive control should appear as plaque or clear results.
  • n-1 (n is the number of assembled fragments) genome fragments, each fragment is 30 fmol, and directly transform into E. coli stellar/pRARE/pKD46;
  • Competent stellar/pRARE/pKD46 the same experimental operation as the experimental group, but without transforming the fragments or genomes, to eliminate the interference of chloroform absorbed after adding chloroform to disrupt the cells on the experimental results.
  • the negative control results should be filled with the natural host bacteria of the phage.
  • A. Streak and isolate monoclonal colonies Take the preserved bacteria in glycerol and streak using the "three-line method".
  • the stellar/pRARE/pKD46 strain needs to be cultured at 30°C, using a solid LB plate with ampicillin and chloramphenicol dual antibiotics (it is better to use a solid SOB plate), and monoclonal colonies of appropriate size can grow in about 2 days;
  • Cultivate the stellar/pRARE strain at 37°C and use a solid LB plate with chloramphenicol antibiotics it is better to use a solid SOB plate).
  • Monoclonal colonies of appropriate size can grow in about 1 day.
  • V. 4°C centrifuge at 2500g for 10 minutes to harvest the bacteria
  • Experimental group 1 30 fmol/fragment, 15 ⁇ L ER Mix, reaction at 42°C for 105 min. Transfer the reaction mixture to E. coli stellar/pRARE competent cells, that is, add the mixture to the competent cells, mix gently, then incubate on ice for 30 minutes, heat shock at 42°C for 90 seconds, then incubate on ice for 3 minutes, add 1 ml containing SOB liquid medium with 5mM Ca 2+ , cultured at 37°C, 220rpm.
  • Experimental group 2 30 fmol/fragment, 15 ⁇ L ER Mix, reaction at 42°C for 105 min. Transfer the reaction mixture to the competent cells of Escherichia coli stellar/pRARE/pKD46, that is, add the mixture to the competent cells, mix gently, then incubate on ice for 30 minutes, heat shock at 42°C for 90 seconds, then incubate on ice for 3 minutes, add 1ml of SOB liquid medium containing 5mM Ca 2+ , cultured at 37°C, 220rpm.
  • Experimental group 3 Transfer 30 fmol/fragment directly into Escherichia coli stellar/pRARE/pKD46 competent cells, that is, add the fragment mixture to the competent cells, mix gently, then incubate on ice for 30 minutes, heat shock at 42°C for 90 seconds, and then ice again Bath for 3 minutes, add 1 ml of SOB liquid medium containing 5mM Ca 2+ , and culture on a shaking table at 37°C and 220rpm.
  • Negative control 1 randomly selected n-1 (n is the number of assembled fragments) genome fragments, each fragment is 30 fmol, directly transformed into E. coli stellar/pRARE/pKD46, that is, the fragment mixture is added to the competent cells, and gently After mixing, incubate on ice for 30 minutes, heat at 42°C for 90 seconds, then incubate on ice for 3 minutes, add 1 ml of SOB liquid culture medium containing 5mM Ca 2+ , and culture on a 37°C, 220rpm shaker.
  • Negative control 2 All genome fragments, 30 fmol of each fragment, are directly transformed into E. coli stellar/pRARE without homologous recombination system, that is, the fragment mixture is added to the competent cells, mixed gently and then incubated on ice for 30 minutes at 42°C. Heat shock for 90 seconds, then incubate on ice for 3 minutes, add 1 ml of SOB liquid culture medium containing 5mM Ca 2+ , and culture on a shaking table at 37°C and 220rpm.
  • Negative control 3 Randomly select a tube of stellar/pRARE/pKD46 competent and do not convert any fragments or genomes. Other operations remain consistent with the experimental group. That is, ice bath for 30 minutes, heat shock at 42°C for 90 seconds, then ice bath for 3 minutes, add 1 ml of SOB medium containing 5mM Ca 2+ , and culture on a shaking table at 37°C and 220 rpm.
  • 200 ⁇ L of supernatant and 300 ⁇ L of the natural host bacteria of the corresponding phage in the logarithmic phase are mixed and incubated at 37°C for 4-5 hours. After that, spread a double-layer agar plate. Add 3ml of soft agar to the incubated mixture, vortex thoroughly and spread it on the solid-containing SOB plate. After solidification, place it at 37°C and incubate for at least 4-6 hours to observe the assembly. result. If the result of clear liquid or plaques is observed, the assembly is successful; if the natural host bacteria of phages are observed on the plate with the naked eye, that is, no plaques appear, it means that the assembly failed. In order to better reflect the situation of plate spots or complete clearing, 300 ⁇ L of logarithmic phase natural host was used to spread a double-layer agar plate as a negative control.

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Abstract

提供了基因组片段组装方法，特别涉及噬菌体基因组片段的表达元件组合、表达载体、宿主、应用及组装方法。在大肠杆菌体内实现噬菌体基因组大片段直接组装，利用该技术可以一步实现噬菌体基因组的改造、组装、激活，极大缩短实验周期、提高改造人造噬菌体的效率。该方法也适用于其他情景下的大片段和/或小片段组装，可用于常规分子克隆实验。

Description

表达元件组合、表达载体、宿主、应用及组装方法 技术领域
本发明涉及基因组片段组装领域，特别涉及噬菌体基因组片段的表达元件组合、表达载体、宿主、应用及组装方法。
背景技术
随着噬菌体疗法的不断应用，有体外实验和临床数据表明，利用天然噬菌体进行噬菌体治疗的效果具有一定的局限性，甚至出现了天然噬菌体耐受的情况。局限性在于噬菌体具有严格的宿主特异性，杀菌谱很窄，一般一种噬菌体只能靶向性的杀死一种细菌，或者一种细菌的一个菌株,这主要是由噬菌体吸附蛋白和宿主细胞膜表面受体的分子结构和其互补性决定的。耐受的原因之一，可能是某些细菌天然的遗传免疫系统(CRISPR-Cas系统)。
