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Complex in vivo Ligation Using Homologous Recombination and High-efficiency Plasmid Rescue from Saccharomyces cerevisiae
通过同源重组进行复合体的体内连接及在酿酒酵母中进行高效质粒拯救   

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Abstract

The protocols presented here allow for the facile generation of a wide variety of complex multipart DNA constructs (tagged gene products, gene fusions, chimeric proteins, and other variants) using homologous recombination and in vivo ligation in budding yeast (Saccharomyces cerevisiae). This method is straightforward, efficient and cost-effective, and can be used both for vector creation and for subsequent one-step, high frequency integration into a chromosomal locus in yeast. The procedure utilizes PCR with extended oligonucleotide “tails” of homology between multiple fragments to allow for reassembly in yeast in a single transformation followed by a method for highly efficient plasmid extraction from yeast (for transformation into bacteria). The latter is an improvement on existing methods of yeast plasmid extraction, which, historically, has been a limiting step in recovery of desired constructs. We describe the utility and convenience of our techniques, and provide several examples.

[Introduction] Homologous recombination (HR) in S. cerevisiae has long been recognized as an extremely convenient method for assembling DNA fragments in vivo (Szostak et al., 1983; Ma et al., 1987; Oldenburg et al., 1997). Given the efficiency of HR in yeast, it has been exploited in ways that have both increased its utility, enhanced its versatility, and permitted its application to a broad range of experimental objectives. Improvements to this general approach include using in vivo ligation as a platform for directed mutagenesis (Muhlrad et al., 1992), introducing counterselection to aid in plasmid creation (Gunyuzlu et al., 2001; Anderson and Haj-Ahmad, 2003), and adapting in vivo assembly to vectors that cannot be propagated in yeast (Iizasa and Nagano, 2006; Joska et al., 2014). However, previous studies have not fully harnessed the power and utility of HR. We have found that HR in vivo allows for very efficient assembly and recovery of plasmids containing numerous (>5 separate pieces) fragments of DNA in a single transformation step. Thereby, we have been readily able to construct a wide variety of gene disruption cassettes and integration cassettes, to clone exceptionally large genes, to assemble multi-part chimeric genes, and to generate multiply-tagged gene products. The primary utility and power of our methods are the capability to correctly and efficiently assemble multiple DNA fragments transformed into yeast in a single step. Our approach (Figure 1A) has been used successfully: (i) to assemble in-frame chimeras between two or more different genes; (ii) to fuse gene products to fluorescent probes and/or epitope tags at either their N- and/or C-termini, or both; (iii) to create gene deletion cassettes with large amounts of untranslated flanking sequence; (iv) to introduce one or more short linker sequences or epitope tag(s) between assembled genes or gene fragments; (v) to utilize a variety of transcriptional promoters and terminators; and, importantly, (vi) in one step, to generate constructs marked with a drug resistance gene cassette or a selectable nutritional gene cassette that integrate into the genome at the desired locus. Because HR in the yeast cell carries out the in vivo construction process (and subsequent integration, if desired), no kit or proprietary system is required and the assembly of collections of plasmids can be done in a massively parallel manner. In this regard, our system is considerably less expensive than the in vitro enzyme-driven “Gibson cloning” (Gibson, 2011) procedure, yet still remarkably efficient. Also, our system (unlike those requiring restriction enzyme digests to insert gene fragments) does not result in the insertion (or loss) of any nucleotides, which can sometimes occur in classical restriction site cloning. In our method, precise control over both the coding sequence and the flanking untranslated regions (UTRs) can be achieved. Lastly, constructs generated using this system can be coupled with the haploid yeast genome deletion collection (Winzeler et al., 1999; Giaever et al., 2002) to allow for simple and efficient one-step integration at any designed locus. The only rate-limiting factor in our method is the need for the transformed yeast cells to grow for a few days before the construct (or genetically altered cell) can be recovered.

Although similar overall methods may exist (Andersen, 2011), in our protocol, we developed several important improvements, which greatly enhance efficient recovery of the DNA constructs from yeast cells, including: (i) a specific yeast genotype that is much easier to lyse than standard laboratory strains, such as S288C (and its derivatives, e.g. BY4741); (ii) a spheroplasting step (to destroy the yeast cell wall); (iii) glass bead beating for better nucleic acid extraction; and, (iv) bacteria chemically treated for ultra-efficient DNA transformation.

