WO2015111248A1 - 単位dna組成物の調製方法及びdna連結体の作製方法 - Google Patents
単位dna組成物の調製方法及びdna連結体の作製方法 Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
Definitions
- the present invention relates to a method for preparing a unit DNA composition and a method for producing a DNA conjugate.
- the base sequence is usually determined by an automatic fluorescence sequencer using the Sanger method, but according to this method, a base sequence having a length of about 800 bases can be confirmed by one base sequence determination. Is possible.
- unit DNA synthesized by chemical synthesis or PCR method is confirmed in the base sequence before gene accumulation, if the number of base sequence determinations is small, time and money costs can be reduced. Therefore, the shorter unit DNA synthesized by chemical synthesis or PCR used for gene accumulation is preferable.
- Patent Document 1 discloses a method for preparing plasmid DNA for Bacillus subtilis cell transformation using the OGAB method.
- the OGAB method uses a so-called multimer plasmid in which a plurality of plasmid units exist in one DNA molecule by homologous recombination between plasmid molecules.
- the DNA molecule for transformation does not need to be circular DNA, but can be tandemly repeated so that one unit of the plasmid and each unit DNA used for accumulation appear repeatedly in the same direction. For example, plasmid transformation is possible.
- the weight is proportional to the length of the unit DNA, and particularly when the measured value of the weight differs by several times or more. In many cases, the value calculated based on the measurement value includes a large error.
- an attempt is made to adjust the molar ratio of each unit DNA to be 1. However, since the length distribution of each unit DNA is wide, the molar ratio should be strictly controlled. I can't.
- the present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for preparing a unit DNA composition in which the number of moles of a plurality of unit DNAs is more uniform and a method for preparing a DNA ligated body.
- the present inventors have found that errors in measurement results are reduced by measuring the number of moles of unit DNA in a state in which an additional sequence is linked to each unit DNA, and the present invention has been completed. . More specifically, the present invention provides the following.
- the unit DNA is used for producing a DNA ligation containing an integrated DNA composed of the unit DNA
- the integrated DNA was equally divided so that each base length was equal when the base length of the sequence of the integrated DNA was divided by the number of types of the unit DNA.
- the preparation of the unit DNA composition according to any one of (1) to (5), comprising the step of designing the unit DNA having a non-palindromic sequence at the end with a non-palindromic sequence in the vicinity of the sequence at the position as a boundary.
- a method for producing a DNA ligation for microbial cell transformation comprising more than one integrated DNA unit comprising a vector DNA containing an effective replication origin in a host microorganism and integrated DNA, A step of preparing a unit DNA composition by the method according to any one of (1) to (6); Preparing the vector DNA; Removing each additional sequence from the unit DNA to which the additional sequence in the solution after preparation has been ligated using a restriction enzyme; After the removing step, the step of connecting the vector DNA and each unit DNA to each other, The vector DNA and each unit DNA have a structure that can be repeatedly linked with each other in the order,
- the integrated DNA is a method for producing a DNA conjugate, wherein the unit DNAs are DNAs linked to each other.
- the present invention it is possible to provide a method for preparing a unit DNA composition in which the number of moles of a plurality of unit DNAs is more uniform, and a method for preparing a DNA conjugate.
- Example 1 of the present invention after transforming the DNA ligation product after ligation of unit DNA and vector DNA into Bacillus subtilis, a plasmid is extracted from the plurality of Bacillus subtilis after the transformation, and the plasmid is restricted. It is a figure which shows the photograph of the electrophoresis after carrying out an enzyme process.
- a plasmid is extracted from a plurality of Bacillus subtilis after transformation, and a result of electrophoresis after the plasmid is treated with a restriction enzyme, a Bacillus subtilis clone containing the target integrated DNA is obtained.
- FIG. 2 is a diagram showing a photograph of electrophoresis after screening and restriction enzyme treatment.
- Example 1 of this invention shows that the selected integrated DNA in Example 1 of this invention formed the plaque of lambda phage DNA. It is a figure in Example 1 of this invention which shows the photograph after carrying out the restriction enzyme process with the genome of the selected integrated DNA and wild type lambda phage with AvaI. It is a figure which shows the photograph of the electrophoresis after carrying out the restriction enzyme process for the plasmid containing the unit DNA after the refinement
- Example 2 of the present invention after transforming the DNA ligation product after ligation of the unit DNA and the vector DNA into Bacillus subtilis, a plasmid is extracted from the plurality of Bacillus subtilis after the transformation, and the plasmid is restricted. It is a figure which shows the photograph of the electrophoresis after carrying out an enzyme process.
- a plasmid was extracted from a plurality of transformed Bacillus subtilis, and from the results of electrophoresis after the plasmid was treated with a restriction enzyme, a Bacillus subtilis clone containing the target integrated DNA was obtained.
- FIG. 2 is a diagram showing a photograph of electrophoresis after screening and restriction enzyme treatment.
- FIG. 1 is a diagram showing a photograph of electrophoresis after screening and restriction enzyme treatment.
- 3 shows photographs of electrophoresis of DNAs (A) to (H) used in Test Example 1.
- 3 is a graph showing the number of transformant appearances of Bacillus subtilis competent cells transformed with DNAs (A) to (H) used in Test Example 1. It is a graph which shows the relationship between the variation CV (%) of a unit DNA fragment density
- FIG. (A) shows a graph for 6 fragment accumulation
- (b) shows a graph for 13 fragment accumulation
- (c) shows a graph for 26 fragments
- (d) shows a graph for 51 fragment accumulation. Indicates.
- (a) is a graph of the ⁇ function for the variation CV (%) of the concentration of the unit DNA fragment derived from the fitting to the exponential distribution curve in Simulation 1
- (b) is the N value of the virtual ligation product. It is a graph of a lambda function about CV (%) of variation of concentration of a unit DNA fragment obtained from an average value. The figure which shows the misligation site about # 1, # 2, # 5, # 7, # 8, # 9, # 10, # 11 among the aggregates obtained in the experiment of ⁇ phage genome reconstruction in simulation 1 It is.
- FIG. 6 is a graph comparing the actual ligated efficiency in simulation 1 and the ligation efficiency by ligation simulation.
- A) is a graph compared with a simulation with 95% ligation capability
- (b) is a graph compared with a simulation with 96% ligation capability
- (c) is compared with a simulation with 97% ligation capability.
- (D) is a graph compared with a simulation with a ligation capability of 98%
- (e) is a graph compared with a simulation with a ligation capability of 99%
- (f) is a graph with a ligation capability of 100%.
- Graphs compared with the simulations are shown respectively.
- the method for preparing a unit DNA composition of the present invention comprises a step of preparing a solution containing a plurality of unit DNAs to which additional sequences are linked for each type of unit DNA, and after each solution is prepared, additional sequences are added to the unit DNA.
- the concentration of the unit DNA in each solution is measured, and based on the result, each solution is fractionated to bring the number of moles of the unit DNA in each solution close to each other.
- the type of “unit DNA” is distinguished for each base sequence.
- the “unit DNA” includes both those with a restriction enzyme recognition site added and those without a restriction enzyme recognition site.
- the concentration of unit DNA in a solution containing the unit DNA when measuring the concentration of each unit DNA in a solution containing the unit DNA, an additional sequence is linked to each unit DNA. Then, since the additional sequence is linked, the distribution of the length of the base sequence becomes small when measuring the concentration of the solution. Therefore, an error in the number of moles of each unit DNA calculated based on the measurement result is reduced. Therefore, by separating each solution based on this measurement result and adjusting the number of moles of unit DNA in each solution to be the same, the molar ratio in each solution can be easily brought close to 1. .
- the “concentration of unit DNA in solution” to be measured refers to the molar concentration of unit DNA.
- the method for measuring the molar concentration of the unit DNA in the solution is not particularly limited.
- the mass% of the unit DNA in the solution is measured, and from the measured numerical value of the mass% of the unit DNA in the solution, The molar concentration of the unit DNA may be calculated.
- a method for measuring the molar concentration of unit DNA in a solution it is preferable to measure by using means capable of measuring the DNA weight concentration with an accuracy within ⁇ 20%, more specifically, by a microspectrophotometer. It is preferable to use an ultraviolet absorption spectrum.
- the step of preparing the solution containing the unit DNA to which the additional sequence is linked is not particularly limited, and for example, the unit DNA may be prepared and then the additional sequence may be linked to the unit DNA.
- the preparation of the unit DNA may be performed by using a previously synthesized one, or may be performed by preparing the unit DNA.
- the unit DNA can be prepared by a conventionally known method, for example, by polymerase chain reaction (PCR) or chemical synthesis.
- PCR polymerase chain reaction
- a primer prepared by adding a restriction enzyme recognition sequence that generates each protruding end to the base sequence on the template DNA is prepared by PCR, or in advance It can be produced by incorporating a restriction enzyme recognition sequence so as to generate an arbitrary protruding sequence at the end and performing chemical synthesis.
- the base sequence of the prepared unit DNA can be confirmed by a conventionally known method. For example, it can be confirmed by incorporating the unit DNA into a plasmid and determining the base sequence by an automatic fluorescent sequencer using the Sanger method. Can do.
- the additional sequence is not particularly limited, and may be linear DNA or a circular plasmid.
- the unit DNA to which the additional sequence is linked has a circular structure, so that it can be transformed into a host such as Escherichia coli.
- the kind of plasmid DNA is not particularly limited, but in order to replicate plasmid DNA in a transformed host, the plasmid DNA sequence preferably has an origin of replication.
- pUC19 which is a high copy plasmid vector of E. coli, or a derivative plasmid thereof is preferable.
- all the unit DNAs are cloned into the same kind of plasmid vector in that the length distribution between the DNAs to which the additional sequences are ligated can be reduced and the number of moles of the unit DNAs can be made closer to each other. Preferably it is.
- the ligation of the additional sequence to the unit DNA may be performed, for example, by ligation using DNA ligase, and may be performed by the TA cloning method when ligating to the plasmid DNA.
- the standard deviation of the total length distribution of each base length of the unit DNA and the base length of the additional sequence linked to each unit DNA is not particularly limited. However, the smaller the standard deviation of the concentration of the DNA in the solution, Since the error in the number of moles of each unit DNA calculated based on the measurement result is reduced, the number of moles of unit DNA in each solution can be made closer to each other.
- the standard deviation of the total length distribution with the base length of the additional sequence linked to each unit DNA is preferably within ⁇ 20% of the average total length, and ⁇ 15 Is more preferably within ⁇ 10%, even more preferably within ⁇ 10%, even more preferably within ⁇ 5%, still more preferably within ⁇ 1%, and within ⁇ 0.5%. Most preferably.
- the average base length of the additional sequence linked to each unit DNA is not particularly limited, but the longer one than the average base length of the unit DNA is calculated based on the measurement result of the concentration of DNA in the solution. Since the error in the number of moles of each unit DNA is reduced, the number of moles of unit DNA in each solution can be made closer to each other.
- the average base length of the additional sequence linked to each unit DNA is preferably 2 times or more, more preferably 5 times or more with respect to the average base length of the unit DNA, The ratio is more preferably 10 times or more, and most preferably 20 times or more.
- the average base length of the additional sequence linked to the unit DNA is too long, it becomes difficult to operate the unit DNA to which the additional sequence is linked.
- the average base length of the additional sequence linked to each unit DNA is 10000 times or less (specifically, 5000 times or less, 3000 times or less, 1000 times or less, 500 times or less) with respect to the average base length of the unit DNA.
- it is preferably 250 or less, 100 times or less, and the like.
- the length of each unit DNA is not particularly limited, but when confirming the base sequence of the unit DNA, it is preferable that the number of times of base sequence determination is smaller, the time and money costs can be reduced. Therefore, the length of each unit DNA is preferably shorter, specifically, it is preferably 1600 bp or less, and more preferably 1200 bp or less. In particular, when base sequence determination is performed by an automatic fluorescent sequencer using the Sanger method, it is possible to confirm a base sequence of about 800 bases in length by one base sequence determination.
- the thickness is most preferably 800 bp or less (specifically, 600 bp or less, 500 bp or less, 400 bp or less, 200 bp or less, 100 bp or less, etc.).
- the length of each unit DNA is preferably 20 bp or more, more preferably 30 bp or more, and further preferably 50 bp or more.
- the unit DNA composition prepared by the preparation method of the present invention is a DNA ligation containing integrated DNA constituted by the unit DNA. Can be used to make a body.
- a unit DNA composition prepared by the method of the present invention is used to prepare a DNA conjugate by the method described below, many unit DNAs (for example, 50 or more types) can be linked. This is considered to be because in the unit DNA composition prepared by the preparation method according to the present invention, the number of moles of each unit DNA is more accurately close to the same.
- the step of preparing a solution containing unit DNA may include a step of designing unit DNA.
- the design of the unit DNA is not particularly limited.
- the base length of the sequence of the integrated DNA is divided by the number of types of the unit DNA.
- the non-pain sequence near the sequence at the position where the integrated DNA is equally divided may be used as a boundary so that each base length becomes equal.
- unit DNA is designed in this way, the length of each unit DNA becomes substantially the same length.
- DNA when used for the preparation of a DNA ligation described below, it appears as a band at substantially the same position in the size fraction after electrophoresis after removing the additional sequence with a restriction enzyme. It is preferable in that DNA can be recovered and work efficiency is improved.
- the “near the sequence in the position where the integrated DNA is equally divided” is not particularly limited, but may be appropriately set based on the length of the base sequence.
- each unit DNA is 1000 bp, from “position where the accumulated DNA is equally divided”, within 100 bp (specifically, within 90 bp, within 80 bp, within 70 bp, within 60 bp, within 50 bp, 30 bp Within 20 bp, within 10 bp, within 5 bp, etc.).