为了解决以上问题，目前较为常见的是天然噬菌体鸡尾酒疗法和天然噬菌体鸡尾酒疗法与抗生素连用，但以上两种方法并没有从根源上解决噬菌体宿主范围狭窄的问题。近几年来，不断有科学家尝试使用基因工程的方法对噬菌体进行改造，尝试合成新型人造噬菌体，拓宽噬菌体宿主范围。目前，人造噬菌体大片段(≥10kb)且多片段(≥4)组装技术主要有体内(酵母)直接组装和体外组装(Gibson Assembly和Golden Gate Assembly)。
2012年Jaschke等人使用PCR产物及合成片段首次在酵母内直接组装5.4kb的噬菌体X174基因组，并在大肠杆菌中激活成功获得噬菌斑。2015年9月cell systems杂志报道了利用酿酒酵母内天然同源重组系统成功组装5-10个1.3-10.1kb大小不等的片段(包含质粒载体YAC 3.0kb)，组装后的噬菌体基因组长度为37-45kb不等。2020年8月American Society for Microbiology杂志报道了利用长片段PCR和Gibson Assembly体外成功组装3-4个8-15kb的噬菌体基因组片段，其总体长度为39-62kb。美国New England Biolabs(NEB)公司研发团队于2020年12月利用体外Golden Gate Assembly方法将长度在148至1735bp之间的52个片段的T7噬菌体基因组(40kb)组装成功。以上两类实验技术，或体外组装噬菌体基因组在大肠杆菌中激活，或酵母内直接组装，获得噬菌体基因组后释放，然后在大肠杆菌中激活。迄今为止，并未有噬菌体基因组大片段在大肠杆菌内直接组装、激活的报道。
此外，山东大学张友明教授团队于2017年自主研发了ExoCET大片段质粒直接克隆法，该方法将最小8kb、最大106kb的单片段细菌或哺乳动物基因组克隆到2.2kb的线性质粒载体上。该团队于2019年使用ExoCET实验技术直接克隆12个长度为1.5-2.5kb的PCR产物到2.1kb的线性载体上，其总长度为25.4kb。美国New England Biolabs(NEB)公司研发有试剂盒
Figure PCTCN2022082446-appb-000001
HiFi DNA Assembly，该试剂盒相较于目前常规的Gibson Assembly Master Mix/Cloning kit更高效更准确，适用于单个大片段(≥10kb)或多个片段(≥4)的分子克隆，目前已成功克隆12kb的DNA片段于7.4kb 的质粒中。美国Thermo Fisher Scientific科技公司研发有Invitrogen TMTOPO TMXL-2完整PCR克隆试剂盒，该试剂盒能简单有效且准确的克隆13kb的长PCR产物。
目前合成人造噬菌体面临基因组组装的技术瓶颈，现阶段多采用体外组装或酵母体内组装的方法，体外组装大片段基因组片段成功率极低；酵母内组装技术实验周期长且技术门槛较高。
1、体外Gibson Assembly组装：
Gibson Assembly组装主要依靠DNA末端同源序列的同源重组，组装效率随着DNA片段的增大、增多而降低，该方法组装噬菌体基因组成功率较低。
2、体外Golden Gate Assembly组装：
Golden Gate Assembly组装利用IIS型限制性内切核酸酶生成具有短单链突出端的DNA片段和DNA连接酶将片段连接在一起。组装依赖目标载体，该载体含有抗性筛选标记用于筛选阳性克隆，但易出现假阳性问题；仍需后续的PCR和测序验证，增加实验周期及成本。
3、酵母体内直接组装：
1)、该方法技术门槛较高，普适性较低；
2)、该方法实验周期长(酵母需30℃生长2-3天；实验需要两个步骤，酵母内组装噬菌体基因组，大肠杆菌内噬菌体激活验证)。
综上，提供基因组片段组装，特别是噬菌体基因组片段组装的方法具有重要的现实意义。
发明内容
有鉴于此，本发明提供了基因组片段的表达元件组合、表达载体、宿主、应用及组装方法。
为了实现上述发明目的，本发明提供以下技术方案：
第一方面，本发明提供了表达元件组合，包括但不限于：
(I)、pRARE质粒中的表达元件；和
(II)、lambda Red同源重组系统的表达元件。
在本发明的一些具体实施方案中，所述表达元件组合中所述lambda Red同源重组系统的表达元件包括但不限于exo、bet和gam。
第二方面，本发明还提供了表达载体，包括如权利要求1或2所述的表达元件组合。
在本发明的一些具体实施方案中，所述表达载体包括但不限于pRARE质粒和pKD46质粒。
第三方面，本发明还提供了转化或转染有所述表达载体的宿主。
在本发明的一些具体实施方案中，所述宿主为原核生物或真核生物，包括但不限于细菌、放线菌、立克次氏体、衣原体、支原体、蓝细菌、古细菌、酵母和霉菌中的一种或多种。
在本发明的一些具体实施方案中，所述细菌包括但不限于革兰氏阴性菌或革兰氏阳性菌；
作为优选，所述革兰氏阴性菌包括但不限于大肠杆菌；更优选的，所述大肠杆菌包括但不限于大肠杆菌DH10B和stellar；
作为优选，所述革兰氏阳性菌包括但不限于细胞壁缺陷型L型细菌；更优选的，细胞壁缺陷型L型细菌包括但不限于L型李斯特菌。
基于上述研究，本发明的第四方面，还提供了所述表达元件组合、所述表达载体，或所述宿主在基因组片段组装中的应用。
第五方面，本发明还提供了所述表达元件组合、所述表达载体，或所述宿主在分子克隆、合成人造噬菌体、噬菌体疗法、制备噬菌体疗法的产品、检测微生物、制备检测微生物的产品、预防和/或治疗微生物引起的感染或疾病，或制备预防和/或治疗微生物引起的感染或疾病的药物中的一种或多种的应用。
在本发明的一些具体实施方案中，所述片段包括但不限于：
(I)、＜2kb的片段；和/或
(II)、≥2kb且＜7kb的片段；和/或
(III)、≥7kb且＜10kb的片段；和/或
(IV)、≥7kb且＜10kb的片段；和/或
(V)、≥10kb的片段。
在本发明的一些具体实施方案中，所述片段的个数包括但不限于：
(I)、＜5个；和/或
(II)、≥5个，且＜10个；和/或
(III)、≥10个且＜15个；和/或
(IV)、≥15个。
第六方面，本发明还提供了基因组片段的组装方法，基于所述表达元件组合、所述表达载体，或所述宿主，对基因组的片段进行组装。
在本发明的一些具体实施方案中，所述组装包括但不限于：
(I)、所述宿主体内直接组装；和/或
(II)、体外Gibson Assembly组装和所述宿主体内组装。
在本发明的一些具体实施方案中，所述组装方法包括但不限于如下步骤：
步骤A：获得待组装的基因组的片段；
步骤B：进行体外Gibson Assembly组装；
步骤C：经宿主体内组装；
其中，所述组装方法可以包括步骤B，也可以不包括步骤B。
在本发明的一些具体实施方案中，所述组装方法中涉及的所述片段包括但不限于：
(I)、末端存在至少40～100bp的同源区域；和/或
(II)、当所述片段的数量较多(>6个)、较大(>2kb)时，增大同源臂长度， 同时保证同源区域在50℃的二级结构自由能绝对值较小(尽可能小，可避免末端形成的二级结构影响末端同源重组，目前成功组装片段的同源臂自由能的绝对值最大值为15.74kcal/mol)。
在本发明的一些具体实施方案中，所述体外Gibson Assembly组装的反应温度包括但不限于37℃～50℃；
作为优选，所述体外Gibson Assembly组装的反应温度包括但不限于：
(I)、42℃或50℃；和/或
(II)、37℃～42℃；和/或
(III)、42℃～50℃。
在本发明的一些具体实施方案中，所述组装方法的步骤B中所述体外Gibson Assembly组装的反应时间包括但不限于15min～105min；
作为优选，所述体外Gibson Assembly组装的反应时间包括但不限于：
(I)、15min、75min或105min；和/或
(II)、15min～75min；和/或
(III)、75min～105min。
在本发明的一些具体实施方案中，所述体外Gibson Assembly组装的体系中，所述片段的用量不少于30fmol；
作为优选，所述片段的用量包括但不限于：
(I)、30fmol～60fmol；和/或
(II)、＞60fmol。
本发明中所述基因组(genome)，是细胞或生物体的全套遗传物质，或载有遗传信息的全体核酸，包括所有染色体上的核酸，对于真核生物，其基因组还包括线粒体上的核酸。各种生物体之间存在着差异，这种差异正是由基因组所决定的。各种生物体基因组的大小变化很大，最小的只有5000bp(base pair)，如最简单的病毒，最大的有1011bp，如一些高等植物。基因组的大小与生物体的复杂性没有直接的关系。基因组中某些成分的位置并非一成不变，而且同种生物的不同个体之间，其基因组大小或基因数目也不是绝对固定的，甚至由于基因组结构变化还会导致功能的变化。尽管如此，各类生物的基因组仍然有着基本的结构特点。人类基因组由3.2′109bp的DNA组成，包括细胞核染色体基因组和细胞质内线粒体基因组。细胞核染色体基因组中大约有一半为不同的重复序列，含基因2.5万～3.5万个。基因组控制着生物体的生长发育，控制着生命活动。要想认识生物的本质，就必须首先认识基因组，掌握基因组的所有核酸序列，解析基因组的全部遗传信息，理解遗传信息的组织结构及其在生物体内的表达。
原核生物的基因组：原核生物的基因组比较小，DNA量低。原核生物基因组的许多信息都是为了维持细胞的基本功能，如构造和复制DNA，产生新蛋白质，以及获得和存储能量。在原核基因组中，基因分布的密度非常高，其中的DNA分子的绝大部分是用来编码蛋白质的，只有非常小的一部分不转录，这点与真核生物DNA分 子不一样。细菌含有染色体和染色体外的质粒DNA。大肠杆菌的基因组DNA是单个双链环状DNA分子，有些细菌还含有存在于细胞质中的小型环状双链DNA。染色体外的DNA也可能含有遗传信息，可以进行自我复制，并将遗传信息传递给子代细胞。
真核生物基因组：真核生物在各个方面都要比原核生物复杂得多，包括丰富的内部膜系统、复杂的遗传信息表达系统等。在基因组方面，真核生物基因组的规模远大于原核生物基因组，组织复杂，信息含量高。在整个DNA序列中，蛋白质编码区域仅占一小部分，而非编码序列则占了很大一部分。真核基因结构也远比原核基因结构复杂、多变。真核细胞含有细胞器，例如线粒体或叶绿体。这些细胞器中也含有DNA，但是它们不同于细胞核基因组DNA，它们通常是很小的环状双链DNA分子。
大多数真核基因都是由蛋白质编码序列和非蛋白质编码序列两部分组成的。基因中的编码序列称为外显子(exon)，而基因中的非编码序列称为内含子(intron)。在一个结构基因中，编码某一蛋白质不同区域的各个外显子并不是连续地排列在一起的，而是常常被长度不同的内含子所隔离，形成镶嵌排列的断裂方式，所以，真核基因有时被称为分裂基因。不同基因拥有内含子的数量和大小相差非常悬殊，如胶原蛋白基因，长约40kb，至少具有40个内含子，其中短的只有50bp，长的可达到2000bp。许多真核基因具有大量的有时是非常大的内含子，例如，与人类囊性纤维症有关的基因，含有24个内含子，加起来的长度超过1Mb。而少数基因，如组蛋白，根本不带内含子。目前尚不清楚内含子的生理功能。
许多外显子为蛋白质分散的结构和功能单位编码，例如，肌红蛋白和血红蛋白基因的中央外显子为一个与血红素结合的区域编码，此区域能可逆地与O2结合。其他外显子专一地编码a螺旋片段，这些片段使蛋白质插在细胞膜中。蛋白质的一个结构域可能是由单个外显子编码。有一种假说认为：在进化过程中，新蛋白质的出现是由于外显子的重新排列，这些外显子编码分散的结构单位、结构部位和催化部位。外显子的混合是产生新基因的迅速而有效的方法。分裂基因有另一个优点，即通过不同方式的剪接而新生的RNA，可能产生一系列相关的蛋白质，这组蛋白质是根据一个发育程序而发生的一个基本特性的变异。
真核基因分裂结构的一个重要特点是外显子-内含子连接区是具有高度保守性和特异性的碱基序列。外显子-内含子连接区是指外显子和内含子的交界，又称边界序列。外显子-内含子连接区虽然很短，但却是高度保守的。这一保守序列(conserved sequence)与剪切机制密切相关，它是RNA剪切的信号序列。
外显子-内含子在连接区的保守序列几乎存在于所有高等真核生物基因中，这表明在这些基因中可能存在着一个共同的剪切机制。但是，在线粒体基因中不存在这类保守序列，这暗示还可能存在不同类型的加工过程。