Keywords: Homologous recombination(同源重组), Plasmid assembly(质粒组装), In vivo ligation(体内结扎), Yeast(酵母), Plasmid rescue(质粒拯救)

Materials and Reagents

  1. Yeast strains: SF838-1Dα (MATα ura3-52 leu2-3,122 his4-519 ade6 pep4-3 gal2; Rothman and Stevens, 1986) and THS4218 (SF838-1Dα; HIS4 his3Δ::HygR)
  2. KOD Hot Start DNA Polymerase (EMD Millipore, distributed by VWR International, catalog number: 80511-384 )
  3. DNA oligonucleotide primers (Integrated DNA Technologies, Inc.)
  4. DpnI restriction enzyme (New England Biolabs, catalog number: R0176S )
  5. Appropriate restriction enzyme(s) for digestion (New England Biolabs)
  6. PEG: Poly (ethylene glycol), BioXtra avg. molecular weight 3,350 (Sigma-Aldrich, catalog number: P4388-1KG )
  7. Lithium acetate dihydrate (Sigma-Aldrich, catalog number: L6883-1KG )
  8. ssDNA: Deoxyribonucleic acid sodium salt from salmon testes (Sigma-Aldrich, catalog number: D1626-1G )
  9. GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific, catalog number: K0503 )
  10. Zymolyase® 100T from Arthrobacter leuteus (Amsbio LLC, catalog number: 120493-1 )
  11. One Shot® TOP10 chemically competent E. coli (Life Technologies, InvitrogenTM, catalog number: C4040-03 )
    Note: These serve as the seed cultures for further chemically competent procedure using CCMB80 buffer. Our competent cells are prepared by inoculating a 1 L culture of SOB medium (Hanahan et al., 1991) with One Shot® TOP10 cells and growing them to A600 nm~0.3 at 23 °C. After harvesting, the TOP10 cells were made chemically competent by treating them, as described (Hanahan et al., 1991), with “CCMB80 buffer” (10 mM KOAc, pH 7.0, 80 mM CaCl2.2H2O, 20 mM MnCl2.4H2O, 10 mM MgCl2.6H2O, 10% glycerol), which was adjusted to pH 6.4 with 0.1 N HCl (if necessary), filter sterilized, and stored at 4 °C. After treatment, the competent cells were stored in aliquots at -80 °C. (Different ultra-chemically competent E. coli strains may be used in place of TOP10 for this procedure).
  12. Ampicillin (final concentration of 100 μg/ml; Research Products International Corp., catalog number: A40040-100.0 ) and Kanamycin (final concentration of 50 μg/ml; Life Technologies, catalog number: 11815-024 )
  13. 1 M sorbitol, 0.1 M Na2EDTA (see Recipes)
  14. YPD liquid media (see Recipes)
  15. SOB medium (see Recipes)
  16. SOC medium (see Recipes)
  17. LB plates (with appropriate drug included) (see Recipes)

Equipment

  1. 0.5 mm glass beads (BioSpec Products, catalog number: 11079105 )
  2. Centrifuge (Eppendorf microcentrifuge model: 5415D , catalog number: 022621408 ; Eppendorf rotor, model: F-45-24-11 for 24 x 1.5/2 ml, catalog number: 022636502 )
  3. Petri dish (100 x 15 mm size; VWR International, catalog number: 25384-088 )
  4. Tube (Axygen Microtubes 1.5 ml clear, homo-polymer, boil-proof, catalog number: MCT-150-C )
  5. Vortexing adaptor (Microtube foam insert for Fisher Vortex Genie 2 mixer, Scientific Industries, Inc.; catalog number: 504-0234-00 )
  6. PCR machine (MJ Research PTC-200 Peltier Thermo Cycler, dual 30-well alpha blocks)