- unit DNA when designing unit DNA as described above, when preparing a DNA ligation containing the target integrated DNA, it is necessary to have a non-palindromic sequence (sequence that is not a palindromic sequence) at the end of the unit DNA. It is preferable to design to.
- the unit DNA designed in this way has a structure that can be repeatedly linked while maintaining the order as will be described later, when the non-papillary sequence is a protruding sequence.
- the present invention also includes a method for producing a DNA conjugate.
- the method for producing a DNA ligated product of the present invention is limited by the above-described method for preparing a unit DNA composition, a step for preparing vector DNA, and a unit DNA to which additional sequences in the prepared solution are linked. A step of removing each additional sequence using an enzyme; and a step of linking the vector DNA and each unit DNA to each other after the removal step.
- the DNA conjugate contains more than one integrated DNA unit and is used for microbial cell transformation.
- the integrated DNA unit is composed of vector DNA and integrated DNA.
- the number of integrated DNA units in the DNA ligation is not particularly limited as long as it exceeds 1, but in order to increase the transformation efficiency, it is preferably 1.5 or more, more preferably 2 or more, and still more preferably 3 Or more, and most preferably 4 or more.
- the vector DNA contains an effective replication origin in the host microorganism to be transformed.
- the vector DNA is not particularly limited as long as it has a sequence that allows DNA replication in a microorganism into which a DNA conjugate can be transformed. For example, it is effective in Bacillus bacteria (Bacillus subtilis) described later.
- the sequence of the replication origin effective in Bacillus subtilis is not particularly limited.
- pTB19 Imanaka, T., et al. J. Gen. Microbioi. 130, 1399 has a ⁇ -type replication mechanism. -1408. (1984)
- pLS32 Tanaka, T and Ogra, M. FEBS Lett. 422, 243-246. (1998)
- pAM ⁇ 1 Singlefield, T. J., et al. Gene 87, 79-90 (1990)
- other sequences such as replication origins.
- Accumulated DNA consists of DNA in which the above unit DNAs are linked to each other.
- the DNA in the present invention is a DNA to be cloned, and the type and size are not particularly limited. Specifically, it may be a naturally occurring sequence such as a prokaryote, a eukaryote, or a virus, or an artificially designed sequence.
- the method of the present invention as described above, since many unit DNAs can be ligated on a plasmid, it is preferable to use DNA having a long base length. Examples of the DNA having a long base length include a group of genes constituting a series of metabolic pathways, the entire genomic DNA such as phage, or a part of the genomic DNA.
- the integrated DNA unit may or may not contain an appropriate base sequence as necessary in addition to vector DNA and integrated DNA.
- a base sequence that controls transcription and translation such as a promoter, an operator, an activator, and a terminator may be included.
- a promoter when Bacillus subtilis is used as a dormitory, specifically, Pspac (Yansura, D. and Henner, D. J. Pro. Natl. Acad.), Whose expression can be controlled by IPTG (isopropyl s-D-thiogalactopyroside). Sci, USA 81, 439-443. (1984)), or Pr promoter (Itaya, M. Biosci. Biotechnol. Biochem. 63, 602-604. (1999)).
- the vector DNA and each unit DNA have a structure that can be repeatedly linked while maintaining the order.
- “join together while maintaining their order” means that unit DNAs or vector DNAs having sequences adjacent to each other on an integrated DNA unit are bonded in the same order and orientation.
- “Repeatedly connect” means that the 5 ′ end of a unit DNA or vector DNA having a base sequence at the 5 ′ end and the 3 ′ end of a unit DNA or vector DNA having a base sequence at the 3 ′ end are combined.
- Specific examples of such unit DNA include, for example, those having ends that can be repeatedly linked to each other while maintaining their order using the complementarity of the base sequences of the protruding ends of the fragments.
- the structure of this overhang is not particularly limited as long as it is a non-batch sequence, including the difference in the shape of the 5 'end overhang and the 3' end overhang.
- the above protruding end is preferably produced by removing each additional sequence from the unit DNA using a restriction enzyme. Therefore, when a DNA conjugate is prepared by the method of the present invention, the unit DNA preferably has a restriction enzyme recognition sequence in order to remove an additional sequence by the restriction enzyme.
- the vector DNA can be prepared, for example, by performing a restriction enzyme treatment so that the vector DNA is provided with a protruding end for repeated ligation with the unit DNA while maintaining the order.
- the restriction enzyme used for the removal of the additional sequence is not particularly limited, but is preferably a type II restriction enzyme, more preferably AarI, BbsI, BbvI, BcoDI, BfuAI, BsaI, BsaXI, BsmAI, BsmBI, BsmFI, BspMI, BspQI , BtgZI, FokI, SfaNI, and the like are type IIS restriction enzymes capable of producing a protruding end of an arbitrary sequence at a predetermined distance away from the recognition sequence.
- a type IIS restriction enzyme is used, the protruding ends of the unit DNA can be made different at each ligation site, so that the order of ligation is maintained.
- type IIS restriction enzyme it is preferable to prepare using type IIS restriction enzyme so as to provide protruding ends for repeated ligation with unit DNA while maintaining the order. preferable.
- each unit DNA is classified into groups for each type of restriction enzyme used to remove the additional sequence, a solution containing two or more types of unit DNA can be mixed for each group before the removal step. .
- the restriction enzyme treatment can be performed once for each group of restriction enzymes.
- the unit DNA is fractionated by electrophoresis, for example, Since the unit DNA can be collected by drawing, the work efficiency is improved.
- an error may occur in the recovery amount between the groups. Errors can occur.
- the number of groups is smaller, that is, it is preferable that the number of types of restriction enzymes used for removal of additional sequences is smaller. Therefore, the number of restriction enzymes to be used is preferably 5 or less, more preferably 3 or less, and most preferably 1 type. That is, if only one type of restriction enzyme is used, a solution containing all the unit DNAs can be mixed, so that the efficiency of the work is dramatically improved and the number of moles aligned between the unit DNAs is not improved. Is unlikely to occur. Since restriction enzymes are mixed in an approximately equimolar state, a large number of unit DNAs can be ligated even if such a mixture is used.
- the restriction enzyme recognition sequence of the unit DNA is designed so as not to recognize the sequence of each unit DNA. That is, when an attempt is made to use a certain type IIS restriction enzyme, if the type IIS restriction enzyme does not recognize the sequence of one unit DNA but recognizes the sequence of the other unit DNA, Design a restriction enzyme recognition sequence for each unit DNA so that a type IIS restriction enzyme different from the type IIS restriction enzyme is used. In such a design, the type IIS restriction enzyme used for each unit DNA is different, but the unit DNA can be used for classification of the above group depending on the type of the type IIS restriction enzyme. If there is a type IIS restriction enzyme that does not recognize any sequence of each unit DNA, the restriction enzyme recognition sequence is added to design the unit DNA, whereby the additional sequence is combined from the unit DNA by one type IIS restriction enzyme. Can be removed.
- the step of linking the vector DNA and each unit DNA to each other is not particularly limited. After the restriction enzyme treatment, the additional sequence after the restriction enzyme treatment and the unit DNA are fractionated, and the fractionated unit DNA and vector are fractionated. It can be performed by ligation using DNA and DNA ligase or the like. Thereby, the DNA coupling body for microorganism transformation can be produced.
- the unit DNA in the ligation step is one to which no restriction enzyme recognition sequence is added.
- the method of fractionating the additional sequence and the unit DNA is not particularly limited, but is preferably a method in which the relationship of the molar ratio of each unit DNA after the restriction enzyme treatment is not disrupted, specifically by agarose gel electrophoresis. Is preferred.
- the method for linking unit DNA and vector DNA is not particularly limited, but it is preferably performed in the presence of polyethylene glycol and a salt.
- the salt is more preferably a monovalent alkali metal salt. More specifically, it is more preferable to carry out with a ligation reaction solution containing 10% polyethylene glycol 6000 and 250 mM sodium chloride.
- the concentration of each unit DNA in the ligation reaction solution is not particularly limited, and is preferably a concentration of 1 fmol / ⁇ l or more.
- the reaction temperature and time for ligation are not particularly limited, but are preferably 30 minutes or more at 37 ° C.
- the vector DNA in the ligation reaction solution is preferably adjusted so that it is equimolar with the unit DNA by measuring the DNA concentration of the solution containing the vector DNA before the reaction.
- the method for preparing a DNA ligation according to the present invention comprises the yield of a DNA fragment having a target ligation number expressed by the product of the number of unit DNAs constituting the accumulation unit and the number of accumulation units, and the concentration of the DNA fragment.
- the concentration of the vector DNA and each unit DNA in the ligation step is determined.
- the step of adjusting the coefficient of variation (hereinafter referred to as “variation coefficient 2” in this specification) may or may not be included, but it is preferable to include it.
- the coefficient of variation 1 is a coefficient of variation used for convenience in the relational expression
- the coefficient of variation 2 is a coefficient of variation in the concentrations of unit DNA and vector DNA in the actual linking process.
- the target ligation number refers to the number of desired DNA fragments to be ligated in the ligation step, and more specifically is represented by the product of the number of unit DNAs constituting the ligated accumulation unit and the number of accumulation units.
- the “yield of DNA fragments of the target ligation number” means the ratio of the number of DNA fragments constituting the integrated DNA unit after ligation to the total number of DNA fragments used for ligation.
- the relational expression according to the present invention is an expression showing the relation between the yield of DNA fragments of the target ligation number and the coefficient of variation 1.
- an expression obtained from a ligation simulation by a computer can be used. More specifically, for example, the relational expression is obtained by performing a ligation simulation in a unit DNA fragment population (for example, 10-30 population) in which the coefficient of variation 1 is changed every 1% of 0 to 20%, and The distribution (for example, exponential distribution) of the number of unit DNA fragments is examined, a fitting curve for this distribution is created, and the fitting curve can be used for setting.
- the tool used for the specific ligation simulation is not particularly limited, and a conventional known means can be used.
- VBA Visual Basic for Applications
- spreadsheet software Excel registered trademark 2007
- the fitting curve can be created by, for example, an exponential approximate curve function of spreadsheet software Excel (registered trademark) 2007 software.
- Adjustment of the coefficient of variation 2 based on the relational expression is performed, for example, by designing the relational expression and then substituting the yield of the desired DNA fragment into the relational expression so that the DNA fragment at the time of ligation becomes the calculated coefficient of variation 1. It can be performed by adjusting the operation in each step before connection.
- the adjustment method is not particularly limited.
- each step such as a step of preparing vector DNA, a step of preparing unit DNA, a step of linking vector DNA and each unit DNA to each other, vector DNA or each unit DNA
- the concentration of the sample measure it in advance so that the coefficient of variation 2 becomes the desired coefficient of variation when selecting the measurement equipment (spectrophotometer, spectrofluorometer, real-time PCR device, etc.) used for the measurement. You may select a measuring instrument with known error.
- the coefficient of variation 2 is not particularly limited, but more unit DNA can be linked if the unit DNA concentration error when linked is smaller. Accordingly, the coefficient of variation 2 is preferably 20% or less, more preferably 15% or less, still more preferably 10% or less, and even more preferably 8% or less. % Is most preferred.
- the method for preparing a DNA conjugate of the present invention may further include a step of inactivating the restriction enzyme after the removing step and before the linking step. If there is a restriction enzyme cleavage site used for cutting off the additional sequence of other unit DNA inside the unit DNA, it is difficult to mix the unit DNA population having the additional sequence before inactivating the restriction enzyme. Therefore, the unit DNA cannot be fractionated at once by integrating them. However, by inactivating the restriction enzyme, it is possible to integrate a unit DNA population having an additional sequence after the inactivation, so that unit DNA can be fractionated in a lump.
- the inactivation of the restriction enzyme can be performed by a conventionally known method, for example, by phenol / chloroform treatment.
- the host microorganism to be transformed is not particularly limited as long as it has a natural transformation ability.
- the natural transformation ability include those that are processed into single-stranded DNA when DNA is incorporated.
- Specific examples include Bacillus genus bacteria, Streptococcus genus bacteria, Haemophilus genus bacteria, and Neisseria genus.
- Bacillus bacteria include B. subtilis, Bacillus subtilis. megaterium (Bacteria), B. stearothermophilus (medium hyperthermophile) and the like.
- the most preferred microorganism is Bacillus subtilis excellent in natural transformation ability and recombination ability.
- the DNA conjugate produced by the method of the present invention can be used for transformation of microbial cells, but the method of making the microorganism to be transformed competent is a known method suitable for each microorganism. You can choose. Specifically, for example, in the case of Bacillus subtilis, Ananostopoulou, C.I. And Spizzen, J.M. J. Bacteriol. 81, 741-746 (1961).
- the transformation method a known method suitable for each microorganism can be used.
- the amount of ligation product to be given to competent cells Preferably, the amount is from 1/20 to the equivalent, more preferably half the amount of the competent cell culture medium.
- a known method for purifying the plasmid from the transformant a known method can be used.
- Whether the plasmid purified from the transformant has accumulated DNA can be confirmed by the size pattern of fragments generated by restriction enzyme cleavage, the PCR method, or the nucleotide sequence determination method. Further, when the inserted DNA has a function such as substance production, it can be confirmed by detecting the function.
- Bacillus subtilis was used as a microbial cell to be transformed.
- Bacillus subtilis RM125 strain (Uozumi, T., et al. Moi. Gene. Genet., 152, 65-69 (1977)) and its derivative strain BUSY9797 were used.
- PGET118 Kaneko, S., et al. Nucleic Acids Res. 31, e112 (2003) was used as a vector DNA replicable in Bacillus subtilis, and pGETS118-AarI-pBR (SEQ ID NO: 1 was constructed as described below) was used.