一个完整的基因，不但包括编码区域，还包括5'-端和3'-端两侧长度不等的特异性序列。虽然这些序列不编码氨基酸，却在基因表达的过程中起着重要的作用。所以，严格的“基因”，这一术语的分子生物学定义是：产生一条多肽链或功能RNA所必需的全部核苷酸序列。
RNA聚合酶在基因表达起始阶段和基因5'-端上游区域的某一段核酸序列结合，然后开始转录，合成基因的RNA拷贝，这一段核酸序列称为启动子(promoter)。启动子决定了DNA转录的方向、速度和准确性。RNA聚合酶必须准确识别基因转录的起始位置。RNA聚合酶在寻找基因的起点时不能寻找任意一个特定的核苷酸，因为在细胞的DNA分子中，单一核苷酸出现的频率非常高。然而，核苷酸间的特定组合却不容易随机出现，并且，涉及的核苷酸的数目越多，该组合出现的几率变得越小。如果所有核苷酸以相同概率出现，那么特定核苷酸串单独出现的概率为P＝(1/4)n，n是核苷酸的数目。原核生物的RNA聚合酶扫描整条DNA，寻找标记基因起点的约为13个核苷酸长度的特定核苷酸序列(一个核苷酸是转录起始位点，6个核苷酸位于距起始位点上游10个碱基处，另6个核苷酸位于起始位点上游35个碱基处)，这些启动子序列之间的相对位置固定。由于多数原核生物的基因组只有几百万个核苷酸，这些启动子序列结合在一起的几率约为七千万分之一，这足以让RNA聚合酶可靠地、唯一地识别基因的起始位置。真核生物的基因组比原核生物大几个数量级，相应地，真核生物的RNA聚合酶要识别更为复杂和更长的启动子序列，以便也能可靠地识别基因的起始位置。
通过对许多基因的启动子进行解析、比较，发现了相当保守的启动子序列。如前面所述，对于原核基因，在转录起始点上游10bp处，有一个嘌呤盒(box)序列，即T80A90T45A60A50T96。这里，字母右下角的数字为相应碱基出现的频率。另外，在转录起始点上游35bp处，有一段保守序列：T82T84G78A65C54A45。对于真核基因，在转录起始点上游30bp处，有TATA盒(TATAbox)，在上游80bp处有一个CAAT盒(CAATbox)，而在上游80bp～110bp附近含有GCCACACCC或GGGCCGGG序列，这称为GC盒(GC BOX)。
启动子的定义是相对于转录起始点上游有相对固定位置的DNA序列，包括TATA盒、CAAT盒和GC盒，一般在从转录起始点到上游150bp这段范围内。这些上游保守序列统称为上游启动子元件(upstream promoter element，UPE)。
TATA盒和其他两个UPE的作用不同。前者的主要作用是使转录精确地起始，如果除去TATA盒或进行突变，转录产物下降的相对值不如CAAT盒或GC盒突变后的明显，但发现所获得的RNA产物的起始点不固定。CAAT盒和GC盒主要控制转录的起始频率，基本不参与起始位点的确定。CAAT盒对转录起始频率的影响最大，该区域任意一个碱基的改变都将极大地影响靶基因的转录强度，而启动区其他序列中一两个碱基的置换对转录强度则没有太大的影响。此外，在TATA盒和相邻的UPE之间插入核苷酸也会使转录减弱。尽管上述三种UPE序列都有着重要的功能，但并不是每个基因的启动子区都包含这三种序列。
启动子对转录固然重要，但是启动子上游部位的序列是否和转录就没有关系呢？人们在研究SV40病毒时发现，启动子上游的某些序列如果发生变化，则可能大大降低转录的活性。这些序列在正常的情况下能对转录起增强作用，所以称为增强子(enhancer)。增强子一般能使转录频率增加10～200倍。
增强子有几个显著的特征：(1)增强子的序列较长，可达数百个碱基对。有时是重复序列，其内部常含有一个核心序列“(G)TGGA/TA/TA/T(G)”。(2)作用距离比较远，可以远离它所作用的基因，达数千个碱基之远。(3)作用没有方向性：序列正反颠倒过来，同样起作用；(4)位置不固定：可以是在某个基因的5'-端上游，也可以是在3'-端下游，甚至可以在基因的内含子内。(5)增强子一般具有组织或细胞特异性。(6)增强子的活性与它在DNA双螺旋结构中的空间方向性有关。
除了启动子和增强子之外，还有一种负调控元件，即沉默子(silencer)。通过沉默子可以抑制基因的表达，或对基因进行有选择性的表达。
单拷贝基因和基因家族：在基因组中仅出现一次的基因称为单拷贝基因。单拷贝基因多是编码蛋白质的基因，一般比较复杂。这些基因往往是断裂基因，即编码区域非连续。在真核生物中也存在如同原核生物操纵子结构的转录单位，其转录出来的初级转录产物可以通过不同的拼接方式产生一种以上的蛋白质，即一段DNA序列可编码多种蛋白质。有25％～50％的真核细胞编码蛋白质的基因是以单个基因存在于基因组中，而其余编码蛋白质的基因都属于两个或两个以上相似基因构成的基因家族。在进化过程中，从一个祖先基因分歧产生多个基因，这些基因构成一族结构和功能上相关的基因群，它们或聚集在一起，或分散在基因组中。这种来自于同一个祖先基因，编码相似而不完全相同的蛋白质的一组基因被称为基因家族。被这些基因编码的蛋白质称为蛋白质家族。基因家族有大有小，少则仅有几个基因，多则有数百个基因。a-珠蛋白和b-珠蛋白对应的基因家族是研究得最多的基因家族。
重复序列：真核生物细胞DNA中存在着大量的重复序列，对于脊椎动物尤其如此。重复序列大致可以分成三类，即低度重复序列、中度重复序列和高度重复序列。
中度重复序列的重复次数在10～105之间，占总DNA的10％～40％，如小鼠基因组的中度重复序列占20％，果蝇的占15％。
高度重复序列的重复次数大于106，基本的重复序列长度从几个碱基到几百个碱基或更长，占基因组的10％～60％。高度重复序列按其长度可分为：(1)卫星DNA(satellite DNA)：重复序列的长度在5～200bp，这些序列聚集在一起，串联排列，总长度可达100mb(mega base)。它们在人群中的多态性不强。(2)小卫星(minisatellite)DNA：重复序列的长度从5个bp到几十个bp，串联排列，总长度在0.5～30kb之间。这类重复序列在人群中存在高度的多态性。(3)微卫星(microsatellite)DNA：重复序列的长度在2～6bp之间，也有高度的多态性。
人们通常将基因组看成是静止的，仅在进化规模上考虑到它的变化，其实，基因组的结构不是完全静止的，是有变化的。生物界普遍存在着变异，每一代都有变异，没有两个生物个体是完全相同的。可遗传的变异包括基因突变、基因重组和染色体畸变。基因突变指的是基因的DNA序列发生突变。目前对分子进化了解最多的是点突变(point mutation)，或者是核酸序列中的单碱基突变。单碱基突变是指一个碱基变化为另一个碱基，或者是在序列中插入一个新的碱基，或者是删除原有的一个碱基。在编码区域中的碱基的插入或删除将导致基因的阅读框移码，使原来的密码子被打 乱，从而使基因的转录出现异常。
许多突变对于生物体的影响并不是很大。一般出现在基因内含子中间部分的突变不造成影响，但是，如果突变发生在内含子边界，则可能影响转录剪切过程。如果突变发生在密码子第三位，则由于密码子的简并性，在蛋白质水平上不会造成多大的影响。即使是一个造成蛋白质序列变化的突变，其影响往往也是中性的。当然，有些点突变是致命的，而有些点突变将导致疾病。点突变很少是有益的。
基因重组和基因移动是生物进化的动力，具有重要的生物学意义。所谓基因重组，是指来源于两个或两个以上不同亲本的DNA序列组合成一条新的基因DNA序列。基因重组是生物界的普遍现象。同源重组是任何具有一段同源序列的两个基因DNA序列之间的交换。发生同源重组的条件是两个DNA分子之间存在同源序列，与实际的碱基序列次序无关。只要它们相似，而且相似区段越长，重组发生的频率就越高。若同源序列太短，则很难发生重组。同源重组也是DNA损伤修复的重要机制。当损伤的一条链需要修复，而又不能用另一条互补链作为修复模板时，就要用另外的同源序列，利用同源重组修复机制来修复被损伤的链。另一种重组方式是位点特异性重组，它发生在位点特异性的短序列区，重组时发生精确的切割反应和连接反应，DNA既不丢失，也不合成。在基因组中还存在一类可移动的基因序列，它们可以从染色体的一个部位转移到同一条染色体或另一条染色体上某一新的位置。一个基因通过各种方式从一处移动到另一处，称为基因移动，可以移动的基因称为移动基因。移动基因一般通过转座子的形式进行移动。重组和移动可产生新的序列和功能，或改变原有序列的功能。
染色体可能发生畸变，从而使得遗传物质发生变化。染色体畸变包括染色体的丢失、易位、移位等。这些变化实际上也是DNA分子的变化，如染色体的部分丢失，实际上就是DNA分子的一部分丢失。变异是产生进化的基本条件。人们已经逐步了解进化的分子机制，通过对相关分子进行序列比较分析，可以得到物种之间的进化关系。另一方面，相关分子之间的相似性和差异提供了关于分子结构和功能的重要信息。分子进化同样包括选择和继承。继承性要求亲代的基因能够传递给子代，而选择性则表明有益的突变将被保留下来。
人类基因组：人类基因组的编码区域不到所有DNA序列的3％，基因数为2.5万～3.5万。人类基因组中重复序列占了很大一部分，约为50％，这些重复序列可能具有一定的功能，但也可能没有功能。可以将人类基因组的重复序列分成4种：(1)短散布元素(SINEs)；(2)长散布元素(LINEs)；(3)长终端重复元素(LTR)；(4)DNA转座子(transposons)。基因组剩余的DNA序列包含启动子、转录调控序列及其他未知特性的序列。
现代生物学很多里程碑式的成果都是基于噬菌体而获得的。在过去数年中，完成测序的噬菌体基因组数目在逐年增加，美国国立生物技术信息中心(NCBI)噬菌体数据库中已登录了530多个噬菌体基因序列，比2005年增加了3倍多。生物圈里拥有数量庞大的噬菌体，而噬菌体的基因组包含了较高比例、未知功能的基因序列，因此对 噬菌体基因组的研究，有助于深入了解噬菌体及其与宿主、环境的相互作用。
综上所述，基因组的片段组装方法均在本发明的保护范围之内。对于基因组的范围，本发明不做限定。
在本发明的一些具体实施方案中，本发明中所述基因组包括但不限于噬菌体基因组。
噬菌体(bacteriophage,phage)是一类侵害细菌(包括放线菌、真菌和原核生物)的病毒，属非细胞生物，具有与其他病毒类似的特性，主要由蛋白质和核酸(DNA或RNA)组成。在自然界中分布广泛，土壤、空气、水中或生物体内都可存在。1977年首次完成了单链DNA噬菌体ψX 174的全基因组测序，其由5386个碱基对构成。第一个被完整测序的双链DNA噬菌体是包含48502个碱基对的λ噬菌体。随后，完成了包含39930个碱基对的T7噬菌体测序。10年后，第一个双链有尾噬菌体被完整测序，其属于L5分枝杆菌噬菌体，且可以侵染非埃希氏菌属大肠杆菌。此后，随着DNA测序技术的发展，大量的噬菌体基因组序列被测定，使得噬菌体在分子生物学的研究进程中扮演了越来越重要角色，取得了许多里程碑式的研究成果。
本领域的技术人员公知，噬菌体基因组大小不一，范围从囊球菌属L5噬菌体的3 435bp到假单胞细菌属201phi2-1噬菌体的316674bp。此外，大多数拥有较大基因组(大于125kbp)的噬菌体是肌尾噬菌体(有收缩性的尾部)，基因组最大的长尾噬菌体是芽抱杆菌噬菌体SPB c2(134416bp)。
噬菌体种群有着显著的遗传多样性。一般来说，无共同宿主的噬菌体，其基因组很少有序列相似性，即便宿主菌具有一定的相关性也是如此。例如，在4个链霉菌属噬菌体基因组和50个分枝杆菌噬菌体基因组之间，虽然宿主分枝杆菌和链霉菌都同属放线菌属，但是它们的核普酸序列并没有明显的相似性。
当不同噬菌体感染同一个细菌宿主时，会在增殖过程中互通信息，可能出现共同的核苷酸序列。有很多关于拥有共同宿主的噬菌体具有相关性序列的例子。例如，分离自假单胞菌(33)、葡萄球菌(48)和结核分枝杆菌(50)的30个噬菌体基因组拥有相关序列。但是新的研究发现，拥有同一宿主的噬菌体也可能很少或根本没有序列相似性。例如，大肠埃希氏菌K-12是λ噬菌体和T4噬菌体的共同宿主，两个噬菌体却具有完全不同的生活方式，而且这两个噬菌体之间的序列相似性也极小。另有研究利用G ep and程序分析分离自共同细菌宿主的30个分枝杆菌噬菌体和50个假单胞菌噬菌体核普酸序列之间的关系，通过比较揭示了几点。首先，在这两个噬菌体各自基因组中都有显著的核酸序列相似性，但是两个基因组之间不存在序列相似性。其次，即使在噬菌体感染同一宿主时，他们之间也存在着巨大的遗传差异性。