Procedure

  1. To begin, oligonucleotides are designed that allow for the amplification of each of the necessary gene fragment(s), epitope or fluorescent protein tags, nutritional markers, and/or drug-resistance cassettes and their recombinational assembly in the desired order (Figure 1B). The latter objective is achieved by "tails" of homology [(typically 30 nucleotides (Baudin et al., 1993)] on each primer that permit the joining of each amplified fragment to its neighbors by HR. Overlap sequences of 30 nucleotides in the 5'-tail on the N-terminal-most fragment and in the 3'-tail of the C-terminal-most fragment with the corresponding flanking sequences in the plasmid DNA also suffice for recombinational insertion into the vector.
  2. The desired fragment sequences can be amplified using as the template either genomic or plasmid DNA (see step 3) as a template. For purified plasmid DNA from most commercial kits (see Materials and Reagents), less than 0.25 μl is required as template DNA; for chromosomal DNA, approximately 2-3 μl is recommended per 50 μL PCR reaction using standard genomic DNA preparations from yeast (Amberg et al., 2006). We have found that optimal production of PCR fragments with overhanging tails is achieved using a two-phase PCR procedure: 5 cycles at a lower annealing temperature, followed by 25 cycles at a higher annealing temperature. To determine the initial temp., the Tm (in °C) for the non-overhanging portion of each of the two primers is determined (for this purpose, we use an algorithm available on-line (http://www.basic.northwestern.edu/biotools/oligocalc.html) and the recommended initial annealing temperature is determined from the formula [(Tm forward + Tm reverse) / 2] - 5. To determine the second temp., Tm's for each of the full primers are determined and the same formula applied. Depending on the DNA polymerase, the sequences being amplified, the thermocycler, and even the tubes used, the PCR reactions may need to be optimized. The critical point is that a single clean PCR product of the expected length needs to be generated. Hence, a sample of the resulting PCR product should be examined on a DNA agarose gel to ensure (i) a product of the correct size was created and (ii) there are not multiple DNA bands corresponding to mispriming.
  3. If any sequence amplification used as the template a yeast-based plasmid propagated in E. coli, subsequent digestion with DpnI (New England Biolabs) allows for destruction of the methylated template DNA and virtually eliminates the recovery of potential false positives in those cases where a marker being selected in the constructed vector of interest is the same as that contained in the template DNA. Digestion with DpnI is performed at 37 °C overnight (no heat-inactivation required) directly in the PCR tube mixture (no buffer required).
  4. The desired yeast vector of interest (Figure 1B) should be digested with a single unique restriction endonuclease; this site should be downstream of any inserted fragment that is already present in the vector. One strategy includes cloning beforehand a promoter sequence (5’-UTR) into the desired yeast-based plasmid (we prefer CEN vectors).
  5. Digestion of the resulting vector on the 3’-end of the promoter should be performed using an appropriate restriction enzyme and incubated overnight.
    Note: This digested product does not have to be extracted or gel purified because even uncut vector will not yield any false positives whenever the marker for selection of the desired construct (e.g., hygromycin-resistance) is distinct from any marker in the parent plasmid (e.g., URA3) (however, nonetheless, it might be prudent to ensure that the plasmid has indeed been linearized by running a small sample on an agarose gel).
  6. A yeast strain that results in efficient assembly and subsequent high yield of the desired plasmid DNA is SF838-1Dα (MATα ura3-52 leu2-3,122 his4-519 ade6 pep4-3 gal2) (Rothman and Stevens, 1986). However, for construction of plasmids in which HIS3 will be the selected marker (and not hygromycin-resistance), a derivative THS4218 (SF838-1Dα HIS4 his3Δ::HygR) is available. We have found that isolation of plasmids from strains of this background reproducibly results in higher plasmid yields and is more reliable than from other laboratory strains, including BY4741 (MATa leu2Δ ura3Δ met15Δ his3Δ) and its derivatives (Brachmann et al., 1998), especially when combined with ultra-chemically competent TOP10 bacterial transformation (see below).
  7. The yeast should be grown overnight to saturation in rich (YPD) medium at 30 °C, back-diluted to A600 nm ~0.25 in 10 ml of YPD and grown at 30 °C with shaking for roughly 4-5 h until A600 nm ~1. For each plasmid construction by this in vivo ligation procedure, a 10 ml culture of yeast is required. Hence, if multiple plasmid constructions are to be done in parallel, then commensurately more culture will be needed.
  8. Yeast should be collected by sedimentation in a clinical centrifuge for 5 min at 6,000 rpm and the YPD medium poured off. Resuspend the resulting pellet in 500 μl of sterile water with gentle pipetting and sediment again in a microfuge for 1 min at 6,000 rpm.