- PGETS151-pBR see SEQ ID NO: 2.
- lambda phage DNA manufactured by Toyobo
- SEQ ID NO: 3 The antibiotic carbenicillin (Wako Pure Chemical Industries, Ltd.) was used for selection of E. coli having plasmid DNA into which unit DNA was incorporated.
- Antibiotic tetracycline (Sigma) was used for selection of Bacillus subtilis.
- type IIS restriction enzymes AarI (Thermo), BbsI (NEB), BsmBI (NEB), and SfiI (NEB) were used.
- HindIII, PvuII, and T4 DNA ligase were manufactured by Takara Bio Inc.
- Takara Ligation Kit (Mighty) (Takara Bio Inc.) was used for general ligation for plasmid construction in E. coli.
- KOD plus polymerase manufactured by Toyobo Co., Ltd. was used for the PCR reaction for preparing unit DNA.
- Ex-Taq HS manufactured by Takara Bio Inc. was used for colony PCR for determining the base sequence of DNA cloned into the plasmid.
- PMD-19 (simple) (Takara Bio Inc.) was used as the plasmid DNA which is an additional sequence for incorporating the unit DNA.
- the enzyme for plasmid purification Plasmid Safe, manufactured by EPICENTER was used.
- As the agarose gel for electrophoresis 2-Hydroxyethyl agarose (Sigma) or UltraPure Agarose (Invitrogen), which is a low melting point agarose gel for DNA electrophoresis, was used.
- phenol: chloroform: isoamyl alcohol 25: 24: 1 and TE saturated phenol (containing 8-quinolinol) manufactured by Nacalai Tesque, Inc. were used.
- Lambda termiase manufactured by EPICENTER was used.
- Gigapack III Plus Packaging Extract from Agilent Technologies was used for packaging of lambda phage.
- the lysozyme manufactured by Wako Pure Chemical Industries, Ltd. was used.
- As the medium components and agar of the LB medium those manufactured by Becton Dickington were used.
- IPTG (isopropyl s-D-thiogalactopyranoside) manufactured by Wako Pure Chemical Industries, Ltd. was used. All other medium components and biochemical reagents other than those described above were manufactured by Wako Pure Chemical Industries.
- any one of E. coli DH5 ⁇ strain, JM109 strain and TOP10 strain was used.
- QIAprep Spin Miniprep Kit from Qiagen was used for small-scale purification of the constructed plasmid from Escherichia coli, and QIAfilter Midi Kit from the company was used for large-scale purification.
- MinElute Reaction Cleanup Kit from Qiagen or QIAquick PCR purification Kit from Qiagen was used.
- MinElute Gel Extraction Kit manufactured by Qiagen was used.
- Nano-drop 2000 manufactured by Thermo was used as the ultra-trace spectrophotometer.
- PGETS118-AarI-pBR (Construction of vector DNA used for accumulation)
- PGETS118-AarI-pBR (SEQ ID NO: 1), which is a vector DNA used for accumulation of lambda phage DNA, is an Escherichia coli-Bacillus subtilis strain having an origin of replication oriS of the F factor of Escherichia coli and an origin of replication repA that functions in Bacillus subtilis. 1 is a plasmid constructed through a multistep process based on the inter-shuttle plasmid vector pGETS118 (Kaneko, et. Al., Nucleic Acids Res., 31, e112. (2003).), And has the structure shown in FIG. .
- the cloning site of the integrated gene is between two AarI cleavage sites, and multiple copies of E. coli are used between these two AarI cleavage sites, which are removed during accumulation, in order to facilitate the acquisition of the vector in E. coli.
- a plasmid pBR322 origin of replication and an ampicillin resistance gene have been introduced.
- the natural AarI cleavage site present in the tetracycline resistance gene inside pGETS118 is deleted by introducing a single base mutation that does not affect the amino acid sequence of the tetracycline resistance gene (tetL). It was.
- PGETS151-pBR (SEQ ID NO: 2), which is a vector DNA used for accumulation of the mevalonate pathway artificial operon, is composed of three sets of primers PartA (5′-TAGGGTCTCaagcggcccccagcttt-3 ′) using the above-described DNA of pGETS118-AarI-pBR as a template. (See SEQ ID NO: 5) and 5'-TAGGGTCTCAGGCggccagagaagcc-3 '(see SEQ ID NO: 6), Part B (5'-TAGGGTCTCACCccCCTCTCCCGGTCCGATAT-3CT (see SEQ ID NO: 7) and 5'-TAGGGTCTCATTGATCATTGATCATTGATCATGT No.
- the diversity of overhangs of 4 bases is 256 to the 4th power.
- the protruding array used in the present invention was selected according to the following criteria. First, all 16 sequences (group 0) (AATT, ATAT, TATA, TTAA, CCGG, CGCG, GCGC, GGCC, ACGT, AGCT, TCGA, TGCA, CATG, CTAG, GATC, GTAC) which become palindrome are these Since the complementary sequence of the sequence also becomes the same sequence and the fragments of the same species can be linked, they are excluded because they cannot be used in the present invention.
- Example 1 the boundary between the vector DNA and the unit DNA was selected from Group 1 from the groups divided above.
- a total of 60 protruding combinations of group III (44 combinations) and group IV (16 combinations) were selected as candidates.
- group III 44 combinations
- group IV 16 combinations
- a complete base sequence of a sequence to be accumulated was first determined, and then an ideal division boundary that equally divides this total length was set.
- the base sequence used in Example 1 will be described as a specific example.
- Example 1 48522 bp obtained by adding a cos site 16 bp and an overhanging sequence 4 bp necessary for accumulation to the total length of lambda phage genome 48502 bp was targeted for reconstruction.
- Table 1 is a table showing ideal divided units, actual divided units, and protruding base sequences of the integrated DNA in Example 1.
- the unit DNA except the accumulation plasmid vector was prepared by dividing it into 50 almost identical sizes, and reconstitution was attempted by ligating them. Although it is desirable to divide all 50 fragments so that they are ideally equal in length, in order not to introduce any base change in the sequence to be accumulated, it depends on the original sequence. Thus, it is necessary to create a 4 base 5 ′ terminal overhang used for accumulation.
- the projection combination assignment simulation is performed so that the length is as close to the ideal division unit as possible.
- 970 bp obtained by dividing the full length (48522 bp) by 50 was an ideal division unit, and the unit DNA of a region having a small absolute base number was named as the 01st fragment, the 02th fragment, the 03th fragment,... .
- Selection of specific protrusions from the protrusion candidates existing in each sequence is performed first from extraction sequences with a low total number of protrusion combination candidates as described above, from protrusion combination sequences with a low frequency of appearance in all extracted sequences. By assigning, a unique protruding combination was assigned to every division unit.
- Lambda phage is a bacteriophage that infects E. coli and is the best studied molecular biological. The genome consists of double-stranded DNA with a total length of 48502 bp, and all base sequences have been clarified. The existence of various mutants has also been revealed. In this example, an attempt was made to produce a lambda phage point mutant from a short unit DNA of about 1 kb.
- lambda phage As lambda phage, ⁇ phage DNA manufactured by Toyobo Co., Ltd. was used. This product is linear at the cos site.
- 6 sites g. 138delG, g.14266_14267insG, g.37589C> T, g with respect to the nucleotide sequences registered in the database (accession number J02459.1).
- SEQ ID NO: 3 (the total length of SEQ ID NO: 3 is a total length of 48526 bp including the above-mentioned 48522 pb and the other four protruding terminal bases) .
- Type IIS restriction enzymes that generate any overhang sequence of 4 bases include AarI (5′-CACCCTGC (N) 4 / ⁇ 3 ′, 5 ′ ⁇ / (N) 8GCAGGGTG-3 ′), BbsI (5′ ⁇ GAAGAC (N) 2 / -3 ′, 5 ′ ⁇ / (N) 6GTCTTC-3 ′), BbvI (5′-GCAGC (N) 8 / ⁇ 3 ′, 5 ′ ⁇ / (N) 12GCTGC-3 ′) BcoDI (5'-GTCTCN / -3 ', 5'-/ (N) 5GAGAC-3 '), BfuAI (5'-ACCCTGC (N) 4 / -3', 5 '-/ (N) 8GCAGGT-3 '), BsaI (5'-GGTCTCN / -3', 5 '-/ (N) 5GAGACC-3'), B
- restriction enzymes are not present in the E. coli plasmid vector (pMD19, Simple, TAKARA) used for subcloning the gene fragment, or the size of the generated fragment is sufficiently larger than the ideal split unit even if it exists.
- pMD19 Simple, TAKARA
- pMD19 Simple, TAKARA
- BsmBI restriction enzyme
- restriction enzyme sites of these candidates the distribution of the restriction enzyme sites in the entire lambda phage from the first fragment to the 50th fragment was examined. As a result, there were 12 sites for AarI, 24 sites for BbsI, and 41 sites for BfuAI. Thus, there were 38 BsmFI, 45 BtgZI, and 14 BsmBI, and there was no restriction enzyme recognition site that was not present in the lambda phage genome for any of the restriction enzymes. Therefore, it was decided to select and use restriction enzymes that do not cleave the interior for each unit DNA.
- BbsI is a total of 33 fragments of Nos. 01-08, 12, 16-22, 24, 27, 28, 33-39, 43, 45-50, -A total of 9 fragments of 13, 23, 25.30, 32, and 44 fragments
- BsmBI was a total of 8 fragments of the 14th, 15th, 26th, 29th, 31th, 40th to 42th fragments.
- the amplified unit DNA is 1 ⁇ TAE buffer containing 2 mg / ml Crystal Bio Red (Wako Pure Chemical Industries, Ltd.) “Tris-acetate-EDTA buffer stock solution (concentration 50 times) pH 8.3 (at concentration)” manufactured by Nacalai Tesque.
- the spin column was transferred to a 5 ml centrifuge tube. 30 ⁇ l of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) was added to the spin column, allowed to stand for 2 min, and centrifuged at 20,000 ⁇ g for 1 min to recover the DNA solution. This recovered DNA was stored at ⁇ 20 ° C. until use. The obtained unit DNA was cloned into an E. coli plasmid vector by the TA cloning method shown below.
- a synonym substitution mutant (g.9515G> C) in the coding region of gene V was present as one of the mutants obtained for the tenth fragment. Due to this mutation, a restriction enzyme AvaI recognition site newly appears in the phage genome (FIG. 2). In this example, for the purpose of clearly showing that the constructed phage was artificially produced, the synonym substitution mutant (g.9515G> C) was not used for the tenth fragment, but the wild type. ).
- Plasmid Safe (Epicenter) 10 ⁇ reaction buffer, 2.4 ⁇ l of 25 mM ATP solution and 2 ⁇ l of Plasmid Safe enzyme solution were added and mixed, and BI-526T (ASTEC), a programmable block incubator, The mixture was incubated at 37 ° C. for 1 h, and then incubated at 75 ° C. for 30 minutes for enzyme deactivation.
- the resulting solution was purified by PCR purification kit (Qiagen). In the final stage of purification of the kit, the DNA adsorbed on the column was eluted with 25 ⁇ l of TE buffer (pH 8.0) instead of the elution buffer attached to the kit to obtain a high purity plasmid solution.
- DNA electrophoresis was performed using the plasmid having the 01st fragment before and after purification and the plasmid having the 21st fragment (UltraPure Agarose, Invitrogen), and it was confirmed that the target fragment (unit DNA) was incorporated (Fig. 3).
- the obtained DNA solution was again measured with an ultra-trace spectrophotometer to determine the concentration of the high purity plasmid solution.
- the concentration of each sample was in the range of approximately 100 ng / ⁇ l to 200 ng / ⁇ l reflecting the degree of purification of the crude plasmid solution against the theoretical maximum of 200 ng / ⁇ l.
- 15 ⁇ l of each plasmid solution is placed in a 1.5 ml tube, TE is added to each solution so that each plasmid has a concentration of 100 ng / ⁇ l, and the obtained high-purity plasmid solution is again added to the ultrapure solution.
- the total volume of the integrated equimolar plasmid solution was about 165 ⁇ l for the BbsI group, about 45 ⁇ l for the AarI group, and about 40 ⁇ l for the BsmBI group. Double sterilized water was added to each group to obtain 495, 135, and 120 ⁇ l of high-purity plasmid solutions, which were cleaved as follows for each type of restriction enzyme.
- the electrophoresis gel was stained with 100 ml of 1 ⁇ TAE buffer containing 1 ⁇ g / ml ethidium bromide (Sigma) for 30 min and visualized by illuminating with long-wave ultraviolet light (366 mn), so that the first to 50th fragments
- the band formed by (about 1 kb) was cut out with a razor and collected in a 1.5 ml tube.
- 1 ⁇ TAE buffer was added to make the total volume about 700 ⁇ l, and this was incubated at 65 ° C. for 10 minutes to dissolve the gel.
- FIG. 6 shows the distribution of the number of molecules of each unit DNA before and after size fractionation
- FIG. 7 shows the change rate of the number of molecules of each unit DNA.
- the DNA weight concentration of the equimolar mixture of the 01st to 50th fragments is 98 ng / ⁇ l, the total base sequence is 48,522 bp, while the vector DNA (pGETS118-AarI / AarI) is 190 ng / ⁇ l with a total length of 15, 139 bp.
- vector DNA was mixed at a ratio of 1.00 ⁇ l to 6.21 ⁇ l of the equimolar mixture of the 01st to 50th fragments.
- plasmid For these four strains, a large amount of plasmid was prepared by cesium chloride-ethidium bromide density gradient ultracentrifugation, and the structure of the plasmid was confirmed by electrophoresis after performing 13 types of restriction enzymes. (Fig. 10). Furthermore, when the nucleotide sequence of the entire region except the vector portion of this plasmid was determined, all of the four plasmids were completely identical to the expected nucleotide sequence.