在过去数年里噬菌体基因组学研究取得了巨大进展，虽然对于噬菌体的多样性和进化途径的研究还没有确切的答案，但是对这些问题的认识已越来越清晰，并且相关研究基础已经奠定。在未来几年，随着序列测定技术的发展，将会获得更多新的噬菌体序列信息。此外，应用噬菌体制剂控制细菌感染已展现出了一定的治疗前景，病原菌广泛的耐药性也迫切需要另辟蹊径解决这种威胁。因此，噬菌体治疗或者联合抗生 素治疗可能会成为一个非常有价值的途径。值得注意的是，为了噬菌体治疗的安全性和可控性，对噬菌体的生物学特性、特异性噬菌体与宿主菌的相互作用关系，需要更加详尽、明确的实验证据。然而，到目前为止，噬菌体对细菌的易感性认知大多是基于体外水平的，噬菌体和细菌在体内是如何作用的，细菌在体内又是如何逃避噬菌体的裂解等方面，还需要更多研究。对新发现的大量噬菌体的功能基因的研究，以及将其在生物学和生物技术等领域应用将是未来噬菌体研究的一个重要方向。
因此，基于本发明提供的噬菌体基因组的片段组装方法，会对噬菌体的序列、功能研究，特别是治疗功能的研究，提供重要的技术支持。
在本发明的一些具体实施方案中，步骤C所述组装后还包括激活的步骤；
所述激活包括但不限于：取所述宿主接种于含有Ca 2+或Mg 2+的培养基中，37℃培养不少于6h。
在本发明的一些具体实施方案中，所述组装方法成功与否的评价标准包括但不限于：出现预期的噬菌斑或平板出现了全清现象，则噬菌体基因组组装成功。
第七方面，本发明还提供了基因组的组装方法，包括上述基因组片段的组装方法。
在本发明的一些具体实施方案中，所述基因组包括但不限于噬菌体基因组。
第八方面，本发明还提供了人造噬菌体的合成方法，包括上述基因组片段的组装方法。
第九方面，本发明还提供了DNA多分子组装的方法，包括上述基因组片段的组装方法。
第十方面，本发明还提供了DNA克隆的方法，包括上述基因组片段的组装方法。
本发明的有益效果包括但不限于：
1、将多个(≥5个)噬菌体基因组大片段(≥10kb)成功组装成完整基因组，克服常规体外Gibson Assembly组装大片段的困难；
2、组装成功的噬菌体基因组具有与天然噬菌体一样的活性，克服目前酵母体内组装的噬菌体基因组难以被激活的技术难题；
3、该发明所实现的基因组组装不依赖于目标载体，可实现噬菌体基因组组装、激活的一步完成，避免了体外Golden Gate Assembly组装和酵母体内组装后的繁琐的筛选步骤，极大减少了实验周期及成本；
4、该发明是合成人造噬菌体的关键步骤之一，该发明可通过与构建噬菌体元件库、定点突变、从头合成等合成生物学技术结合，合成具有临床治疗病原菌潜力的人造噬菌体；
5、该发明可实现的基因组组装不仅只局限于噬菌体基因组，可延伸用于其他基因组的组装。
综上，本发明可以实现通过PCR或从头合成得到的噬菌体基因组片段在大肠杆菌内的直接组装，从而为有临床治疗病原菌潜力的人造噬菌体的合成奠定技术基础。可以用于合成人造噬菌体，噬菌体疗法改良，治疗细菌特别是临床多重耐药菌引起的感染。本发明在噬菌体基因组组装上实验周期短、普适性好、成功率高，有利于推动 人造噬菌体的合成，改进噬菌体疗法，市场前景好。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1示本发明提供的噬菌体基因组片段组装流程图；
图2示使用本发明的PH-Kp7215-A、PH-Kp5295 4～5个～10kb基因组片段组装结果示例(全清)；其中，A示大肠杆菌体内组装与体外Gibson Assembly辅助相结合；B示大肠杆菌体内组装与体外Gibson Assembly辅助相结合；C示大肠杆菌体内直接组装基因组；
图3示阴性对照示例，宿主菌Kp7215铺板(长满宿主菌)；
图4示对比例：仅体外Gibson Assembly的PH-Kp5295 5个～10kb基因组片段组装结果示例(长满宿主菌)；
图5示噬菌体PH-Kp5295基因组片段组装阴性对照示例，随机选取1个片段(长满宿主菌)；
图6示对比例：体外Gibson Assembly的PH-Kp5295 5个～10kb基因组片段组装结果胶图；图中maker是1kb extent；A-为未发生Gibson Assembly的基因组片段；A1是基因组片段Gibson Assembly 15min结果，gDNA1是gDNA Gibson Assembly 15min结果，即基因组环化，起组装效果的对照作用；依次A1-5，gDNA1-5分别是基因组片段和gDNA Gibson Assembly反应15min,30min,45min,60min,75min；可看到，片段组装的结果和完整基因组的大小存在明显差异，完整基因组在50kb左右，而Gibson组装产物在20-30Kb左右，存在未组装上去的DNA片段；
图7示对比例：体外Gibson Assembly的PH-Kp9495-AB1 10个2kb～7kb基因组片段组装结果胶图；图示同图6，其中使用的marker除1kb extent外前2个分别是1kb DNA Ladder和DL15000DNA marker；基因组片段和gDNA Gibson Assembly反应时间为45min,60min,75min,90min,105min；可看到，片段组装的结果和完整基因组的大小存在明显差异，完整基因组在50kb左右，而Gibson组装产物最大不超过15kb，存在大量未组装上去的DNA片段；
图8示使用本发明的Palz-36个～15kb基因组片段组装结果示例(全清和长有噬菌斑)；其中，A示大肠杆菌体内组装与体外Gibson Assembly辅助相结合；B示大肠杆菌体内组装与体外Gibson Assembly辅助相结合；C示大肠杆菌体内直接组装基因组；
图9示对比例：仅体外Gibson Assembly的Palz-36个～15kb基因组片段组装结果示例(长满宿主菌)；
图10示阴性对照示例，宿主菌Pao1铺板(长满宿主菌)；
图11示Palz-3噬菌体基因组片段组装阴性对照示例，随机选取5个片段大肠杆菌体内组装(长满宿主菌)；
图12示使用该发明的10个2kb～7kb PH-Kp9495-AB1基因组片段组装结果示例(全清和长有噬菌斑)；其中，A示大肠杆菌体内直接组装基因组；B示大肠杆菌体内组装与体外Gibson Assembly辅助相结合；C示大肠杆菌体内组装与体外Gibson Assembly辅助相结合；
图13示对比例：仅体外Gibson Assembly的10个2kb～7kb PH-Kp9495-AB1基因组片段组装结果示例(长满宿主菌)；
图14示噬菌体PH-Kp9495-AB1基因组片段组装阴性对照示例，随机选取6个片段大肠杆菌体内组装(长满宿主菌)；
图15示阴性对照示例，宿主菌Kp9495铺板(长满宿主菌)；
图16示使用本发明psgRNA N20 GFP质粒克隆1kb片段结果示例；其中，A示大肠杆菌体内组装与体外Gibson Assembly辅助相结合；B示大肠杆菌体内直接组装质粒片段；
图17示阴性对照示例，质粒片段和载体分别作为阴性对照；
图18示使用该发明的12个2.7～5kb、13个2.2～5kb、14个2.6～4.7kb、15个2.5～4.5kb PH-Kp9495-AB1基因组片段组装结果示例(全清)；其中，A示大肠杆菌体内组装与体外Gibson Assembly辅助相结合组装12个2.7～5kb PH-Kp9495-AB1基因组片段；B示大肠杆菌体内组装与体外Gibson Assembly辅助相结合组装13个2.2～5kb PH-Kp9495-AB1基因组片段；C示大肠杆菌体内组装与体外Gibson Assembly辅助相结合组装14个2.6～4.7kb PH-Kp9495-AB1基因组片段；D示示大肠杆菌体内组装与体外Gibson Assembly辅助相结合组装15个2.5～4.5kb PH-Kp9495-AB1基因组片段
图19对比例：A、B、C、D依次是仅体外Gibson Assembly的12个2.7～5kb、13个2.2～5kb、14个2.6～4.7kb和15个2.5～4.5kb PH-Kp9495-AB1基因组片段组装结果示例(长满宿主菌)；
图20示阴性对照示例，15个2.5～4.5kb连续完整的PH-Kp9495-AB1基因组片段用不含同源重组系统的stellar/pRARE激活(长满宿主菌)。
具体实施方式
本发明公开了基因组片段的表达元件组合、表达载体、宿主、应用及组装方法，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明采用了以下技术方案：
1、待组装的噬菌体基因组片段制备：先通过设计引物将噬菌体基因组拆分成多个片段，相邻可拼接起来的基因组片段末端存在至少40-100bp的同源区域，若组装的 片段数量较多、较大，可适当增大同源臂长度，同时保证同源区域及其加上下游20bp的区域在50℃的二级结构自由能的绝对值较小(二级结构自由能小，理论上组装效果更好，NUPACK预测)；通过PCR和胶回收制备待组装的噬菌体基因组片段；
2、体外较优Gibson组装反应条件测试：通过设置37℃，42℃，50℃三个不同反应温度及15min、30min、45min、60min、75min 5种不同反应时间(反应时间可根据组装片段长度和数量适当调整)来选择较佳的反应温度和反应时间；
3、体外Gibson Assembly组装：利用上一步骤测试出来的较好的反应温度和反应时间完成Gibson Assembly组装。每一个反应体系里包含所有的基因组片段，每个片段60fmol。Gibson Assembly反应所使用的预混合液是国家基因库合成与编辑平台提供的ER mix。
4、大肠杆菌体内组装及噬菌体激活：体外组装DNA大片段的效率很低，最终反应混合物中可能只有极少量的所有片段组装成完整基因组，大部分还是未组装成功的片段或2-4个拼接起来的中间产物。体内组装DNA大片段的效率是远高于体外组装的，而影响体内组装效率的一个关键因素就是所有片段进入同一个感受态细胞的概率问题。所以为了进一步提高组装效率、提高完整组装的产物量，考虑将体外组装和体内组装联合起来。先体外组装确保较多数量的大片段相互间能拼接起来，再在此基础上进行体内组装时就大大增加了所有片段进入同一个感受态细胞进行体内重组的概率。同时，考虑到组装完整的噬菌体基因组需要经中间宿主大肠杆菌的的激活验证，所以直接在大肠杆菌体内组装成了我们目前的首选目标，因为这样可以直接跳过繁琐的筛选完整基因组的步骤，实现组装和激活的一步完成。
本课题组实验室有含有表达稀有密码子的pRARE质粒的大肠杆菌DH10B，该菌株常用于激活噬菌体基因组，还有含lambda Red同源重组系统的pKD46质粒。Lambda Red同源重组系统具有同源序列短(40-60bp)，重组效率高的特点，由lambda噬菌体exo、bet、gam三个基因组成，分别编码Exo、Beta、Gam三种蛋白质(原理示意图见附图Lambda Red同源重组系统)。Exo蛋白是一种核酸外切酶，是一种环状三聚物分子，中间有一中空的通道，通道的一端可容纳双链DNA分子，另一端只可容纳单链DNA。Exo蛋白可结合在双链DNA的末端，从DNA双链的5’端向3’端降解DNA，使DNA分子形成3’粘性末端；Beta蛋白是一种退火蛋白，在Red同源重组中起着决定性作用，其紧紧结合3’突出端，防止DNA被单链核酸酶降，同时介导互补单链DNA的退火。待组装的噬菌体基因组片段间存在40-100bp的同源区域，在Exo蛋白消化5’端露出3’突出时，相邻基因组片段的同源区域露出的3’突出会在Beta蛋白介导下实现退火，双链DNA退火完成后，Beta蛋白从DNA双链上解离下来。Gam蛋白是16kD的多肽分子，可与RecBCD核酸外切酶结合，抑制其对外源DNA的疆界作用，防止外源DNA进入细胞后被宿主降解。
pRARE和pKD46两质粒可以在大肠杆菌DH10B中共存，将表达同源重组系统的pKD46质粒化转到常用于噬菌体基因组激活的DH10B/pRARE大肠杆菌中，这样就构建了能一步实现组装、激活的中间宿主大肠杆菌。