- each integrated plasmid of # 3, # 4, # 6, and # 12 is cleaved with lambda terminase (Lambda terminator, Epicenter) to divide it into a vector and an integrated gene portion, and this is divided into lambda packaging extract. (Gigapack III Plus Packing Extract, Agilent Technologies). When this was infected with Escherichia coli (VCS257 strain), spread on an LB plate and cultured at 37 ° C. overnight, plaques were confirmed.
- the shape of the obtained plaque was confirmed to be the same form as that obtained with TOYOBO lambda phage DNA performed in parallel (FIG. 11).
- Phage DNA was purified from plaques obtained from each plasmid, and it was confirmed whether the introduced mutation was present by digestion with restriction enzyme AvaI.
- FIG. 12 it was different from lambda phage DNA manufactured by TOYOBO.
- the cleavage pattern was shown, and it was confirmed that the AvaI site was present in all the phages as scheduled.
- the lambda phage genome produced by accumulating all 50 fragments of the 01 to 50 fragments was complete in both base sequence and plaque forming ability.
- IPP isopentenyl diphosphate
- this size is an ideal division unit, and a specific sequence (two in total of A and T is C and C and 44 combinations excluding 8 combinations of palindrome out of all 52 combinations in which the total of G is 2 (Group III above), and 32 combinations in which the total of A and T is 1 and the total of C and G is 3 Among them, it was examined whether any one kind of array appears in all ideal division units among all 16 combinations (total 60 types of the above-mentioned group IV) in which C and G are not 3 consecutive. It was found that any one kind of specific sequence appeared within a range of ⁇ 7 bp from the unit. Based on this, the entire length was divided into 55 98-115 bp fragments.
- Table 2 is a table showing the division units and protruding base sequences of the integrated DNA in Example 2.
- the protrusion (ATTA and AAAA) which consists only of A and T was utilized.
- PCR prepared to hybridize to the AarI recognition sites at both ends in order to amplify the double-stranded unit DNA obtained by hybridization of these two synthetic DNAs and subsequent template-dependent extension reaction by the PCR method.
- the concentration of 1 ⁇ l of these was measured with an ultra-trace spectrophotometer, the concentration was 108 to 213 ng / ⁇ l. From this, 20 ⁇ l of high-purity plasmid solution was taken in a separate tube, and TE was added to the tube so that the concentration of each plasmid was calculated to be 100 ng / ⁇ l for dilution. The concentration of this purified plasmid solution is again calculated with a microspectrometer, and the volume of each high-purity plasmid based on this concentration is calculated with a precision of 1 ⁇ l of 2 digits after the decimal point. About 5 ⁇ l) was taken from each plasmid solution and pooled into a single tube.
- ⁇ Batch size fraction of 55 unit DNA> An equal amount of phenol, chloroform, and isoamyl alcohol (25: 24: 1) was added to the reaction solution to inactivate AarI, followed by centrifugation. The supernatant was purified by ethanol precipitation, and 20 ⁇ l of the precipitate was purified. Dissolved in TE. To this, xylene cyanol was added as a dye for electrophoresis, and electrophoresis was performed with 2.5% agarose gel using TAE as a buffer at 100 V for 30 minutes to separate vector DNA pMD19 and insert unit DNA. (FIG. 13). The electrophoresed gel was divided with a razor, a portion thereof was stained with ethidium bromide, and the target DNA band was cut out from the unstained gel while confirming the positions of the target 55 equimolar mixed fragment bands. .
- DNA was purified from the obtained gel fragment using MiniElute Gel Extraction Kit (QIAGEN) as shown below.
- Example 1 the standard deviation of the distribution of the total length of each base length of the unit DNA and the base length of the additional sequence linked to each unit DNA is 3691.4 ⁇ 6.6 bp. , ⁇ 0.18% with respect to the average total length.
- Example 2 the standard deviation of the distribution of the total length of each base length of the unit DNA and the base length of the additional sequence linked to each unit DNA is 2828.2 ⁇ 4.5 bp, and the average The total length is ⁇ 0.16%.
- Examples 1 and 2 since the standard deviation is thus small with respect to the average total length, there is an error in the number of moles of each unit DNA calculated based on the measurement result of the concentration of DNA in the solution. It is thought that it was less.
- the ratio of the average base length of the additional sequence linked to each unit DNA to the average base length of the unit DNA is about 2.7 in Example 1 and about 27 in Example 2.
- the integrated DNA is equally divided so that each base length is equal when the unit DNA design is divided by the number of types of unit DNA. Since the unit DNA was designed in this way, the length of each unit DNA was substantially the same because the non-pain sequence near the sequence at the position was used as a boundary. Therefore, in the size fraction after electrophoresis after the additional sequence is removed by the restriction enzyme, it appears as a band at substantially the same position, and unit DNA can be recovered by one size fraction, which improves working efficiency. Indicated.
- Example 1 the restriction enzymes used for removal of additional sequences are classified into groups (3 types in Example 1 and 1 type in Example 2). Therefore, before the removal step, a solution containing two or more types of unit DNA can be mixed for each group, and it is not necessary to perform restriction enzyme treatment for each unit DNA.
- the restriction enzyme treatment was completed in one round. That is, it was confirmed that this improves the efficiency of the production of the DNA ligated product. Moreover, even when using such a mixture, it was confirmed that a large number of unit DNAs can be linked because they are mixed in an approximately equimolar state as described above.
- pGETS118-t0-Pr-SfiI-pBR was transformed into E. coli.
- the plasmid obtained from this transformant is mainly the DNA of (A), but contains a small amount of multimer (multimer).
- this plasmid was subjected to low melting point agarose gel electrophoresis.
- the DNA of (A) was prepared by cutting out and purifying only the monomer plasmid DNA region from the gel by the DNA size fractionation according to 1.
- the DNA of (B) was prepared by treating the DNA of (A) with the restriction enzyme BlpI (recognition site is (5′-GC / TNAGC-3 ′)).
- the DNA in (C) is a tandem repeat linear multimeric plasmid DNA with a redundancy r> 1.
- BlpI used when preparing the DNA of (B) above forms a non-palindromic 3-base overhang at the 5 ′ end. Therefore, the DNA of (B) was prepared by ligating the DNA of (B) with DNA ligase to prepare the DNA of (C), which is a linear multimeric plasmid DNA in which plasmid units are continuous in the same direction.
- the DNA of (D) was prepared by treating the DNA of (A) with the restriction enzyme EcoRI (recognition site is (5′-G / AATTC-3 ′)).
- the DNA of (E) is a linear multimeric plasmid DNA in which the DNA of (D) is linked in a random orientation, partially including a portion of redundancy r> 1.
- EcoRI used when preparing the DNA of (D) above forms a palindromic 3-base overhang at the 5 ′ end.
- plasmid DNA cleaved with EcoRI is ligated, it is possible to prepare multimeric plasmid DNA in which plasmid units are ligated in random orientations.
- the DNA of (E) was prepared by ligating the DNA of (D) with a DNA ligase.
- ⁇ Preparation of DNA of (F)> The DNA of (F) is cleaved with the restriction enzyme KasI that cleaves the DNA of (A) only at one position, dephosphorylated and then cleaved with BlpI in the vicinity thereof, and the DNA of (A) is converted to 1 It is an r ⁇ 1 linear quasi-monomer mixture obtained by cleaving with a restriction enzyme AfeI that cleaves only at a site, dephosphorylated, and then mixed with an equal amount of DNA cleaved with BlpI nearby. In any of the DNA fragments in the mixture of (F), the redundancy r is slightly less than 1.
- the cutting sites of (B) and (D) are separated from each other.
- the DNA of (H) is a mixture in which the DNAs of (B) and (D) are mixed without being linked so as to be equimolar.
- the DNAs (C) and (E) are distributed in a wide range on the lane, so that the bands are difficult to distinguish.
- N indicates the number of unit DNA fragments contained in one molecule of the ligated DNA fragment in the virtual ligation
- L indicates the sequence of the protruding end on the left side of the ligation product expressed as an arbitrary natural number
- R like “L” represents the value of the sequence of the protruding end on the right side of the ligation product expressed by an arbitrary natural number.
- the simulation of ligation was performed as follows.
- a random number j satisfying i ⁇ j is multiplied by a uniform random number between 0 and 1 generated by the RAND () method and m (described later). This was generated by rounding to an integer, thereby selecting an F j (N j , L j , R j ) fragment. Whether or not the two fragments can be linked is determined using the following discriminant. If they can be linked, the parameters of the fragments are changed as shown below.
- the sort function of VBA command Excel2007 (Sort method), by performing the rearrangement of F i fragment as L i value is in descending order, F (0,0,0) fragment The total number of other Fi fragments was counted, and the total number was inserted as new m, and virtual ligation of the next cycle was performed.
- Virtual ligation cycle there is no protruding pieces that are complementary to, and carried to the minimum fragment number m min can not perform any more virtual ligation.
- the mmin value was obtained by the following calculation using information of the initial unit DNA fragment before the start of ligation.
- ⁇ Ligation simulation> A hypothesis having a coefficient of variation (CV) set in increments of 1% in the range of 0 to 20% with an average number of DNA fragments of the same unit 640 in each integration scale up to 6 fragments, 13 fragments, 26 fragments, and 51 fragments.
- the unit DNA fragment population was prepared as follows.
- each temporary unit DNA fragment population was prepared by adding the average value of the number of fragments to the value obtained by multiplying (fragment average value * CV (%) / 100) by The random number population for each CV (%) values for each of the integrated scale, to prepare a separate 20 population, by the simulation for each random population, was carried out virtual ligation to m min.
- FIG. 19 shows the distribution of the size of the initial unit DNA fragment incorporated into the ligation product.
- (a) shows a graph for 6 fragment accumulation
- (b) shows a graph for 13 fragment accumulation
- (c) shows a graph for 26 fragments
- (d) shows for 51 fragment accumulation. The graph of is shown.
- the patterns of each bar graph in each redundancy (0 ⁇ r ⁇ 1, 1 ⁇ r ⁇ 2, 2 ⁇ r ⁇ 5, 5 ⁇ r ⁇ 10) are represented by N of each redundancy for each pattern.
- the histogram of the number of molecules of the ligation product for each N value showed an index decreasing tendency as the N value increased as a whole, and at first glance showed a distribution close to a geometric distribution which is one type of discrete probability distribution.
- this histogram shows a periodic structure with a period of the number of gene accumulations or one redundancy (6 in the case of 6-fragment accumulation), and in particular, the N value is an integral multiple of the gene accumulation scale. In the part corresponding to, the characteristic structure that no fragment appears was shown. This feature does not completely match the exponential distribution when considered as a geometric distribution or a continuous probability distribution.
- each component distinguished by the remainder obtained by dividing N by the integration scale is linear from the molecular value obtained by logarithmic transformation of about three cycles.
- the slope of the straight line obtained by approximation was determined as - ⁇ , and ⁇ was calculated from this value.
- ⁇ was calculated from this value.
- ⁇ (CV (%)) for each CV (%) was calculated from the reciprocal.
- the ligation products are sampled at various reaction times. A qualitative analysis was performed on the 51 connected portions.
- the average concentration of the unit DNA fragment used for ligation is about 0.2 fmol / ⁇ l.
- T4 DNA ligase After adding T4 DNA ligase to the solution of the unit DNA fragment, 37 ° C., 0, 1.25, 2.5, 5 After 10, 20, 40, 80, 160, and 320 minutes, a part of each reaction solution is sampled, and DNA is designed to be amplified across the ligation part of two unit DNA fragments that are normally ligated.
- a commercially available ⁇ phage genomic DNA (Toyobo) or an already constructed PCR primer set for quantitative PCR and a primer set for quantitative PCR designed so that only the inside of each unit DNA fragment is amplified.
- the integrated plasmid was cleaved with a restriction enzyme, and the degree of progress of ligation of each fragment was examined using a dilution series of linearized DNA as an index. As a result, under these reaction conditions, the ligation was completed in about 10 minutes in any connected part, and the ligation was almost completely completed in the actual reaction time of 4 hours (240 minutes). Was confirmed.
- a ligation simulation was performed. In this simulation, not only m min indicating a reaction rate of 100% but also 100 independent values for m values when ligation was possible at 95%, 96%, 97%, 98%, and 99%.
- a random number group was prepared, and the reaction was continued to each designated m value.
- the obtained 100 ligation product distributions were integrated, and the length of the DNA of each virtual ligation product of each unit DNA fragment was determined in bp using parameters of F (N, L, R).
- the actual molecular weight distribution of the DNA is 0.5 ⁇ TBE, 5 V / cm, 30 sec using a CHEF type pulse field gel electrophoresis apparatus (manufactured by Biocraft). Electrophoresis was performed for 16 hours under periodic electrophoresis conditions. A photograph of the electrophoresis is shown in FIG.
- the DNA density distribution of the obtained electrophoretic photograph was obtained by NIHimage software, and the resulting DNA distribution map for each ligation efficiency obtained by the simulation was compared with the obtained figure for comparison.
- the result is shown in FIG. According to FIG. 24, the molecular weight distribution of the DNA by electrophoresis substantially coincided with the expected DNA distribution diagram in the case of ligation efficiency of 98% -100%, and in particular, the ligation efficiency where the maximum molecular weight showing the maximum concentration is 98%. And a good match.
- the simulation results showed that almost all ligations were completed in a 4-hour ligation reaction, and although approximately 2% misligation could occur, the entire ligation state could be reproduced.
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Abstract
Description
前記各々の溶液の準備後、前記単位DNAに付加配列が連結された状態で、前記各々の溶液中の単位DNAの濃度を測定し、その結果に基づいて、前記各々の溶液を分取して、各々の溶液中の単位DNAのモル数を互いに同一に近づける工程と、を有する単位DNA組成物の調製方法。
前記単位DNAを含む溶液を準備する工程は、前記集積DNAの配列の塩基長を前記単位DNAの種類の数で割った場合に各々の塩基長が等しくなるように、前記集積DNAを等分した位置にある配列近傍の非回文配列を境界として、端部に非回文配列を有する前記単位DNAを設計する工程を含む、(1)から(5)いずれか記載の単位DNA組成物の調製方法。
(1)から(6)いずれかに記載の方法によって、単位DNA組成物を調製する工程と、
前記ベクターDNAを準備する工程と、
調製後の前記溶液中の付加配列が連結された単位DNAから制限酵素を用いて各々の付加配列を除去する工程と、
前記除去工程後、前記ベクターDNA及び前記各々の単位DNAを互いに連結する工程と、を有し、
前記ベクターDNA及び前記各々の単位DNAは互いに順序を保ったまま繰り返し連結し得る構造を有し、
前記集積DNAは、前記各々の単位DNAが互いに連結したDNAからなる、DNA連結体の作製方法。
本発明の単位DNA組成物の調製方法は、付加配列が連結された複数の単位DNAを該単位DNAの種類毎に含む溶液を準備する工程と、各々の溶液の準備後、単位DNAに付加配列が連結された状態で、各々の溶液中の単位DNAの濃度を測定し、その結果に基づいて、各々の溶液を分取して、各々の溶液中の単位DNAのモル数を互いに同一に近づける工程とを有する。本明細書において、「単位DNA」の種類は、それぞれの塩基配列毎に区別する。また、「単位DNA」には、制限酵素認識部位を付加したものと、付加していないもののいずれも含むものとする。
本発明は、DNA連結体の作製方法も包含する。本発明のDNA連結体の作製方法は、上述の方法による、単位DNA組成物を調製する工程と、ベクターDNAを準備する工程と、調製後の溶液中の付加配列が連結された単位DNAから制限酵素を用いて各々の付加配列を除去する工程と、除去工程後、ベクターDNA及び各々の単位DNAを互いに連結する工程と、を有する。
形質転換の対象となる微生物細胞として枯草菌を使用した。枯草菌としては、RM125株(Uozumi, T., et al. Moi. Gen. Genet., 152, 65-69(1977))と、その派生株のBUSY9797株を用いた。枯草菌で複製可能なベクターDNAとして、pGET118(Kaneko, S., et al. Nucleic Acids Res. 31, e112 (2003))を用い、後述のとおりに構築したpGETS118-AarI-pBR(配列番号1を参照)、pGETS151-pBR(配列番号2を参照)を使用した。集積DNAとしては、ラムダファージDNA(東洋紡社製)(配列番号3を参照)と、後述するメバロン酸経路人工オペロン(配列番号4を参照)を用いた。単位DNAを組み込んだプラスミドDNAを有する大腸菌の選択には、抗生物質カルベニシリン(和光純薬工業社)を用いた。枯草菌の選択には、抗生物質テトラサイクリン(シグマ社)を用いた。タイプIIS制限酵素は、AarI(Thermo社)、BbsI(NEB社)、BsmBI(NEB社)、SfiI(NEB社)を用いた。制限酵素HindIII、PvuII、T4 DNA Ligaseは、タカラバイオ社製のものを使用した。大腸菌のプラスミド構築用の一般的なライゲーションには、Takara Ligation Kit (Mighty)(タカラバイオ社)を用いた。単位DNA調製用のPCR反応には、東洋紡社製のKOD plus polymeraseを使用した。プラスミドにクローニングされたDNAの塩基配列決定のためのコロニーPCRには、タカラバイオ社製のEx-Taq HSを用いた。単位DNAを組み込む付加配列であるプラスミドDNAとしては、pMD-19(simple)(タカラバイオ社)を用いた。環状プラスミド精製用酵素Plasmid Safeは、EPICENTER社製のものを使用した。電気泳動用アガロースゲルは、DNA電気泳動用の低融点アガロースゲルである2-Hydroxyethyl agarose(シグマ社)、又はUltraPure Agarose(インビトロジェン社)を使用した。制限酵素の失活には、フェノール:クロロフォルム:イソアミルアルコール 25:24:1と、TE飽和フェノール(8-キノリノール含有)は、ナカライテスク社製のものを使用した。ラムダターミアーゼは、EPICENTER社製のものを使用した。ラムダファージのパッケージングには、アジレント・テクノロジーズ社のGigapack III Plus Packaging Extractを用いた。リゾチームは和光純薬工業社製のものを使用した。LB培地の培地成分及び寒天には、ベクトンディッキントン社製のものを使用した。IPTG(isopropyl s-D-thiogalactopyranoside)は、和光純薬工業社製のものを使用した。上記以外の他の全ての培地成分及び生化学試薬は、和光純薬工業社製のものを使用した。特記以外のプラスミドの構築には、大腸菌DH5α株、JM109株又はTOP10株のいずれかを使用した。構築したプラスミドの大腸菌からの少量精製には、キアゲン社のQIAprep Spin Miniprep Kitを用い、大量精製には、同社のQIAfilter Midi Kitを用いた。酵素反応液からのDNAのクリーンアップには、キアゲン社のMinElute Reaction Cleanup Kit、又はキアゲン社のQIAquick PCR purification Kitを用いた。通常のアガロースゲル電気泳動で分離して得られたゲルブロックを精製する場合は、キアゲン社のMinElute Gel Extraction Kitを用いた。超微量分光光度計は、Thermo社のnano-drop 2000を用いた。塩基配列決定には、アプライドバイオシステムズ社製の蛍光自動シーケンサーの3130xlジェネティックアナライザーを用いた。他の一般的なDNAの操作については、標準プロトコール(Sambrook, J., et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989))にしたがって行った。枯草菌の形質転換とプラスミド抽出は、既法のとおり行った(Tsuge, K., et al., Nucleic Acids Res. 31, e133.(2003))。
ラムダファージDNAの集積に用いたベクターDNAであるpGETS118-AarI-pBR(配列番号1)は、大腸菌のF因子の複製開始点oriSと、枯草菌で機能する複製開始点repAを有する大腸菌―枯草菌間シャトルプラスミドベクターpGETS118(Kaneko, et. al., Nucleic Acids Res., 31,e112. (2003).)を元に多段階の過程を経て構築されたプラスミドであり、図1に示す構造である。集積遺伝子のクローニングサイトは、2つのAarI切断部位間となっており、集積の際に取り除くこの2つのAarI切断部位間には、大腸菌中でのベクターの取得を容易にする目的で大腸菌の多コピープラスミドのpBR322の複製開始点と、アンピシリン耐性遺伝子が導入されている。また、pGETS118内部のテトラサイクリン耐性遺伝子中に存在する天然のAarI切断部位については、テトラサイクリン耐性遺伝子(tetL)のアミノ酸配列に影響を与えないような1塩基の変異を導入することにより認識部位を消失させた。メバロン酸経路人工オペロンの集積に用いたベクターDNAであるpGETS151-pBR(配列番号2)は、上述のpGETS118-AarI-pBRのDNAを鋳型に、3組のプライマーPartA(5’-TAGGGTCTCAaagcggccgcaagctt-3’(配列番号5を参照)と5‘-TAGGGTCTCAGCggccaagaaggcc-3’(配列番号6を参照))、PartB(5’-TAGGGTCTCAccGCCCTTCCCGGTCGATAT-3’(配列番号7を参照)と5’-TAGGGTCTCAtaTTAGCTTAATTGTTATCCGCTCACAATTCC-3’(配列番号8を参照))、PartC(5’-TAGGGTCTCAAAtaactggaaaaaattagtgtctcatggttcg-3’(配列番号9を参照)と5’-TAGGGTCTCAgcttaagtggtgggtagttgacc-3’(配列番号10を参照))により増幅した断片を連結することにより作製したベクターDNAであり、元のプラスミドに比較して大腸菌中でしか機能しない遺伝子領域(cat~oriS間、parA~parC間)を除去している(図1)。このベクターDNAは、遺伝子を集積した際には、枯草菌中でしか複製できないが、遺伝子集積においては、pGETS118-AarI-pBRと同一の性質を示す。これらのプラスミド溶液約10μl(5μg相当)に、滅菌水29μl、制限酵素に付属する10×Buffer_for_AarIを5μlと、同じく制限酵素に付属する切断活性化用の50×Oligoncleotideを1μl、制限酵素AarI(Thermo社)5μlを添加し37℃で2時間反応を行った。得られた液体を、低融点アガロースゲル電気泳動により分離後、ベクター本体の約15kbの断片(pGETS118-AarI-pBRの場合)、又は4.3kbの断片(pGETS151-pBRの場合)をゲルから切り出し、目的のベクターDNAを精製し、20μlのTEに溶解した。ベクターDNAの濃度の測定は、このTE溶液1μlを取り、超微量分光光度計により測定することにより行った。
4塩基の突出の多様性は、4の4乗で、256とおり存在する。これらのうち本発明に使用する突出配列を以下の基準で選定した。まず、パリンドロームとなる全16配列(グループ0)(AATT,ATAT,TATA,TTAA,CCGG,CGCG,GCGC,GGCC,ACGT,AGCT,TCGA,TGCA,CATG,CTAG,GATC,GTAC)は、これらの配列の相補配列も同一配列となり、同一種断片同士の連結が可能となるため、本発明では使用できない為除外した。残りの240配列については、一方の配列(例えば、CCTA)に対して、その相補配列(TAGG)を包含するため、DNA連結に使用可能な突出配列の組み合わせは理論上、240÷2=120組合せである。これらのうち、GC含量の違いとそのGC塩基の出現順序の違いから、以下の基準で突出組合せのグループ化を行った。
(グループII)AとTの合計が3個でCとGの合計が1個となる全32組合せ(CAAA/TTTG,ACAA/TTGT,AACA/TGTT,AAAC/GTTT,GAAA/TTTC,AGAA/TTCT,AAGA/TCTT,AAAG/CTTT,CAAT/ATTG,ACAT/ATGT,AACT/AGTT,AATC/GATT,GAAT/ATTC,AGAT/ATCT,AAGT/ACTT,AATG/CATT,CATA/TATG,ACTA/TAGT,ATCA/TGAT,ATAC/GTAT,GATA/TATC,AGTA/TACT,ATGA/TCAT,ATAG/CTAT,CTTA/TAAG,TCTA/TAGA,TTCA/TGAA,TTAC/GTAA,GTTA/TAAC,TGTA/TACA,TTGA/TCAA,TTAG/CTAA)。
(グループIII)AとTの合計が2つでCとGの合計が2つとなる全52組合せ中パリンドロームの8組合せを除く44組合せ(AACC/GGTT,AACG/CGTT,AAGC/GCTT,AAGG/CCTT,ACAC/GTGT,ACAG/CTGT,ACCA/TGGT,ACCT/AGGT,ACGA/TCGT,ACTC/GAGT,ACTG/CAGT,AGAC/GTCT,AGAG/CTCT,AGCA/TGCT,AGGA/TCCT,AGTC/GACT,AGTG/CACT,ATCC/GGAT,ATCG/CGAT,ATGC/GCAT,ATGG/CCAT,CAAC/GTTG,CAAG/CTTG,CACA/TGTG,CAGA/TCTG,CATC/GATG,CCAA/TTGG,CCTA/TAGG,CGAA/TTCG,CGTA/TACG,CTAC/GTAG,CTCA/TGAG,CTGA/TCAG,CTTC/GAAG,GAAC/GTTC,GACA/TGTC,GAGA/TCTC,GCAA/TTGC,GCTA/TAGC,GGAA/TTCC,GGTA/TACC,GTCA/TGAC,GTGA/TCAC,TCCA/TGGA)。
(グループIV)AとTの合計が1個でCとGの合計が3個となる全32組合せ中、CとGが3連続で並ばない全16組合せ(CACC/GGTG,CCAC/GTGG,CTCC/GGAG,CCTC/GAGG,CACG/CGTG,CCAG/CTGG,CTCG/CGAG,CCTG/CAGG,CAGC/GCTG,CGAC/GTCG,CTGC/GCAG,CGTC/GACG,GAGC/GCTC,GGAC/GTCC,GTGC/GCAC,GGTC/GACC)。
(グループV)AとTの合計が1個でCとGの合計が3個となる全32組合せ中、CとGが3連続する全16組合せ(ACCC/GGGT,CCCA/TGGG,TCCC/GGGA,CCCT/AGGG,ACCG/CGGT,CCGA/TCGG,TCCG/CGGA,CCGT/ACGG,ACGC/GCGT,CGCA/TGCG,TCGC/GCGA,CGCT/AGCG,AGGC/GCCT,GGCA/TGCC,TGGC/GCCA,GGCT/AGCC)。
(グループVI)突出がCとGのみからなる全6組合せ(CCCC/GGGG,GCCC/GGGC,CGCC/GGCG,CCGC/GCGG,CCCG/CGGG,CGGC/GCCG)。
<ラムダファージ>
ラムダファージは、大腸菌に感染するバクテリオファージで、分子生物学的に最も良く研究されているファージである。ゲノムは全長48502bpの二本鎖DNAからなり、全ての塩基配列が明らかになっている。また、様々な変異体の存在も明らかになっている。本実施例では、1kb程度の短い単位DNAからラムダファージ点突然変異体の作製を行うことを試みた。
ラムダファージは、東洋紡社製のλphageDNAを使用した。本製品は、cosサイトで直鎖状になっている。ファージゲノムの全長の塩基配列を調べたところ、データベースに登録されている塩基配列(アクセッションナンバーJ02459.1)に対して6か所(g.138delG,g.14266_14267insG,g.37589C>T,g.37743C>T,g.43082G>A,g.45352G>A)が異なっていた(配列番号3)(配列番号3の全長は、上記48522pbに更にもう一方の突出末端4塩基を含む全長48526bp)。得られた塩基配列を用いて、全長48522bp(cosサイトの重複を含む)をほぼ均等の長さに分割するために、理想分割境界を970bp毎に設定し、上述の(単位DNA分割領域の設定方法)により行った結果、表1に示す切断部位の右側4塩基により構成される5’末端突出が単位DNA集団中でそれぞれ特異的になるように割り振ることができた。