所以基于以上的实验设计思路，本发明旨在实现通过PCR或从头合成得到的噬菌体基因组片段在大肠杆菌内的直接组装，该目标可由2条技术路线实现：1、大肠杆菌体内直接组装噬菌体基因组；2、大肠杆菌体内组装与体外Gibson Assembly辅助相结合的组装方法，即在完成体外Gibson Assembly组装反应后，将反应完成后得到的混合液直接化转到含lambda Red同源重组系统的pKD46质粒的大肠杆菌DH10B感受态细胞内进行大肠杆菌体内组装，此大肠杆菌中还有表达稀有密码子的pRARE质粒，该质粒有助于噬菌体基因组在中间宿主大肠杆菌中的激活。化转后的感受态细胞在含Ca 2+的LB培养基中孵育6h，孵育结束后取出用5％氯仿涡旋震荡破碎中间宿主大肠杆菌，释放出激活的噬菌体颗粒。
5、组装成功与否的验证：所有组装测试的噬菌体基因组都是之前验证过的能够成功激活得到有活性噬菌体颗粒的。所以如果噬菌体基因组组装完整，其也能被成功激活得到有活性的噬菌体颗粒，具备侵染其本身宿主的能力。将上一步涡旋震荡后的混合液离心后小心吸取上清，与处于生长对数期的宿主菌在37℃共培养4-5h,铺双层平板(下层固体LB平板，上层软琼脂，上下层根据不同噬菌体激活的需要添加Ca 2+或Mg 2+)37℃培养至少6h，若出现预期的噬菌斑或平板出现了全清现象则说明噬菌体基因组组装成功。
本发明以组装肺炎克雷伯菌噬菌体的基因组为例介绍了该方法的实施，还可以应用到其他噬菌体基因组的组装，如铜绿假单胞菌、鲍曼不动杆菌等。
目前尝试了多至10个2kb～7kb噬菌体基因组片段的组装，之后欲实现至少15个～10kb噬菌体基因组片段的组装。
目前尝试了组装宿主为革兰氏阴性菌的噬菌体基因组，对于宿主为革兰氏阳性菌的噬菌体，目前报道的其高效激活中间宿主有细胞壁缺陷型L型细菌如L型李斯特菌。之后的噬菌体基因组组装可以不仅仅只局限于大肠杆菌体内组装，在对该类菌进行改造后如转入同源重组系统，可尝试在该类菌中组装，同样有望于实现改造、组装和激活的一步完成。
本发明的有益效果包括但不限于：
本发明可用于改造、合成人造噬菌体，这些噬菌体可用于进一步改良的噬菌体疗法。
本发明相比现有的噬菌体基因组组装技术实现了组装和激活一步完成的效果。
①相比体外Gibson组装成功率大大提高(尝试过直接用体外Gibson组装基因组，未成功，无论是跑胶结果还是噬菌体基因组激活验证均说明组装失败了)；
②相比体外Golden Gate Assembly组装，该组装技术不依赖于目标载体，可实现噬菌体基因组组装、激活的一步完成，避免了繁琐的筛选步骤，极大减少了实验周期及成本；
③相比于酵母体内组装，该组装技术在基因组片段制备好的情况下，完成组装仅需2天时间，而酵母至少需要1周，且酵母组装后续还需要PCR验证和阳性筛选，以及释放噬菌体后在大肠杆菌内激活；
该方法实验周期短、普适性好、成功率较高。
本发明首次在大肠杆菌体内实现噬菌体基因组大片段直接组装，利用该技术可以一步实现噬菌体基因组的改造、组装、激活，极大缩短实验周期、提高改造人造噬菌体的效率。该发明也适用于其他情景下的大片段(小片段也适用)组装，可用于常规分子克隆实验。
本发明提供的基因组片段的表达元件组合、表达载体、宿主、应用及组装方法中所用原料及试剂均可由市场购得。下面结合实施例，进一步阐述本发明：
下面结合实施例，进一步阐述本发明：
实施例1肺炎克雷伯菌(Klebsiella pneumoniae,Kp)噬菌体PH-Kp9495-AB1、PH-Kp5295、PH-Kp7215-A基因组，4-5个～10kb片段组装。
片段制备所用到的引物：
表1
Figure PCTCN2022082446-appb-000002
Figure PCTCN2022082446-appb-000003
PCR所用反应混合液：
Figure PCTCN2022082446-appb-000004
Hot Start High-Fidelity 2X Master Mix。
表2.PH-Kp7215-A4个片段：
Figure PCTCN2022082446-appb-000005
表3.PH-Kp9495-AB1 5个片段：
Figure PCTCN2022082446-appb-000006
表4.PH-Kp5295 5个片段：
Figure PCTCN2022082446-appb-000007
Gibson Assembly预混合液：国家基因库合成与编辑平台生产的ER mix
体外Gibson Assembly反应条件：反应温度42℃，反应时间75min
体外Gibson组装反应体系：每个基因组片段60fmol+ddH2O补齐5μL
(片段总体积和水的总体积5μL)+15μL ER mix
大肠杆菌菌株：课题组保存的菌株DH10B/pRARE；DH10B/pRARE/pKD46
每个噬菌体的组装实验组包括三种:
1.仅体外Gibson Assembly组装，再将反应混合液化转到用于激活的大肠杆菌DH10B/pRARE菌株中，即测试体外Gibson Assembly组装效果，作为对比例说明；
2.先体外Gibson Assembly组装，再将反应混合液化转到含有同源重组系统的大肠杆菌DH10B/pRARE/pKD46菌株中，即测试本发明中大肠杆菌体内组装和体外Gibson Assembly组装辅助相结合的组装效果；
3.不进行体外Gibson Assembly组装，直接将所有片段化转到含有Lambda Red同源重组系统的大肠杆菌DH10B/pRARE/pKD46菌株中，即测试本发明中大肠杆菌体内直接组装噬菌体基因组片段的组装效果；
其中3种不同实验组所用到的片段量均为60fmol/片段。
每个实验组设置3个平行实验。
对照设置：
1、阳性对照：提取的噬菌体基因组DNA，用中间宿主DH10B/pRARE和中间宿主DH10B/pRARE/pKD46激活，理论上阳性对照结果中应出现噬菌斑或全清的结果。
2、阴性对照：
a.随机选取的基因组片段(例如PH-Kp5295第1个片段；PH-Kp9495-AB1第4个片段)；
b.噬菌体的宿主菌Kp9495、Kp7215、Kp5295；
c.感受态DH10B/pRARE/pKD46,和实验组同样实验操作，但不转化片段或基因组，排除添加氯仿破碎细胞后吸取到氯仿对实验结果的干扰。
理论上阴性对照结果中应长满噬菌体的天然宿主菌。
实验操作：
1、DH10B/pRARE/pKD46、DH10B/pRARE化转感受态细胞制备：
A.划线分离得到单克隆菌落：甘油中取保存菌，用“三线法”划线。其中DH10B/pRARE/pKD46菌株需30℃下培养，使用带有氨苄和氯霉素双抗生素的固体LB平板，培养2天左右可长出合适大小的单克隆菌落；DH10B/pRARE菌株37℃下培养，使用带有氯霉素抗生素的固体LB平板，培养1天左右可长出合适大小的单克隆菌落。
B.过夜菌制备：分别挑取上述得到的单克隆菌落至液体LB培养基中，其中DH10B/pRARE/pKD46菌株使用的LB液体培养基中需加入终浓度10mM阿拉伯糖以诱导Lambda Red同源重组系统的表达，此外仍需含有氨苄和氯霉素2种抗生素，30℃培养；DH10B/pRARE菌株使用的LB液体培养基中需含有氯霉素抗生素,37℃培养。
C.对数菌制备：根据实验所需的100X感受态细胞量计算所需制备的对数菌体积。DH10B/pRARE菌株37℃下培养至OD600在0.5-0.55；DH10B/pRARE/pKD46菌株30℃下培养至OD600在0.5-0.55；两种菌使用的液体LB培养基同上一步。
D.感受态制备：“氯化钙法”制备以上两种不同的大肠杆菌感受态，2种感受态细胞均为200μL 100X。
2、大肠杆菌DH10B/pRARE/pKD46、DH10B/pRARE转化：
实验组1:60fmol/片段,15μL ER Mix,42℃反应90min。将反应后的混合液化转到大肠杆菌DH10B/pRARE感受态细胞中，即混合液加入感受态细胞，轻轻混匀后冰浴30min,42℃准确热激90s,再冰浴3min,加1ml含有5mM Ca 2+的LB培养基，37℃,220rpm培养。
实验组2:60fmol/片段,15μL ER Mix,42℃反应90min。将反应后的混合液化转到大肠杆菌DH10B/pRARE/pKD46感受态细胞中，即混合液加入感受态细胞，轻轻混匀后冰浴30min,42℃准确热激90s,再冰浴3min,加1ml含有5mM Ca 2+的LB培养基，37℃,220rpm培养。
实验组3:将60fmol/片段直接转到大肠杆菌DH10B/pRARE/pKD46感受态细胞中，即片段混合液加入感受态细胞，轻轻混匀后冰浴30min,42℃准确热激90s,再冰浴3min,加1ml含有5mM Ca 2+的LB培养基，37℃,220rpm摇床培养。
阴性对照1：随机选取一个基因组片段，取60fmol直接转到大肠杆菌DH10B/pRARE/pKD46感受态细胞中，即片段混合液加入感受态细胞，轻轻混匀后冰浴30min,42℃热准确激90s,再冰浴3min,加1ml含有5mM Ca 2+的LB培养基，37℃,220rpm摇床培养。
阴性对照2:随机选取一管DH10B/pRARE/pKD46感受态不转化任何片段或基因组，其他操作与实验组保持一致。即冰浴30min,42℃热准确激90s,再冰浴3min,加1ml含有5mM Ca 2+的LB培养基，37℃,220rpm摇床培养
阳性对照：提取的噬菌体基因组DNA，取60fmol直接转到大肠杆菌DH10B/pRARE/pKD46和DH10B/pRARE感受态细胞中，即基因组DNA加入感受态细胞，轻轻混匀后冰浴30min,42℃热准确激90s,再冰浴3min,加1ml含有5mM Ca 2+的LB培养基，37℃,220rpm摇床培养。
3、基因组的一步组装和激活：分别在培养6h,8h,和过夜这三个时间点取样，每个时间点约取350μL，其中取完8h的样品后，将摇床温度调至22℃。向样品中加入终浓度5％的氯仿，涡旋震荡至少40s，14000rpm离心5min。取离心后的上清，用较小量程的移液器少量多次吸取上清。约可以取到200μL的上清，200μL上清和300μL的对数期相应噬菌体的天然宿主菌混匀后在37℃孵育4-5h。之后，铺双层琼脂平板，即将完成孵育后的混合液加入3ml含5mM Mg 2+的软琼脂，充分涡旋混匀后铺到含5mM Mg 2+的固体LB平板上，待凝固后放至37℃培养至少4-6h即可观察组装结果。若观察到全清或噬菌斑的结果即可证明组装成功；若肉眼观察到平板上长满了噬菌体的天然宿主菌，即无噬菌斑的出现，说明组装失败。为了更好体现平板出斑或全清的情况，同时用300μL对数期的天然宿主铺双层琼脂平板，作为1种阴性对照。
组装结果：
对比例：Gibson Assembly组装结果胶图(附结果图4)片段组装的结果和完整基因组的大小存在明显差异，完整基因组在50kb左右，而Gibson Assembly组装产物在20-30Kb左右，存在未组装上去的DNA片段，胶图结果显示组装失败。
PH-Kp5295、PH-Kp7215-A对比例中体外Gibson Assembly组装结果和本发明中大肠杆菌体内直接组装基因组和大肠杆菌体内组装与体外Gibson Assembly辅助相结合的组装方法差异不大，3次平行实验中均出现全清或噬菌斑的结果。但PH-Kp5295对比例中体外Gibson Assembly组装方法，3次平行实验均失败了，而本发明中大肠杆菌体内直接组装基因组和大肠杆菌体内组装与体外Gibson Assembly辅助相结合的组装方法中，3个平行实验组中1个实验组出现了全清的平板，这也说明了本发明较常规的体外Gibson Assembly组装在成功率(使用本发明3个噬菌体基因组全部成功组装，而体外Gibson Assembly成功组装2个，失败1个)上表现更优异。阴性对照均长满了宿主菌，阳性对照也是全清平板。
表5
Figure PCTCN2022082446-appb-000008
Figure PCTCN2022082446-appb-000009
实施例2铜绿假单胞菌噬菌体Palz-3基因组，6个～15kb片段组装。
片段制备所用到的引物：
表6