4塩基の任意の突出配列を生成するタイプIIS制限酵素には、AarI(5’-CACCTGC(N)4/-3’,5’-/(N)8GCAGGTG-3’)、BbsI(5’-GAAGAC(N)2/-3’,5’-/(N)6GTCTTC-3’)、BbvI(5’-GCAGC(N)8/-3’,5’-/(N)12GCTGC-3’)、BcoDI(5’-GTCTCN/-3’,5’-/(N)5GAGAC-3’)、BfuAI(5’-ACCTGC(N)4/-3’,5’-/(N)8GCAGGT-3’)、BsaI(5’-GGTCTCN/-3’,5’-/(N)5GAGACC-3’)、BsmAI(BcoDIのイソジマー)、BsmBI(5’-CGTCTCN/-3’,5’-/(N)5GAGACG-3’)、BsmFI(5’-GGGAC(N)10/-3’,5’-/(N)14GTCCC-3’)、BspMI(BfuAIのイソジマー)、BtgZI(5’-GCGATG(N)10/-3’,5’-/(N)14CATCGC-3’)、FokI(5’-GGATG(N)9/-3’5’-/(N)13CATCC-5’)、SfaNI(5’-GCATC(N)9/-3’,5’-/(N)13GATGC-5’)等が挙げられる。これらの制限酵素のうち、遺伝子断片をサブクローニングするために使用する大腸菌プラスミドベクター(pMD19,Simple,TAKARA)に存在しないか、あるいは、存在しても発生する断片の大きさが理想分割単位よりも十分に大きい断片と十分に小さい断片とを発生させる制限酵素を調べたところ、全く切断サイトのないものが、5種類(AarI,BbsI,BfuAI,BsmFI,BtgZI)と、ベクター内部に認識配列が存在するが、理想分割単位よりも十分に大きい断片と十分に小さい断片とを発生させる制限酵素1種類(BsmBI)とが存在し、合計6種類の候補が存在した。これらの候補の制限酵素サイトについて、第01断片から第50断片のラムダファージ全体に対してその制限酵素サイトの分布を調べたところ、AarIは12か所、BbsIは24か所、BfuAIは41か所、BsmFIは38か所、BtgZIは45か所、BsmBIは14か所であり、いずれの制限酵素についても、ラムダファージゲノムに存在しない制限酵素認識部位はなかった。そこで、単位DNA毎に内部を切断しない制限酵素をそれぞれ選択して用いることにした。使用する制限酵素の種類を出来るだけ少なくするために制限酵素の組合せを検討したところ、BbsI、AarI、BsmBIの3種類のみの使用で十分であることを確認した。各単位DNAの切り出しに用いるタイプIIS制限酵素の割り振りは、以下のようになった。
BbsIで切断するグループは、第01~08・12・16~22・24・27・28・33~39・43・45~50断片の合計33断片、AarIで切断するグループは、第09~11・13・23・25.30・32・44断片の合計9断片、BsmBIで切断するグループは、第14・15・26・29・31・40~42断片の合計8断片とした。
第01断片から第50断片までの全50断片については、PCR法を用いてラムダファージゲノム全長から増幅した。まず、上記で決定した突出組合せ間のDNA配列を増幅するためのプライマーの5’末端に上記で決定した制限酵素認識部位を望ましい突出を切り出す位置に付加し、更に5’末端にTAGの配列を付加したプライマーを使用した。これらのプライマー組を用いて、ラムダファージゲノム全長から指定領域のDNA断片を増幅した。PCRの反応条件は、1反応(50μl)につき、KOD Plus10×buffer Ver.25μl、25mM MgSO43μl、dNTP(2mM each)5μl、KOD Plus(1unit/μl)1μl、ラムダファージDNA(TOYOBO)48pg、プライマー(FプライマーとRプライマーのそれぞれ)15pmol、滅菌水を添加して作製し、GeneAmp PCR System 9700(Applied Biosystems社)により、以下のプログラムにより行った。
望ましい配列を有する第01~50の断片をクローンするプラスミドを有する大腸菌形質転換体全50種類をそれぞれ50mlの100μg/mlのカルベニシリン入りLB培地において37℃、120spmで一晩終夜培養し、得られた菌体を、QIAfilter Plasmid Midi Kit(キアゲン社)を用い、精製した。得られた粗プラスミド溶液50μlに、5μlの3M酢酸カリウム-酢酸緩衝液(pH5.2)と、125μlのエタノールを添加して、20000×gで10min遠心し、DNAをエタノール沈殿させ、得られた沈殿を70%エタノールでリンスした後、残渣を取り除き、50μlのTE(pH8.0)に再溶解した。濃度測定のためにこの粗プラスミド溶液1μlをとり、超微量分光光度計(ND-2000、サーモ社)で、DNA濃度を測定した。この時点で粗プラスミド溶液のDNA量は、概ね0.5~4μg/μlであった。測定値を参考に、各粗プラスミド溶液から、5μgのDNAを1.5mlチューブに採取し、これに全体積が50μlになるように滅菌水を添加した。これにPlasmid Safe(エピセンタ社)の10×反応バッファーを6μl、25mM ATP溶液を2.4μl、Plasmid Safe酵素溶液を2μl添加して混合し、プログラマブルブロックインキュベーターであるBI-526T(ASTEC社)により、37℃で1h恒温し、続いて酵素失活のために75℃、30min恒温した。得られた溶液を、PCR purification kit(キアゲン社)により精製した。本キット精製最終段階では、カラムに吸着したDNAをキット付属の溶出バッファーではなく、25μlのTEバッファー(pH8.0)により溶出することで、高純度プラスミド溶液を得た。精製前後の第01断片を有するプラスミドと、第21断片を有するプラスミドとを用いDNA電気泳動を行い(UltraPure Agarose、インビトロジェン社)、目的断片(単位DNA)が組み込まれていることを確認した(図3)。
得られたDNA溶液を再度、超微量分光光度計で測定することで、高純度プラスミド溶液の濃度を求めた。各サンプルの濃度は、理論上の最大値の200ng/μlに対して粗プラスミド溶液の精製度合を反映しておおよそ100ng/μl~200ng/μlの範囲になった。測定結果に基づいて各プラスミド溶液15μlを1.5mlチューブにとり、それぞれの溶液に、それぞれのプラスミドが100ng/μlの濃度になるようにTEを添加し、得られた高純度プラスミド溶液を再度、超微量分光光度計で濃度を測定すると、目標値の100ng/μlに対して数パーセント程度の範囲でズレが存在したので、各高純度プラスミドについて、500ngのDNAの体積量を小数点以下2ケタのμlの精度で計算し、この体積量(約5μl)でそれぞれのDNA溶液を分取し、後の切り出しに用いる制限酵素の種類別(BbsIグループ、AarIグループ、BsmBIグループ)に、それぞれの単位DNAのモル数が略等モルになるように統合した。
統合した等モルプラスミド溶液の合計体積は、BbsIグループが約165μl、AarIグループが約45μl、BsmBIグループが約40μlとなった。各グループに2倍の滅菌水を添加して、それぞれ495、135、120μlの高純度プラスミド溶液を得て、制限酵素の種類毎に以下のように切断した。
確認後、各グループに等量のフェノール・クロロフォルム・イソアミルアルコール(25:24:1)(ナカライテスク社)を添加し、よく混合することで制限酵素を失活した。ここで各グループのフェノール・クロロフォルム・イソアミルアルコール(25:24:1)混合物を1つのチューブに統合したのち、遠心分離(20,000×g、10min)によりフェノール相と水相に分離し、水相(約900μl)を別の1.5mlチューブに回収した。ここに1-ブタノール(和光純薬工業社)を500μl添加し、よく混合し、遠心分離(20,000×g、1min)により分離し、水分を飽和した1-ブタノールを取り除くという操作を水相の体積が450μl以下になるまで繰り返すことで、水相の体積を減少させた。これに、3M酢酸カリウム-酢酸緩衝液(pH5.2)を50μlと、エタノール900μlを添加し、遠心分離(20,000×g、10min)することにより、DNAを沈殿させ、これを70%エタノールでリンスして、20μlのTEに溶解した。これに電気泳動用の10×Dyeを2μl添加し、その全量を0.7%の低融点アガロースゲル(2-Hydroxyethyl Agarose TypeVII,シグマ社)で、1×TAE(Tris-Acetate-EDTA Buffer)バッファー存在下で、汎用アガロースゲル電気泳動装置(i-MyRun.N 核酸用電気泳動システム、コスモバイオ社)で、35V(約2V/cm)の電圧を印加し、4h泳動することにより第01~50断片とプラスミドベクターとを分離した(図5)。この電気泳動ゲルを、1μg/mlの臭化エチジウム(シグマ社)を含む1×TAEバッファー100mlで30min染色し、長波長の紫外線(366mn)で照らすことにより可視化することで、第01~50断片がなすバンド(約1kb付近)をカミソリで切り出し、1.5mlチューブに回収した。回収した低融点アガロースゲル(約300mg程度)に、1×TAEバッファーを添加することにより全体積を約700μlとし、これを65℃で10min恒温することにより、ゲルを溶解した。得られたゲル溶液に、500μlの1-ブタノールを添加し、遠心分離(20,000×g、1min)により水相とブタノール相を分離し、水飽和ブタノールを捨てることを水相の体積が450μl以下になるまで繰り返した。得られた液体に、50μlの3M酢酸カリウム-酢酸緩衝液(pH5.2)と、900μlのエタノールを添加して、遠心分離(20,000×g、1min)によりDNAの沈殿を得て、これを70%エタノールでリンスした後に、20μlのTEに溶解した。そのうちの1μlを取り、超微量分光光度計で濃度を測定した。
第01~50断片の等モル混合物のDNA重量濃度は、98ng/μlであり、合計塩基配列は48,522bpで、一方ベクターDNA(pGETS118-AarI/AarI)は、190ng/μlで全長は15,139bpである。この長さの重量の比率にしたがって、両DNAの等モル混合物を得るために、第01~50断片の等モル混合物6.21μlに対して、ベクターDNAを1.00μlの比率で混合した。得られた等モル混合溶液7.2μlに2×ライゲーションバッファーを8.2μl添加し、全体を37℃で5min間恒温した後、1μlのT4DNAリガーゼ(Takara)を添加して、37℃で4h恒温した。その一部を取って電気泳動することにより、ライゲーションされていることを確認した(図8)。これを8μl新しいチューブに採取し、枯草菌コンピテントセルを100μl添加し、37℃で30minダックローターで回転培養した。その後、300μlのLB培地を添加して、37℃で1hダックローターで回転培養し、その後、培養液を10μg/mlのテトラサイクリン入りLBプレートに広げ、37℃で一晩培養した。コロニーは、250個得られた。
ランダムに12株のコロニーを選択して、2mlの10μg/mlのテトラサイクリン入りLB培地で一晩培養し、内部のプラスミドのコピー数を増幅するためにIPTGを終濃度1mMとなるように添加して更に37℃で3h培養した。得られた菌体からプラスミドを抽出して、制限酵素HindIIIとSfiIによるダブルダイジェスチョンし、電気泳動により確認したところ、12株中4株について望ましい切断パターンを示した(図9)。この4株については、塩化セシウムーエチジウムブロマイド密度勾配超遠心法によりプラスミドを大量調製し、プラスミドの構造を13種類の制限酵素を行った後、電気泳動により確認したところ、いずれも予想される断片と一致した(図10)。更に、本プラスミドのベクター部分を除く全領域について塩基配列決定したところ、4株のプラスミドとも予想塩基配列と完全に一致した。
4株のプラスミドについて、ラムダファージとしての機能を確認するために、プラーク形成能の確認を以下のように行った。まず、♯3、♯4、♯6、♯12の各集積プラスミドをラムダターミアーゼ(Lambda terminase,Epicenter社)により切断することで、ベクターと集積遺伝子部分に分割し、これをラムダパッケージングエキストラクト(Gigapack III Plus Packaging Extract、アジレント・テクノロジー社)に添加した。これを大腸菌(VCS257株)に感染させて、LBプレートに広げ37℃で一晩培養したところ、プラークが確認された。得られたプラークの形状は、並行して行ったTOYOBO社製ラムダファージDNAで得られるものと同様の形態であることが確認された(図11)。各プラスミドから得られたプラークからファージDNAを精製し、制限酵素AvaIで切断することにより導入した変異が存在するかどうかを確認したところ、図12に示すようにTOYOBO社製ラムダファージDNAとは異なる切断パターンを示し、全てのファージに予定どおりAvaIサイトが存在していることを確認した。これにより、第01~50断片の全50断片を集積して作製したラムダファージゲノムが塩基配列とプラーク形成能においてともに完全であることが確認された。
イソプレンの単位を骨格として持つ物質のイソプレノイドには、多くの物質が知られているが、これらはイソペンテニル二リン酸(IPP)という共通の材料から合成される。解糖系からIPPに至る経路には2つの経路、すなわち、メバロン酸経路と非メバロン酸経路とが存在することが知られており、1つの生物の中に両経路を有する生物も存在するが、大腸菌には非メバロン酸経路しか存在しない。大腸菌のIPP生産能を増強する目的で、真核生物である酵母のメバロン酸経路の一部の遺伝子について、大腸菌のコドン使用頻度に適合した人工遺伝子の構築を、合成DNA断片から集積することにより作製することを試みた。
酵母のメバロン酸経路の前半部分となるアセチルCoA~メバロン酸までの代謝経路に必要な3つの遺伝子(ERG10(1.2kb)、ERG13(1.5kb)、HMG1(3.2kb))を大腸菌のコドン使用頻度に基づいてコドンを変換した3つの人工遺伝子を並べた人工オペロン(5,951bp)(配列番号4)(ただし配列番号4の全長は、突出に用いる4塩基の配列を含む5,955pb)の作製を試みた。酵母遺伝子の大腸菌コドンへの変換は、酵母の同義語コドン中で、酵母全遺伝子中の出現頻度に順位を付け、同様に大腸菌の同義語コドン中で大腸菌全遺伝子中の出現頻度の順位を付け、同じ順位の者同士で変換することにより行った。
同義語コドン変換を行った5,951bpのDNA配列について、実施例1と同様にこれを切断しない制限酵素サイトを検索したところ、制限酵素AarIの認識配列がなく、AarIでは切断されないことが判明したので、全てのクローンはAarIを利用して調製することにした。全長5,951bpを55個の断片に分割すると、平均108bpの断片となるので、この大きさを理想分割単位とし、この分割単位近傍で特定の配列(AとTの合計が2つでCとGの合計が2つとなる全52組合せ中パリンドロームの8組合せを除く44組合せ(前述のグループIII)と、AとTの合計が1個でCとGの合計が3個となる全32組合せ中、CとGが3連続しない全16組合せ(前述のグループIV)との合計60種類)のうち全ての理想分割単位でいずれか1種類の配列が出現するかどうかを調べたところ、理想分割単位から±7bpの範囲で何れか一種の特定配列が出現することが判明した。これを元に全長を55個の98~115bpの断片に分割した。表2は、実施例2における集積DNAの分割単位及び突出塩基配列を示す表である。なお、メバロン酸遺伝子群と遺伝子集積ベクターの境界については、AとTからのみなる突出(ATTAとAAAA)を利用した。
分割して得られた各断片は、Rossiらの方法(Rossi, J.J., and Itakura, K. 1982. J. Biol. Chem. 257, 9226-9229(1982))により、80塩基の化学合成DNA2本により作製した。具体的には、2つの化学合成DNAが3’末端で数10bpがハイブリダイズするようにし、5’末端には、AarI切断部位よりも5’末端側に上記で設計した突出がAarI消化により出現するように認識部位を付加した。これらの2つの合成DNAのハイブリダイゼーションと続く鋳型依存的伸長反応の結果得られる二本鎖の単位DNAをPCR法により増幅するために、両末端のAarI認識部位にハイブリダイズするように作製したPCRプライマー1種類の計3種類のDNAを添加して、PCRの反応によりAarIの切断サイトに囲まれた単位DNAを得て、これをTAクローニング法により、大腸菌プラスミドベクターpMD19に連結し、大腸菌に形質転換することによりクローニングした。これをシーケンシングすることにより各断片について塩基配列の望ましいクローンを選択した。
得られた望ましいクローンを持つ大腸菌55株を培養し、Plasmid mini-prep(QIAGEN社)によりそれぞれから粗プラスミド溶液50μlを得た。これから各1μlを取り超微量分光光度計によりDNA濃度を測定したところ、82~180ng/μlであった。概ね各5μgのプラスミドを取りPlasmid Safeにより処理し、酵素の熱失活後、Mini-elute PCR purification Kit(QIAGEN社)により精製して、25μlの高純度プラスミド溶液を得た。このうちの1μlを超微量分光光度計により濃度を測定したところ、108~213ng/μlの濃度であった。ここから各々20μlの高純度プラスミド溶液を別のチューブに取り、このチューブに各プラスミドの濃度が計算上100ng/μlになるようにTEを添加して希釈した。この精製プラスミド溶液を再度微量分光光度計で濃度を算出し、この濃度に基づいて各高純度プラスミドの重量が500ngになる体積量を小数点以下2ケタのμlの精度で算出し、その体積量(約5μl)をそれぞれのプラスミド溶液から分取し、を一本のチューブにプールした。合計で約275μlの等モルプラスミド混合溶液に2倍溶の滅菌水と137.5μlの10×Buffer_for_AarIと、67.5μlの制限酵素AarIを添加して、37℃で一晩反応させた。
この反応液に等量のフェノール・クロロフォルム・イソアミルアルコール(25:24:1)を添加してAarIを失活させた後、遠心し、その上清をエタノール沈殿により精製して、沈殿を20μlのTEに溶解した。これに電気泳動用の色素としてキシレンシアノールを添加して、2.5%アガロースゲルでTAEをバッファーとして100Vで、30分間電気泳動することにより、ベクターDNAのpMD19とインサートの単位DNAとを分離した(図13)。泳動したゲルをカミソリで分割し、その一部をエチジウムブロマイドで染色し、目的の55個の等モル混合断片のバンドの位置を確認しながら、無染色のゲルから目的のDNAのバンドを切り出した。
得られたゲル断片からDNAの精製は、MiniElute Gel Extraction Kit(QIAGEN社)を用いて以下に示すように行った。
このDNA濃度を超微量分光光度計により濃度測定を行ったところ、20ng/μlであった。並行して調製したpGET151/AarIの濃度は、67ng/μlであったので、これらの長さの比率(5955bp:4306bp)を考慮に入れて4.63:1の比率になるように55断片等モル混合溶液とpGETS151/AarIとを混合した。
得られた等モル混合溶液5.63μlに2×ライゲーションバッファーを6.63μl添加し、全体を37℃、5min間恒温した後、1μlのT4DNAリガーゼ(Takara)を添加して、37℃で4h恒温した。一部をとって電気泳動することにより、単位DNAとベクターDNAがタンデムリピート状にライゲーションされているか否かを確認した(図14)。ライゲーション後の溶液8μlを別のチューブに採取し、これに枯草菌コンピテントセルを100μl添加し、37℃で30minダックローターで回転培養した。その後、300μlのLB培地を添加して、37℃で1hダックローターで回転培養し、その後、培養液を10μg/mlのテトラサイクリン入りLBプレートに広げた。
得られたコロニー154個から、ランダムに24個のクローンを選択し、10μg/mlのテトラサイクリン入りLBに植菌した。対数増殖期に終濃度1mMとなるようにIPTGを添加して、定常期まで培養後、プラスミドDNAを抽出して制限酵素PvuIIで処理し、電気泳動により切断パターンを調べた(図15)。その結果、2クローン(♯10と#20)について予想される塩基配列と一致することが確認されたので、このプラスミドについて他の制限酵素処理を行い、電気泳動により詳細な構造確認を行ったところ、目的の構造と一致した(図16)。このプラスミドについてシーケンシングすることで、最終的にクローン10と20は、設計とおりの塩基配列を有していたことを確認した。
枯草菌プラスミド形質転換において必要とされる、集積DNAユニットの数の繰り返し数(リダンダンシー)rを確認するために、以下の試験を行った。
(A)のDNAは、リダンダンシーr=1の環状モノマープラスミドDNAである。まず、pGETS118-t0-Pr-SfiI-pBRを大腸菌に形質転換した。この形質転換体から得られるプラスミドは、(A)のDNAが主ではあるが、若干量の多量体(マルチマー)が含まれるので、これらを除去する目的で、このプラスミドを低融点アガロースゲル電気泳動によるDNAサイズ分画により、モノマープラスミドDNAの領域のみゲルより切り出し、精製することによって、(A)のDNAを調製した。
(B)のDNAは、リダンダンシーr=1の直線上のモノマープラスミドDNAである。この(B)のDNAは、上記(A)のDNAを制限酵素BlpI(認識部位は(5’-GC/TNAGC-3‘))により処理することで、調製した。
(C)のDNAは、リダンダンシーr>1のタンデムリピートの直線状マルチマープラスミドDNAである。上記(B)のDNAを調製した際に使用したBlpIは、5‘末端に非パリンドロームの3塩基突出を形成する。よって、上記(B)のDNAを、DNAリガーゼにより連結することによって、プラスミド単位が同一方向に連続した直線状マルチマープラスミドDNAである(C)のDNAを調製した。
(D)のDNAは、リダンダンシーr=1の直線上のモノマープラスミドDNAである。この(D)のDNAは、上記(A)のDNAを制限酵素EcoRI(認識部位は(5’-G/AATTC-3‘である))により処理することで、調製した。
(E)のDNAは、部分的にリダンダンシーr>1の部分を含む、上記(D)のDNAがランダムな向きで連結した直線状マルチマープラスミドDNAである。上記(D)のDNAを調製した際に使用したEcoRIは、5’末端にパリンドロームの3塩基突出を形成する。EcoRIで切断したプラスミドDNAを連結すると、プラスミド単位が、ランダムな向きで連結したマルチマープラスミドDNAを作製することが可能となる。(E)のDNAは、上記(D)のDNAを、DNAリガーゼにより連結することによって調製した。
(F)のDNAは、(A)のDNAを1か所のみ切断する制限酵素KasIで切断し、脱リン酸化した後にその近傍のBlpIで切断したDNA断片と、(A)のDNAを1か所でのみ切断する制限酵素AfeIで切断し、脱リン酸化した後にその近傍のBlpIで切断したDNAとを等量混合した、r≒1直鎖状準モノマー混合物である。この(F)の混合物中のいずれのDNA断片もリダンダンシーrは、1を若干下回っている。
(G)のDNAは、上記(F)の2つのDNA断片をDNAリガーゼにより連結することによって、BlpIサイトのみで方向性を指定した連結されたリダンダンシーr=1.98の直線上の準ダイマープラスミドDNAである。
上記(B)と(D)は、互いに切断サイトが離れている。(H)のDNAは、これら(B)と(D)のDNAを等モルとなるように、連結せずに混合した混合物である。
上記(A)~(H)のDNAを、枯草菌コンピテントセルに形質転換して、得られたテトラサイクリン耐性株の出現数を指標に1μg当たりの形質転換体を求めた。(A)~(H)のDNAはライゲーション反応の有無にかかわらず、ライゲーションバッファー中に溶解し、形質転換に用いた。(A)~(H)のDNAの電気泳動の写真を図17に示し、(A)~(H)のDNAの枯草菌コンピテントセルの形質転換体出現数を、図18に示す。なお、図17の電気泳動写真において、(C)と(E)のDNAは、様々なサイズのものがレーン上の広い範囲で分布しているため、バンドが判別しにくくなっている。また、図17の「G」において、上のバンドが、リダンダンシーr=1.97の(G)のDNAであり、下のバンドが、(G)のDNAに混ざっていたリダンダンシーr=0.95のDNAである。
<ライゲーションシミュレーションアルゴリズムの設定>
シミュレーションプログラミングは、表計算ソフトExcel(登録商標)2007のVBAを用いて行った。仮想ライゲーション中のDNA断片Fは、3つのパラメーターFi(Ni,Li,Ri)を用いて表現した。ここで、「i」は断片識別番号を意味し、より具体的には、Excel上のi列セルを意味する。「N」は、仮想ライゲーション中のライゲーションDNA断片1分子中に含まれる単位DNA断片数を示し、「L」はライゲーション産物の左側の突出末端の配列を任意の自然数で数値化したものを示し、「R」は、「L」と同じくライゲーション産物の右側の突出末端の配列を任意の自然数で数値化したものを示す。ここで、L=Rである場合、2つの突出配列は相補関係にあり、L=Rは、ライゲーション可能であることを定義する。ライゲーションのシミュレーションは以下のように行った。
mmin=(総単位DNA断片数)―(L=Rを満たす関係にある2種類の単位DNA断片のうち少ない単位DNAの断片数の、系全体における合計数)
6断片、13断片、26断片、51断片集積までの各集積規模において、同一単位DNA断片数の平均640で、0~20%の範囲で1%刻みに設定した変動係数(CV)を持つ仮想単位DNA断片集団は以下のように作製した。
上記のライゲーションシミュレーションの数値解析から、ライゲーションのメカニズムについて一般化された式を求めるにあたって、各集積規模の各CV値におけるライゲーション産物の分布のフィッティングカーブを算出可能か否かを検証した。6断片集積平均640断片の場合についての、CV=20%におけるライゲーション産物の単位DNA断片含有数の分布を図20に示す。図20において、各リダンダンシー(0<r<1、1<r<2、2<r<5、5<r<10)中の各棒グラフの模様は、各模様毎にそれぞれのリダンシーのNをrで割った際に区別される成分(6断片集積の場合、余りが0、1、2、3、4、5の6成分のうち、N値が0のため対数変換が出来ない余り0の成分を除いた5成分)の種類を示し、異なるリダンダンシーにおいて、同一の模様である棒グラフは、Nをrで割った際に区別される成分が同一の種類であることを示す。また、図20のグラフ中の線形近似曲線は、それぞれの模様の成分の種類毎に求められたものである。
上記のライゲーションシミュレーションは、正規な突出組合せ同士のライゲーションが全て完了した状態を想定したものである。これに対し、実際に遺伝集積する際のライゲーション反応条件がどの程度シミュレーションの反応条件に近いのかを検討するために、ライゲーション反応のキネティックスを調べた。
より詳細にライゲーションの状況を確認するために、上記のλファージゲノム再構成の実験において得られた集積体のうち、全塩基配列を完全決定した#3、#4、#6、#12を除く全てのクローンについて(#1、#2、#5、#7、#8、#9、#10、#11)、誤った組み合わせでライゲーションされている箇所の同定を目的として塩基配列決定を行った。それぞれのクローンについてのミスライゲーションサイトを、図22に示す。この結果から、#11クローンを除く7クローンについては、その内部に1個もしくは2個のミスライゲーションが存在していることが確認され、全てのミスライゲーションを同定できた。一方、#11クローンについては、集積DNA内に同じ単位DNA断片が繰り返し存在していることが確認され、完全な構造確認を行うことはできなかったが、全6か所のミスライゲーションサイトが存在していることが確認された。#11は、詳細な単位DNA断片数が不明であるため、#11は除外して、全てのクローンのミスライゲーション出現頻度を算出した結果、連結点46箇所に1箇所程度の割合でミスライゲーションが存在していることが確認され、2.2%程度という比較的低い割合でミスライゲーションが出現することが確認された。この結果は、上記の定量PCRの結果と矛盾しない結果であった。
上記ミスライゲーション割合の推定と、ライゲーションの反応速度の定性的解析の2つの検証によって、実際のライゲーション反応においては、4時間という十分な時間が経過した後では、略全てのライゲーションが完了しており、またミスライゲーションが生じる確率は少ないと推定された。そこで、初発の単位DNA断片に濃度のバラつきが存在する実際の単位DNA断片集団について、シミュレーションによって実際のライゲーション産物の大きさ分布が予測可能か否かを以下の方法で検証した。
実際に集積に用いる単位DNA断片は、濃度のバラつきが避けられないが、実際にはどの程度の単位DNA断片の濃度のバラつきに抑えなければならないのかを、上記で求められた一般式f(N)=0.0058*CV(%)*exp(-0.0058*CV(%)*N)を用いて総括した図を、図25に示す。現在の遺伝子集積実験では、概ねDNA濃度のバラつきがCV(%)=6.6程度であるが、図25によると、CV(%)=6.6の場合、51断片の集積において、用いた単位DNA断片の約40%が、r値が1より大きいライゲーション産物に取り込まれていることが示される。また、図25によると、仮に、これの2倍の集積規模の102断片集団による新規な遺伝子集積を計画する場合、51断片と同程度の集積効率を期待するなら、CV(%)=3.3を実現する必要があることが示された。また、一般式f(N)=0.0058*CV(%)*exp(-0.0058*CV(%)*N)を用いて、単位DNA断片の濃度のバラつきと1ライゲーション産物の平均単位DNA断片数との関係を求めた。その結果を図26に示す。これにより、一般式f(N)=0.0058*CV(%)*exp(-0.0058*CV(%)*N)を用いることで、CV(%)値による、1ライゲーション産物中の平均単位DNA断片含有数を容易に推測することが可能となることが示された。