Figure PCTCN2022082446-appb-000010
PCR所用反应混合液：
Figure PCTCN2022082446-appb-000011
Hot Start High-Fidelity 2X Master Mix
Palz-3 6个片段：
表7
Figure PCTCN2022082446-appb-000012
Gibson Assembly预混合液：国家基因库合成与编辑平台生产的ER mix。
体外Gibson Assembly反应条件：反应温度42℃，反应时间75min
体外Gibson组装反应体系：每个基因组片段60fmol+ddH2O补齐5μL
(片段总体积和水的总体积5μL)+15μL ER mix
大肠杆菌菌株：课题组保存的菌株DH10B/pRARE；DH10B/pRARE/pKD46
每个噬菌体的组装实验组包括三种:
1.仅体外Gibson Assembly组装，再将反应混合液化转到用于激活的大肠杆菌DH10B/pRARE菌株中，即测试体外Gibson Assembly组装效果，作为对比例说明；
2.先体外Gibson Assembly组装，再将反应混合液化转到含有同源重组系统的大肠杆菌DH10B/pRARE/pKD46菌株中，即测试本发明中大肠杆菌体内组装和体外Gibson Assembly组装辅助相结合的组装效果；
3.不进行体外Gibson Assembly组装，直接将所有片段化转到含有Lambda Red同源重组系统的大肠杆菌DH10B/pRARE/pKD46菌株中，即测试本发明中大肠杆菌体内直接组装噬菌体基因组片段的组装效果；
其中3种不同实验组所用到的片段量均为60fmol/片段。
每个实验组设置3个平行实验。
对照设置：
1、阳性对照：提取的噬菌体基因组DNA，用中间宿主DH10B/pRARE和中间宿主DH10B/pRARE/pKD46激活，理论上阳性对照结果中应出现噬菌斑或全清的结果。
2、阴性对照：
a.随机选取的5个噬菌体基因组片段；
b.噬菌体的宿主菌Pao1；
c.感受态DH10B/pRARE/pKD46,和实验组同样实验操作，但不转化片段或基因组，排除添加氯仿破碎细胞后吸取到的氯仿对实验结果的干扰。
理论上阴性对照结果中应长满噬菌体的天然宿主菌。
实验操作：
1、DH10B/pRARE/pKD46、DH10B/pRARE化转感受态细胞制备：
A.划线分离得到单克隆菌落：甘油中取保存菌，用“三线法”划线。其中DH10B/pRARE/pKD46菌株需30℃下培养，使用带有氨苄和氯霉素双抗生素的固体LB平板，约2天左右可长出合适大小的单克隆菌落；DH10B/pRARE菌株37℃下培养，使用带有氯霉素抗生素的固体LB平板，约1天左右可长出合适大小的单克隆菌落。
B.过夜菌制备：分别挑取上述得到的单克隆菌落至液体LB培养基中，其中DH10B/pRARE/pKD46菌株使用的LB液体培养基中需加入终浓度10mM阿拉伯糖以诱导Lambda Red同源重组系统的表达，此外仍需含有氨苄和氯霉素2种抗生素，30℃培养过夜；DH10B/pRARE菌株使用的LB液体培养基中需含有氯霉素抗生素,37℃培养过夜。
C.对数菌制备：根据实验所需的100X感受态细胞量计算所需制备的对数菌体积。DH10B/pRARE菌株37℃下培养至OD600在0.5-0.55；DH10B/pRARE/pKD46菌株30℃下培养至OD600在0.5-0.55；两种菌使用的液体LB培养基同上一步。
D.感受态制备：“氯化钙法”制备以上两种不同的大肠杆菌感受态，2种感受态细胞均为200μL 100X。
2、大肠杆菌DH10B/pRARE、DH10B/pRARE/pKD46转化：
实验组1:60fmol/片段,15μL ER Mix,42℃反应90min。将反应后的混合液化转到大肠杆菌DH10B/pRARE感受态细胞中，即混合液加入感受态细胞，轻轻混匀后冰浴30min,42℃准确热激90s,再冰浴3min,加1ml含有5mM Ca2+的LB培养基，37℃,220rpm培养。
实验组2:60fmol/片段,15μL ER Mix,42℃反应90min。将反应后的混合液化转到大肠杆菌DH10B/pRARE/pKD46感受态细胞中，即混合液加入感受态细胞，轻轻混匀后冰浴30min,42℃准确热激90s,再冰浴3min,加1ml含有5mM Ca2+的LB培养基，37℃,220rpm培养。
实验组3:将60fmol/片段直接转到大肠杆菌DH10B/pRARE/pKD46感受态细胞中，即片段混合液加入感受态细胞，轻轻混匀后冰浴30min,42℃准确热激90s,再冰浴3min,加1ml含有5mM Ca2+的LB培养基，37℃,220rpm摇床培养。
阴性对照1：随机选取一个基因组片段，取60fmol直接转到大肠杆菌DH10B/pRARE/pKD46感受态细胞中，即片段混合液加入感受态细胞，轻轻混匀后冰浴30min,42℃热准确激90s,再冰浴3min,加1ml含有5mM Ca2+的LB培养基，37℃,220rpm摇床培养。
阴性对照2:随机选取一管DH10B/pRARE/pKD46感受态不转化任何片段或基因组，其他操作与实验组保持一致。即冰浴30min,42℃热准确激90s,再冰浴3min,加1ml含有5mM Ca 2+的LB培养基，37℃,220rpm摇床培养。
阳性对照：提取的噬菌体基因组DNA，取60fmol直接转到大肠杆菌DH10B/pRARE/pKD46和DH10B/pRARE感受态细胞中，即基因组DNA加入感受态细胞，轻轻混匀后冰浴30min,42℃热准确激90s,再冰浴3min,加1ml含有5mM Ca2+ 的LB培养基，37℃,220rpm摇床培养
3、基因组的一步组装和激活：分别在培养6h,8h这2个时间点取样，每个时间点约取350μL，其中取完8h的样品后，将摇床温度调至22℃。向样品中加入终浓度5％的氯仿，涡旋震荡至少40s，14000rpm离心5min。取离心后的上清，用较小量程的移液器少量多次吸取上清。约可以取到200μL的上清，200μL上清和300μL的对数期相应噬菌体的天然宿主菌混匀后在37℃孵育4-5h。之后，铺双层琼脂平板，即将完成孵育后的混合液加入3ml含5mM Mg2+的软琼脂，充分涡旋混匀后铺到含5mM Mg2+的固体LB平板上，待凝固后放至37℃培养至少4-6h即可观察组装结果。若观察到全清或噬菌斑的结果即可证明组装成功；若肉眼观察到平板上长满了噬菌体的天然宿主菌，即无噬菌斑的出现，说明组装失败。为了更好体现平板出斑或全清的情况，同时用300μL对数期的天然宿主铺双层琼脂平板，作为1种阴性对照。
组装结果：
本发明中大肠杆菌体内直接组装基因组和大肠杆菌体内组装与体外Gibson Assembly辅助相结合的组装方法在3次平行实验结果中均得到了噬菌斑或全清的平板。相比较之下，纯体外Gibson Assembly组装3次平行实验中出现了未成功的情况，说明本发明的结果稳定性上优于传统的Gibson Assembly组装，可较稳定的实现多至6个15kb噬菌体基因组大片段的成功组装。
表8
Figure PCTCN2022082446-appb-000013
Figure PCTCN2022082446-appb-000014
实施例3肺炎克雷伯菌(Klebsiella pneumoniae,Kp)噬菌体PH-Kp9495-AB1基因组，6、8、10个片段的组装。
片段制备所用到的引物：
表9
Figure PCTCN2022082446-appb-000015
Figure PCTCN2022082446-appb-000016
PCR所用反应混合液：NEB
Figure PCTCN2022082446-appb-000017
Start High-Fidelity 2X Master Mix PH-Kp9495-AB1 6个片段：
表10
Figure PCTCN2022082446-appb-000018
PH-Kp9495-AB1 8个片段：
表11
Figure PCTCN2022082446-appb-000019
PH-Kp9495-AB1 10个片段：
表12
Figure PCTCN2022082446-appb-000020
Figure PCTCN2022082446-appb-000021
Gibson Assembly预混合液：国家基因库合成与编辑平台生产的ER mix
体外Gibson Assembly反应条件：反应温度42℃，反应时间105min
体外Gibson组装反应体系：每个基因组片段60fmol+ddH2O补齐5μL
(片段总体积和水的总体积5μL)+15μL ER mix
大肠杆菌菌株：课题组保存的菌株DH10B/pRARE；DH10B/pRARE/pKD46
每个噬菌体的组装实验组包括三种:
1.仅体外Gibson Assembly组装，再将反应混合液化转到用于激活的大肠杆菌DH10B/pRARE菌株中，即测试体外Gibson Assembly组装效果，作为对比例说明；
2.先体外Gibson Assembly组装，再将反应混合液化转到含有同源重组系统的大肠杆菌DH10B/pRARE/pKD46菌株中，即测试本发明中大肠杆菌体内组装和体外Gibson Assembly组装辅助相结合的组装效果；
3.不进行体外Gibson Assembly组装，直接将所有片段化转到含有Lambda Red同源重组系统的大肠杆菌DH10B/pRARE/pKD46菌株中，即测试本发明中大肠杆菌体内直接组装噬菌体基因组片段的组装效果；
其中3种不同实验组所用到的片段量均为60fmol/片段。
每个实验组设置3个平行实验。
对照设置：
1、阳性对照：提取的噬菌体基因组DNA，用中间宿主DH10B/pRARE和中间宿主DH10B/pRARE/pKD46激活，理论上阳性对照结果中应出现噬菌斑或全清的结果。
2、阴性对照：
a.随机选取的5个基因组片段直接转化到中间宿主DH10B/pRARE/pKD46中；
b.噬菌体的宿主菌Kp9495；
c.感受态DH10B/pRARE/pKD46,和实验组同样实验操作，但不转化片段或基因组，排除添加氯仿破碎细胞后吸取到氯仿对实验结果的干扰。
理论上阴性对照结果中应长满噬菌体的天然宿主菌。
实验操作：
1、DH10B/pRARE/pKD46、DH10B/pRARE化转感受态细胞制备：
A.划线分离得到单克隆菌落：甘油中取保存菌，用“三线法”划线。其中DH10B/pRARE/pKD46菌株需30℃下培养，使用带有氨苄和氯霉素双抗生素的固体LB平板，约2天左右可长出合适大小的单克隆菌落；DH10B/pRARE菌株37℃下培养，使用带有氯霉素抗生素的固体LB平板，约1天左右可长出合适大小的单克隆菌落。
B.过夜菌制备：分别挑取上述得到的单克隆菌落至液体LB培养基中，其中DH10B/pRARE/pKD46菌株使用的LB液体培养基中需加入终浓度10mM阿拉伯糖以诱导Lambda Red同源重组系统的表达，此外仍需含有氨苄和氯霉素2种抗生素，30℃培养过夜；DH10B/pRARE菌株使用的LB液体培养基中需含有氯霉素抗生素,37℃培养过夜。
C.对数菌制备：根据实验所需的100X感受态细胞量计算所需制备的对数菌体积。DH10B/pRARE菌株37℃下培养至OD600在0.5-0.55；DH10B/pRARE/pKD46菌株30℃下培养至OD600在0.5-0.55；两种菌使用的液体LB培养基同上一步。
D.感受态制备：“氯化钙法”制备以上两种不同的大肠杆菌感受态，2种感受态细胞均为200μL 100X。
2、大肠杆菌DH10B/pRARE、DH10B/pRARE/pKD46转化：
实验组1:60fmol/片段,15μL ER Mix,42℃反应105min。将反应后的混合液化转到大肠杆菌DH10B/pRARE感受态细胞中，即混合液加入感受态细胞，轻轻混匀后冰浴30min,42℃准确热激90s,再冰浴3min,加1ml含有5mM Ca 2+的LB培养基，37℃,220rpm培养。
实验组2:60fmol/片段,15μL ER Mix,42℃反应105min。将反应后的混合液化转到大肠杆菌DH10B/pRARE/pKD46感受态细胞中，即混合液加入感受态细胞，轻轻混匀后冰浴30min,42℃准确热激90s,再冰浴3min,加1ml含有5mM Ca 2+的LB培养基，37℃,220rpm培养。