Claims (12)
- 付加配列が連結された複数の単位DNAを該単位DNAの種類毎に含む溶液を準備する工程と、
前記各々の溶液の準備後、前記単位DNAに付加配列が連結された状態で、前記各々の溶液中の単位DNAの濃度を測定し、その結果に基づいて、前記各々の溶液を分取して、各々の溶液中の単位DNAのモル数を互いに同一に近づける工程と、を有する単位DNA組成物の調製方法。 - 前記付加配列が連結された単位DNAが環状構造を有し、前記付加配列が複製開始点を有するプラスミドDNA配列である請求項1記載の単位DNA組成物の調製方法。
- 前記単位DNAの各々の塩基長と、各々の単位DNAに連結された付加配列の塩基長との合計長さの分布の標準偏差が、平均の前記合計長さに対して±20%以内である請求項1又は2記載の単位DNA組成物の調製方法。
- 前記各々の単位DNAに連結された付加配列の平均塩基長が、前記単位DNAの平均塩基長の2倍以上である、請求項1から3いずれか記載の単位DNA組成物の調製方法。
- 前記単位DNAの各々の長さが1600bp以下である請求項1から4いずれか記載の単位DNA組成物の調製方法。
- 前記単位DNAは、該単位DNAによって構成される集積DNAを含むDNA連結体を作製するために用いられるものであり、
前記単位DNAを含む溶液を準備する工程は、前記集積DNAの配列の塩基長を前記単位DNAの種類の数で割った場合に各々の塩基長が等しくなるように、前記集積DNAを等分した位置にある配列近傍の非回文配列を境界として、端部に非回文配列を有する前記単位DNAを設計する工程を含む、請求項1から5いずれか記載の単位DNA組成物の調製方法。 - 宿主微生物中で有効な複製開始点を含むベクターDNAと集積DNAとからなる集積DNAユニットを、1を超えて含む微生物細胞形質転換用DNA連結体の作製方法であって、
請求項1から6いずれかに記載の方法によって、単位DNA組成物を調製する工程と、
前記ベクターDNAを準備する工程と、
調製後の前記溶液中の付加配列が連結された単位DNAから制限酵素を用いて各々の付加配列を除去する工程と、
前記除去工程後、前記ベクターDNA及び前記各々の単位DNAを互いに連結する工程と、を有し、
前記ベクターDNA及び前記各々の単位DNAは互いに順序を保ったまま繰り返し連結し得る構造を有し、
前記集積DNAは、前記各々の単位DNAが互いに連結したDNAからなる、DNA連結体の作製方法。 - 集積ユニットを構成する単位DNAの数と集積ユニットの数との積で表される標的連結数のDNA断片の収率と、該DNA断片の濃度の変動係数との関係式に基づいて、前記連結工程における前記ベクターDNA及び前記各々の単位DNAの濃度の変動係数を調節する工程を含む、請求項7記載のDNA連結体の作製方法。
- 前記制限酵素は、タイプII制限酵素である請求項7又は8記載のDNA連結体の作製方法。
- 前記除去工程の前に、調製後の単位DNAを含む溶液のうち、2種以上の単位DNAを含む溶液を混合する工程を更に有する、請求項7から9いずれかに記載のDNA連結体の作製方法。
- 前記除去工程後、前記連結工程の前に、前記制限酵素を失活させる工程を更に有する、請求項7から10いずれかに記載のDNA連結体の作製方法。
- 前記微生物が枯草菌である請求項7から11いずれか記載のDNA連結体の作製方法。
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WO2021241584A1 (ja) * | 2020-05-26 | 2021-12-02 | Spiber株式会社 | I型ポリケチドシンターゼ遺伝子を含むプラスミドの調製方法 |
WO2022097646A1 (ja) | 2020-11-04 | 2022-05-12 | 株式会社シンプロジェン | 枯草菌におけるウイルスベクタープラスミド生産 |
WO2022097647A1 (ja) | 2020-11-04 | 2022-05-12 | 株式会社シンプロジェン | 統合型プラスミド |
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CN109817277B (zh) * | 2018-12-29 | 2022-03-18 | 北京百迈客生物科技有限公司 | 基于PacBio全长转录组测序数据的质控方法 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2004129654A (ja) * | 2002-09-19 | 2004-04-30 | Mitsubishi Chemicals Corp | 挿入dnaユニットを含むプラスミドの製造方法 |
JP2006503583A (ja) * | 2002-10-24 | 2006-02-02 | バイオジェン・アイデック・エムエイ・インコーポレイテッド | フェリチン重鎖遺伝子座に基づいた高発現遺伝子座ベクター |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1297172B1 (en) * | 2000-06-28 | 2005-11-09 | Glycofi, Inc. | Methods for producing modified glycoproteins |
CA2418317A1 (en) * | 2000-08-11 | 2002-02-21 | Genencor International, Inc. | Bacillus transformation, transformants and mutant libraries |
BR0115245A (pt) * | 2000-10-30 | 2005-05-10 | Pharmacia Corp | 11 alfa-hidroxilase e óxido redutase de aspergillus ochraceus |
DK2175018T3 (da) * | 2008-10-08 | 2011-08-22 | Icon Genetics Gmbh | Fremgangsmåde til ren kloning |
US10017794B2 (en) * | 2012-01-09 | 2018-07-10 | The Research Foundation For The State University Of New York | Engineered strain of Escherichia coli for production of poly-R-3-hydroxyalkanoate polymers with defined monomer unit composition and methods based thereon |
CN106170547B (zh) * | 2014-01-21 | 2020-03-27 | 株式会社辛普罗根 | 单元dna组合物的制备方法及dna连结体的制作方法 |
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2004129654A (ja) * | 2002-09-19 | 2004-04-30 | Mitsubishi Chemicals Corp | 挿入dnaユニットを含むプラスミドの製造方法 |
JP4479199B2 (ja) | 2002-09-19 | 2010-06-09 | 三菱化学株式会社 | 挿入dnaユニットを含むプラスミドの製造方法 |
JP2006503583A (ja) * | 2002-10-24 | 2006-02-02 | バイオジェン・アイデック・エムエイ・インコーポレイテッド | フェリチン重鎖遺伝子座に基づいた高発現遺伝子座ベクター |
Non-Patent Citations (19)
Title |
---|
ANAGNOSTOPOULOU, C.; SPIZIZEN, J., J. BACTERIOL., vol. 81, 1961, pages 741 - 746 |
HIROMICHI SAWAKI ET AL.: "Development of quantitative PCR array for studying human glycogens expression profiling", JAPAN JOURNAL OF MOLECULAR TUMOR MARKER RESEARCH, vol. 24, 2009, pages 29, XP055359025 * |
IMANAKA, T. ET AL., J. GEN. MICROBIOI., vol. 130, 1984, pages 1399 - 1408 |
ITAYA, M. ET AL.: "Construction and Manipulation of Giant DNA by a Genome Vector", METHODS IN ENZYMOLOGY, vol. 498, 2011, pages 427 - 447, XP055355825 * |
ITAYA, M., BIOSCI. BIOTECHNOL. BIOCHEM., vol. 63, 1999, pages 602 - 604 |
KANEKO, NUCLEIC ACIDS RES., vol. 31, 2003, pages ELL2 |
KANEKO, S. ET AL., NUCLEIC ACIDS RES., vol. 31, 2003, pages ELL2 |
KENJI TSUGE ET AL.: "Genome Builder Oyobi Genome Designer to shite no Kosokin", JOURNAL OF THE SOCIETY FOR BIOSCIENCE AND BIOENGINEERING, vol. 90, no. 6, 2012, JAPAN, pages 281 - 284, XP008183902 * |
MITSUHIRO ITAYA ET AL.: "Chosa DNA no Gosei to Gosei Seibutsu Kogaku deno Katsuyo", JOURNAL OF THE SOCIETY FOR BIOSCIENCE AND BIOENGINEERING, vol. 91, no. 6, 2013, JAPAN, pages 319 - 321, XP008183906 * |
ROSSI, J. J.; ITAKURA, K., J. BIOL. CHEM., vol. 257, 1982, pages 9226 - 9229 |
SAMBROOK, J. ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS |
SWINFIELD, T. J. ET AL., GENE, vol. 87, 1990, pages 79 - 90 |
TAKAAKI TAMURA: "4 DNA no Kenshutsu, Kaitei Idenshi Kogaku Jikken Note 1st volume, DNA o Eru", TORIATSUKAI NO KIHON TO CHUSHUTSU SEISEI BUNRI, 2006, pages 28 - 29, XP008183907 * |
TANAKA, T; OGRA, M., FEBS LETT., vol. 422, 1998, pages 243 - 246 |
TSUGE, K. ET AL., NUCLEIC ACIDS RES., vol. 31, 2003, pages E133 |
TSUGE, K. ET AL.: "Production of the non- ribosomal peptide plipastatin in Bacillus subtilis regulated by three relevant gene blocks assembled in a single movable DNA segment", JOURNAL OF BIOTECHNOLOGY, vol. 129, 2007, pages 592 - 603, XP022034270 * |
UOZUMI, T. ET AL., MOI. GEN. GENET., vol. 152, 1977, pages 65 - 69 |
YANSURA, D.; HENNER, D., J. PRO. NATL. ACAD. SCI, USA, vol. 81, 1984, pages 439 - 443 |
YASUKO NISHINO: "Researcher Interview No.3 Koshi Kenji TSUGE", KEIO IAB RESEARCH DIGEST, vol. 2, 2009, pages 8 - 11, XP008183908 * |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPWO2020203496A1 (ja) * | 2019-04-01 | 2020-10-08 | ||
WO2020203496A1 (ja) * | 2019-04-01 | 2020-10-08 | 国立大学法人神戸大学 | キメラプラスミドライブラリーの構築方法 |
JP7101431B2 (ja) | 2019-04-01 | 2022-07-15 | 国立大学法人神戸大学 | キメラプラスミドライブラリーの構築方法 |
US11643648B2 (en) | 2019-04-01 | 2023-05-09 | National University Corporation Kobe University | Method for constructing chimeric plasmid library |
WO2021241593A1 (ja) * | 2020-05-26 | 2021-12-02 | Spiber株式会社 | マルチモジュール型生合成酵素遺伝子のコンビナトリアルライブラリーの調製方法 |
WO2021241584A1 (ja) * | 2020-05-26 | 2021-12-02 | Spiber株式会社 | I型ポリケチドシンターゼ遺伝子を含むプラスミドの調製方法 |
WO2022097646A1 (ja) | 2020-11-04 | 2022-05-12 | 株式会社シンプロジェン | 枯草菌におけるウイルスベクタープラスミド生産 |
WO2022097647A1 (ja) | 2020-11-04 | 2022-05-12 | 株式会社シンプロジェン | 統合型プラスミド |
JPWO2022250068A1 (ja) * | 2021-05-25 | 2022-12-01 | ||
WO2022250068A1 (ja) * | 2021-05-25 | 2022-12-01 | Spiber株式会社 | プラスミドの製造方法及びプラスミド |
JP7370121B2 (ja) | 2021-05-25 | 2023-10-27 | Spiber株式会社 | プラスミドの製造方法及びプラスミド |
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