实验组3:将60fmol/片段直接转到大肠杆菌DH10B/pRARE/pKD46感受态细胞中，即片段混合液加入感受态细胞，轻轻混匀后冰浴30min,42℃准确热激90s,再冰浴3min,加1ml含有5mM Ca 2+的LB培养基，37℃,220rpm摇床培养。
阴性对照1：随机选取一个基因组片段，取60fmol直接转到大肠杆菌DH10B/pRARE/pKD46感受态细胞中，即片段混合液加入感受态细胞，轻轻混匀后冰浴30min,42℃热准确激90s,再冰浴3min,加1ml含有5mM Ca 2+的LB培养基，37℃,220rpm摇床培养。
阴性对照2:随机选取一管DH10B/pRARE/pKD46感受态不转化任何片段或基因组，其他操作与实验组保持一致。即冰浴30min,42℃热准确激90s,再冰浴3min,加1ml含有5mM Ca 2+的LB培养基，37℃,220rpm摇床培养。
阳性对照：提取的噬菌体基因组DNA，取60fmol直接转到大肠杆菌DH10B/pRARE/pKD46和DH10B/pRARE感受态细胞中，即基因组DNA加入感受态 细胞，轻轻混匀后冰浴30min,42℃热准确激90s,再冰浴3min,加1ml含有5mM Ca 2+的LB培养基，37℃,220rpm摇床培养。
3、基因组的一步组装和激活：分别在培养6h,8h,和过夜这三个时间点取样，每个时间点约取350μL，其中取完8h的样品后，将摇床温度调至22℃。向样品中加入终浓度5％的氯仿，涡旋震荡至少40s，14000rpm离心5min。取离心后的上清，用较小量程的移液器少量多次吸取上清。约可以取到200μL的上清，200μL上清和300μL的对数期相应噬菌体的天然宿主菌混匀后在37℃孵育4-5h。之后，铺双层琼脂平板，即将完成孵育后的混合液加入3ml含5mM Mg 2+的软琼脂，充分涡旋混匀后铺到含5mM Mg 2+的固体LB平板上，待凝固后放至37℃培养至少4-6h即可观察组装结果。若观察到全清或噬菌斑的结果即可证明组装成功；若肉眼观察到平板上长满了噬菌体的天然宿主菌，即无噬菌斑的出现，说明组装失败。为了更好体现平板出斑或全清的情况，同时用300μL对数期的天然宿主铺双层琼脂平板，作为1种阴性对照。
组装结果：
体外Gibson Assembly组装6个6kb～9kb大小的片段成功率较高(3次平行实验全部全清)，但当片段数量增加到8个3kb～7kb、甚至10个2kb～7kb大小的片段组装效果大大降低(3次平行实验中只有1个出现噬菌斑，甚至3次平行实验均失败的结果)。相比较之下，本发明中大肠杆菌体内直接组装基因组和大肠杆菌体内组装与体外Gibson Assembly辅助相结合的组装方法成功率较高，6、8、10个片段组装均成功组装且3次平行实验均出现了全清或噬菌斑的结果。进一步说明了该发明可实现多至10个噬菌体基因组片段的成功组装，且在成功率和结果稳定性上优于对比例中常规使用的体外Gibson Assembly组装。
表13
Figure PCTCN2022082446-appb-000022
Figure PCTCN2022082446-appb-000023
实施例4大肠杆菌体内psgRNA(N20_GFP_CmR)质粒1个～1kb片段的克隆
片段制备所用到的引物：
表14
Figure PCTCN2022082446-appb-000024
PCR所用反应混合液：NEB
Figure PCTCN2022082446-appb-000025
Hot Start High-Fidelity 2X Master Mix
片段与载体：
表15
Figure PCTCN2022082446-appb-000026
Gibson Assembly预混合液：国家基因库合成与编辑平台生产的ER mix
体外Gibson Assembly反应条件：反应温度50℃，反应时间15min
体外Gibson组装反应体系：每个片段30fmol+ddH2O补齐5μL
(片段总体积和水的总体积5μL)+15μL ER mix
大肠杆菌菌株：课题组保存的菌株DH10B/pRARE/pKD46
质粒克隆实验组包括2种:
1.先体外Gibson Assembly反应，再将反应混合液化转到DH10B/pRARE/pKD46，即测试本发明中大肠杆菌体内组装与体外Gibson Assembly辅助相结合的组装方法
2.不进行体外Gibson Assembly反应，直接将所有片段化转到含有Lambda Red同源重组系统的大肠杆菌DH10B/pRARE/pKD46菌株中，即测试本发明中大肠杆菌体内直接克隆质粒片段的效果；
其中2种不同实验组所用到的片段量均为30fmol/片段。
每个实验组设置3个平行实验。
对照设置：
1、阳性对照：提取的psgRNA N20 GFP质粒，理论上阳性对照结果中应出现密集的大肠杆菌单克隆菌落。
2、阴性对照：质粒载体和片段分别作为阴性对照；理论上阴性对照结果中应无或较少大肠杆菌单克隆菌落。
实验操作：
1、DH10B/pRARE/pKD46化转感受态细胞制备：
A.划线分离得到单克隆菌落：甘油中取保存菌，用“三线法”划线。其中DH10B/pRARE/pKD46菌株需30℃下培养，使用带有氨苄和氯霉素双抗生素的固体LB平板，约2天左右可长出合适大小的单克隆菌落。
B.过夜菌制备：分别挑取上述得到的单克隆菌落至液体LB培养基中，其中DH10B/pRARE/pKD46菌株使用的LB液体培养基中需加入10mMol/L阿拉伯糖以诱导Lambda Red同源重组系统的表达，此外仍需含有氨苄和氯霉素2种抗生素，30℃培养过夜；
C.对数菌制备：根据实验所需的100X感受态细胞量计算所需制备的对数菌体积。30℃下培养至OD600在0.5-0.55，使用的液体LB培养基同上一步。
D.感受态制备：“氯化钙法”制备以上大肠杆菌感受态，感受态为200μL 100X。
2、大肠杆菌DH10B/PRARE/PKD46转化：
实验组1:30fmol/片段,15μL ER Mix,50℃反应15min。将反应后的混合液化转到大肠杆菌DH10B/pRARE/pKD46感受态细胞中，即混合液加入感受态细胞，轻轻混匀后冰浴30min,42℃准确热激90s,再冰浴3min,加1ml LB培养基，37℃,220rpm培养1h。之后将培养液14000rpm离心3min，吸取并弃掉上清～800ml培养基，用剩下的培养基将菌体沉淀混匀后涂布固体LB平板。
实验组2:30fmol/片段直接转到大肠杆菌DH10B/pRARE/pKD46感受态细胞中，即片段混合液加入感受态细胞，轻轻混匀后冰浴30min,42℃准确热激90s,再冰浴3min, 加1ml LB培养基，37℃,220rpm摇床培养1h。之后将培养液14000rpm离心3min，吸取并弃掉上清～800ml培养基，用剩下的培养基将菌体沉淀混匀后涂布固体LB平板。
阴性对照：质粒片段或载体，取30fmol直接转到大肠杆菌DH10B/pRARE/pKD46感受态细胞中，即片段混合液加入感受态细胞，轻轻混匀后冰浴30min,42℃准确热激90s,再冰浴3min,加1ml LB培养基，37℃,220rpm摇床培养1h之后将培养液14000rpm离心3min，吸取并弃掉上清～800μL培养基，用剩下的培养基将菌体沉淀混匀后涂布固体LB平板。
阳性对照：提取的psgRNA N20 GFP质粒，取30fmol直接转到大肠杆菌DH10B/pRARE/pKD46和DH10B/pRARE感受态细胞中，即psgRNA N20 GFP质粒加入感受态细胞，轻轻混匀后冰浴30min,42℃热准确激90s,再冰浴3min,加1ml LB培养基，37℃,220rpm摇床培养1h之后将培养液14000rpm离心3min，吸取并弃掉上清～800μL培养基，用剩下的培养基将菌体沉淀混匀后涂布固体LB平板。
结果：
本发明中大肠杆菌体内直接组装基因组和大肠杆菌体内组装与体外Gibson Assembly辅助相结合的组装方法均能实现载体和片段的连接以构建完整质粒，说明本发明同样适用于常规分子克隆实验。
表16
Figure PCTCN2022082446-appb-000027
实施例5肺炎克雷伯菌(Klebsiella pneumoniae,Kp)噬菌体PH-Kp9495-AB1基因组，12、13、14、15个片段的组装。
片段制备所用到的引物：
表17
Figure PCTCN2022082446-appb-000028
Figure PCTCN2022082446-appb-000029
Figure PCTCN2022082446-appb-000030
Figure PCTCN2022082446-appb-000031
PCR所用反应混合液：NEB
Figure PCTCN2022082446-appb-000032
Start High-Fidelity 2X Master Mix PH-Kp9495-AB1 12个片段：
表18
Figure PCTCN2022082446-appb-000033
Figure PCTCN2022082446-appb-000034
PH-Kp9495-AB1 13个片段：
表19
Figure PCTCN2022082446-appb-000035
PH-Kp9495-AB1 14个片段：
表20
Figure PCTCN2022082446-appb-000036
Figure PCTCN2022082446-appb-000037
PH-Kp9495-AB1 15个片段：
表21
Figure PCTCN2022082446-appb-000038
Gibson Assembly预混合液：国家基因库合成与编辑平台生产的ER mix
体外Gibson Assembly反应条件：反应温度42℃，反应时间105min
体外Gibson组装反应体系：每个基因组片段30fmol+ddH2O补齐5μL
(片段总体积和水的总体积5μL)+15μL ER mix
大肠杆菌菌株：课题组保存的菌株stellar/pRARE；stellar/pRARE/pKD46
每个噬菌体的组装实验组包括三种:
1.仅体外Gibson Assembly组装，再将反应混合液化转到用于激活的大肠杆菌stellar/pRARE菌株中，即测试体外Gibson Assembly组装效果，作为对比例说明；
2.先体外Gibson Assembly组装，再将反应混合液化转到含有同源重组系统的大肠杆菌stellar/pRARE/pKD46菌株中，即测试本发明中大肠杆菌体内组装和体外Gibson Assembly组装辅助相结合的组装效果；
3.不进行体外Gibson Assembly组装，直接将所有片段化转到含有Lambda Red同源重组系统的大肠杆菌stellar/pRARE/pKD46菌株中，即测试本发明中大肠杆菌体内直接组装噬菌体基因组片段的组装效果；
其中3种不同实验组所用到的片段量均为30fmol/片段。
每个实验组设置3个平行实验。
对照设置：
1、阳性对照：提取的噬菌体基因组DNA，用中间宿主stellar/pRARE和中间宿主stellar/pRARE/pKD46激活，理论上阳性对照结果中应出现噬菌斑或全清的结果。
2、阴性对照：
a.随机选取的n-1(n为组装片段个数)个基因组片段，每个片段30fmol,直接转化到大肠杆菌stellar/pRARE/pKD46中；
b.噬菌体的宿主菌Kp9495；
c.所有的基因组片段，每个片段30fmol,直接转化到没有同源重组系统的大肠杆菌stellar/pRARE中。
d.感受态stellar/pRARE/pKD46,和实验组同样实验操作，但不转化片段或基因组，排除添加氯仿破碎细胞后吸取到氯仿对实验结果的干扰。
理论上阴性对照结果中应长满噬菌体的天然宿主菌。
实验操作：
1、stellar/pRARE/pKD46、stellar/pRARE化转感受态细胞制备：
A.划线分离得到单克隆菌落：甘油中取保存菌，用“三线法”划线。其中stellar/pRARE/pKD46菌株需30℃下培养，使用带有氨苄和氯霉素双抗生素的固体LB平板(使用固体SOB平板更佳)，约2天左右可长出合适大小的单克隆菌落；stellar/pRARE菌株37℃下培养，使用带有氯霉素抗生素的固体LB平板(使用固体SOB平板更佳)，约1天左右可长出合适大小的单克隆菌落
B.过夜菌制备：分别挑取上述得到的新鲜的(未放置冰箱保存)单克隆菌落至 液体SOB培养基中，其中stellar/pRARE/pKD46菌株使用的SOB液体培养基中需加入终浓度10mM阿拉伯糖以诱导Lambda Red同源重组系统的表达，此外仍需含有氨苄和氯霉素2种抗生素，30℃培养过夜；stellar/pRARE菌株使用的SOB液体培养基中需含有氯霉素抗生素,37℃培养过夜。
C.对数菌制备：根据实验所需的50X感受态细胞量计算所需制备的对数菌体积。stellar/pRARE菌株37℃下培养至OD600在0.5-0.55；stellar/pRARE/pKD46菌株30℃下培养至OD600在0.5-0.55；两种菌使用的液体SOB培养基同上一步。
D.Inoue法制备stellar超级感受态:
I.将上述得到的OD600在0.5-0.55间的对数菌分装到50ml预冷无菌的聚丙烯离心管中，置冰上冷却10min，至培养物冷却至0℃；
II.4℃，2500g离心10min，收获细菌；
III.倒掉培养基，使用200μL移液枪移除残留在离心管壁上的液滴；
IV.每管加入冰冷的10ml Inoue转化缓冲液在冰上轻轻重悬细菌；
V.4℃，2500g离心10min，收获细菌；
VI.倒掉转化液，使用200μL移液枪移除残留在离心管壁上的液滴；
VII.每管用1ml冰冷的Inoue转化缓冲液温和悬浮沉淀，然后合并至其中的一个50ml管中；
VIII.缓慢加入终浓度7％的DMSO，同时轻轻旋转混匀，之后冰上存放10min；
IX.200μL每管分装待用(新鲜制备而非冷冻保存的感受态更利于组装)。
2、大肠杆菌stellar/pRARE、stellar/pRARE/pKD46转化：
实验组1:30fmol/片段,15μL ER Mix,42℃反应105min。将反应后的混合液化转到大肠杆菌stellar/pRARE感受态细胞中，即混合液加入感受态细胞，轻轻混匀后冰浴30min,42℃准确热激90s,再冰浴3min,加1ml含有5mM Ca 2+的SOB液体培养基，37℃,220rpm培养。
实验组2:30fmol/片段,15μL ER Mix,42℃反应105min。将反应后的混合液化转到大肠杆菌stellar/pRARE/pKD46感受态细胞中，即混合液加入感受态细胞，轻轻混匀后冰浴30min,42℃准确热激90s,再冰浴3min,加1ml含有5mM Ca 2+的SOB液体培养基，37℃,220rpm培养。
实验组3:将30fmol/片段直接转到大肠杆菌stellar/pRARE/pKD46感受态细胞中，即片段混合液加入感受态细胞，轻轻混匀后冰浴30min,42℃准确热激90s,再冰浴3min,加1ml含有5mM Ca 2+的SOB液体培养基，37℃,220rpm摇床培养。
阴性对照1：随机选取的n-1(n为组装片段个数)个基因组片段，每个片段30fmol,直接转化到大肠杆菌stellar/pRARE/pKD46中，即片段混合液加入感受态细胞，轻轻混匀后冰浴30min,42℃热准确激90s,再冰浴3min,加1ml含有5mM Ca 2+的SOB液体培养基，37℃,220rpm摇床培养。
阴性对照2:所有的基因组片段，每个片段30fmol,直接转化到没有同源重组系统的大肠杆菌stellar/pRARE中,即片段混合液加入感受态细胞，轻轻混匀后冰浴 30min,42℃热准确激90s,再冰浴3min,加1ml含有5mM Ca 2+的SOB液体培养基，37℃,220rpm摇床培养。
阴性对照3:随机选取一管stellar/pRARE/pKD46感受态不转化任何片段或基因组，其他操作与实验组保持一致。即冰浴30min,42℃热准确激90s,再冰浴3min,加1ml含有5mM Ca 2+的SOB培养基，37℃,220rpm摇床培养
阳性对照：提取的噬菌体基因组DNA，取30fmol直接转到大肠杆菌stellar/pRARE/pKD46和stellar/pRARE感受态细胞中，即基因组DNA加入感受态细胞，轻轻混匀后冰浴30min,42℃热准确激90s,再冰浴3min,加1ml含有5mM Ca 2+的SOB液体培养基，37℃,220rpm摇床培养。
3、基因组的一步组装和激活：分别在培养6h,8h,和过夜这三个时间点取样，每个时间点约取350μL，其中取完8h的样品后，将摇床温度调至22℃。向样品中加入终浓度5％的氯仿，涡旋震荡至少40s，14000rpm离心5min。取离心后的上清，用较小量程的移液器少量多次吸取上清。约可以取到200μL的上清，200μL上清和300μL的对数期相应噬菌体的天然宿主菌混匀后在37℃孵育4-5h。之后，铺双层琼脂平板，即将完成孵育后的混合液加入3ml软琼脂，充分涡旋混匀后铺到含固体SOB平板上，待凝固后放至37℃培养至少4-6h即可观察组装结果。若观察到全清或噬菌斑的结果即可证明组装成功；若肉眼观察到平板上长满了噬菌体的天然宿主菌，即无噬菌斑的出现，说明组装失败。为了更好体现平板出斑或全清的情况，同时用300μL对数期的天然宿主铺双层琼脂平板，作为1种阴性对照。
组装结果：
体外Gibson Assembly组装12个2.7～5kb、13个2.3～5kb、14个2.6～4.7kb、15个2.5～4.5大小片段时3次平行实验均失败。相比较之下，本发明中大肠杆菌体内组装与体外Gibson Assembly辅助相结合的组装方法成功率较高，12、13、14、15个片段组装均成功，且12、13个片段组装的3次平行实验均出现了全清或噬菌斑的平板，方法的结果稳定很好进一步说明了该发明可实现多至15个噬菌体基因组片段的成功组装，且在成功率和结果稳定性上优于对比例中常规使用的体外Gibson Assembly组装。
表22
Figure PCTCN2022082446-appb-000039
Figure PCTCN2022082446-appb-000040
以上对本发明所提供的基因组片段的表达元件组合、表达载体、宿主、应用及组装方法进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (25)

  1. 表达元件组合，其特征在于，包括但不限于：
    (I)、pRARE质粒中的表达元件；和
    (II)、lambda Red同源重组系统的表达元件。
  2. 如权利要求1所述的表达元件组合，其特征在于，所述lambda Red同源重组系统的表达元件包括但不限于exo、bet和gam。
  3. 表达载体，其特征在于，包括如权利要求1或2所述的表达元件组合。
  4. 如权利要求3所述的表达载体，其特征在于，包括但不限于pRARE质粒和pKD46质粒。
  5. 转化或转染有如权利要求3或4所述表达载体的宿主。
  6. 如权利要求5所述的宿主，其特征在于，所述宿主为原核生物或真核生物，包括但不限于细菌、放线菌、立克次氏体、衣原体、支原体、蓝细菌、古细菌、酵母和霉菌中的一种或多种。
  7. 如权利要求6所述的宿主，其特征在于，所述细菌包括但不限于革兰氏阴性菌或革兰氏阳性菌；
    作为优选，所述革兰氏阴性菌包括但不限于大肠杆菌；更优选的，所述大肠杆菌包括但不限于大肠杆菌DH10B；
    作为优选，所述革兰氏阳性菌包括但不限于细胞壁缺陷型L型细菌；更优选的，细胞壁缺陷型L型细菌包括但不限于L型李斯特菌。
  8. 如权利要求1或2所述表达元件组合、如权利要求3或4所述表达载体，或如权利要求5至7任一项所述宿主在基因组片段组装中的应用。
  9. 如权利要求1或2所述表达元件组合、如权利要求3或4所述表达载体，或如权利要求5至7任一项所述宿主在分子克隆、合成人造噬菌体、噬菌体疗法、制备噬菌体疗法的产品、检测微生物、制备检测微生物的产品、预防和/或治疗微生物引起的感染或疾病，或制备预防和/或治疗微生物引起的感染或疾病的药物中的一种或多种的应用。
  10. 如权利要求9所述的应用，其特征在于，所述片段包括但不限于：
    (I)、＜2kb的片段；和/或
    (II)、≥2kb且＜7kb的片段；和/或
    (III)、≥7kb且＜10kb的片段；和/或
    (IV)、≥7kb且＜10kb的片段；和/或
    (V)、≥10kb的片段。
  11. 如权利要求9或10所述的应用，其特征在于，所述片段的个数包括但不限于：
    (I)、＜5个；和/或
    (II)、≥5个，且＜10个；和/或
    (III)、≥10个且＜15个；和/或
    (IV)、≥15个。
  12. 基因组片段的组装方法，其特征在于，基于如权利要求1或2所述表达元件组合、如权利要求3或4所述表达载体，或如权利要求5至7任一项所述宿主，对基因组的片段进行组装。
  13. 如权利要求12所述的组装方法，其特征在于，所述组装包括但不限于：
    (I)、所述宿主体内直接组装；和/或
    (II)、体外Gibson Assembly组装和所述宿主体内组装。
  14. 如权利要求12或13所述的组装方法，其特征在于，包括但不限于如下步骤：
    步骤A：获得待组装的基因组的片段；
    步骤B：进行体外Gibson Assembly组装；
    步骤C：经宿主体内组装；
    其中，所述组装方法可以包括步骤B，也可以不包括步骤B。
  15. 如权利要求13或14所述的组装方法，其特征在于，所述片段包括但不限于：
    (I)、末端存在至少40～100bp的同源区域；和/或
    (II)、当所述片段的数量较多、较大时，增大同源臂长度，同时保证同源区域及其加上下游20bp的区域在50℃的二级结构自由能较小。
  16. 如权利要求13至15任一项所述的组装方法，其特征在于，所述体外Gibson Assembly组装的反应温度包括但不限于37℃～50℃；
    作为优选，所述体外Gibson Assembly组装的反应温度包括但不限于：
    (I)、42℃或50℃；和/或
    (II)、37℃～42℃；和/或
    (III)、42℃～50℃；
    步骤B中所述体外Gibson Assembly组装的反应时间包括但不限于15min～105min；
    作为优选，所述体外Gibson Assembly组装的反应时间包括但不限于：
    (I)、15min、75min或105min；和/或
    (II)、15min～75min；和/或
    (III)、75min～105min。
  17. 如权利要求13至16任一项所述的组装方法，其特征在于，所述体外Gibson Assembly组装的体系中，所述片段的用量不少于30fmol；
    作为优选，所述片段的用量包括但不限于：
    (I)、30fmol～60fmol；和/或
    (II)、＞60fmol。
  18. 如权利要求14至17任一项所述的组装方法，其特征在于，所述基因组包括但不限于噬菌体基因组。
  19. 如权利要求18所述的组装方法，其特征在于，步骤C所述组装后还包括激 活的步骤；
    所述激活包括但不限于：取所述宿主接种于含有Ca 2+或Mg 2+的培养基中，37℃培养不少于6h。
  20. 如权利要求18或19所述的组装方法，其特征在于，所述组装方法成功与否的评价标准包括但不限于：出现预期的噬菌斑或平板出现了全清现象，则噬菌体基因组组装成功。
  21. 基因组的组装方法，其特征在于，包括如权利要求12至20任一项所述的组装方法。
  22. 如权利要求21所述的组装方法，其特征在于，所述基因组包括但不限于噬菌体基因组。
  23. 人造噬菌体的合成方法，其特征在于，包括如权利要求12至20任一项所述的组装方法。
  24. DNA多分子组装方法，其特征在于，包括如权利要求12至20任一项所述的组装方法。
  25. DNA克隆方法，其特征在于，包括如权利要求12至20任一项所述的组装方法。
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