  9. For yeast transformation, a modified lithium acetate-based high-efficiency protocol should be used (Eckert-Boulet et al., 2012). For this purposed, remove the water with a pipet and resuspend the yeast pellet in 500 μl of sterile 100 mM lithium acetate solution and re-collect the cells for 1 min at 6,000 rpm and remove the supernatant solution and keep the yeast pellet.
  10. To each yeast pellet, add the following to the tube: 240 μl of 50% polyethylene glycol (PEG; autoclaved and sterile), 36 μl of sterile 1 M lithium acetate, 50 μl of 10 mg/ml single-stranded salmon sperm DNA (ssDNA in water). A high amount of ssDNA is included to (i) serve as an efficient carrier of the transformed DNA and (ii) the ssDNA is extremely viscous (and can clump into globs that resist transferring via micropipette) at concentrations of 10 mg/ml and therefore, a significant amount is lost in pipetting the solution into each tube (or a master mix solution). Therefore, to ensure that adequate ssDNA is included, we suggest the approximate amount of 50 μl with the understanding that some amount will be lost in solution preparation. Ingredients should be added in this order with the PEG solution added to the yeast first. Alternatively, should multiple transformations be performed at the same time, a “master mix” of the PEG, lithium acetate, and ssDNA can be mixed together and vortexed vigorously for 30 sec prior to adding to each yeast pellet. Two aspects of these reagents are important. First, the stock of salmon sperm DNA should be boiled for 10 min and cooled on ice for 10 min every time that it is used. Second, if the "master mix" is used, it must be made fresh for each experiment (it cannot be stored and reused).
  11. Add the appropriate DNA fragments (15-20 μl from each PCR reaction; the concentration will be dependent on the efficiency of the PCR reaction the length of the fragment, but generally this corresponds to roughly 3-5 μg of each PCR reaction) and cleaved vector (15-20 μl from the restriction enzyme digest; and approximately 2 μg total DNA content depending on the size of the parent vector being used) to the tube of lithium acetate- and PEG-treated yeast. Vortex the tube for 30 sec at maximum speed to resuspend the yeast into solution and incubate at exactly 42 °C for 45 min.
  12. Collect the yeast for 1 min at 6,000 rpm in a microfuge, remove the supernatant solution, and gently resuspend the yeast pellet in 550 μl sterile YPD medium and incubate overnight standing in a rack in a 30 °C incubator. This overnight incubation step is absolutely required for any constructs using a drug-resistance cassette (KanR, NatR or HygR); yeast require a sufficient amount of time to express the drug-resistance cassette and become resistant before they are challenged with the drug present in the selection medium. Standing overnight incubation, as opposed to aeration by shaking or rotation, allows for slow and largely anaerobic growth reducing the rate of production of CO2 and other volatiles, which, in our experience, can pop the lids off of standard Eppendorf tubes.
  13. Plate the full tube of yeast on the appropriate agar medium (YPD + drug, or synthetic drop-out medium), and incubate the plates for at least two days at 30 °C. Optional controls include transforming the same yeast strains with (i) only digested vector and/or (ii) only each PCR fragment-both of these should result in no plasmid constructs being generated and no growth on the selection plates.
  14. For recovery of plasmid DNA, scrape yeast off the plate (avoid agar)-use at least 25% of the surface area of the entire Petri dish (150 mm diameter dish; assuming there is a full lawn of coverage). Ideally, this high-efficiency in vivo ligation technique coupled with the high-efficiency yeast transformation should result in a complete lawn on the selective medium, although other factors (such as the complexity of the assembly) can reduce the final colony count. It is advised to only proceed with the protocol with constructs that resulted in ample coverage on the selection plate. If there are only single colonies on the initial selection plate, it is recommended to collect all of them and replate onto a fresh (selective) plate to grow up a sufficiently dense lawn of yeast for use in this protocol.
  15. The yeast scraped from the plate are resuspended in 500 μl of 1 M sorbitol, 0.1 M Na2EDTA (pH 7.5) (Amberg et al., 2006), briefly vortexed (maximum speed) to ensure full resuspension (expect that the solution will be very thick and concentrated), and then 5-7.5 μl of a stock (final concentration of 25 mg/ml in 50% glycerol) of Zymolyase 100T (stored at 4 °C) is added. After incubation at 37 °C on rotator for at least 1 h (but not more than 2 h), the resulting spheroplasts are collected at 12,000 rpm in a microfuge.
  16. After removal of the supernatant solution, the spheroplast pellet is resuspended in 250 μl of the proprietary bacterial plasmid isolation solution (see Materials and Reagents). Triturate by drawing the solution in and out of a pipet; at this point, the solution of lysed spheroplasts is extremely viscous and it takes significant effort to ensure very thorough resuspension.
  17. Add a roughly equal volume (roughly 100-250 μl) of glass beads (0.5 mm diameter). Briefly spin in microfuge at 2,000 rpm for 5 sec (to ensure all glass beads are mixed with the yeast solution rather than stuck to the walls of the tube). Vortex on maximum for 5 min (if you are doing multiple constructions in parallel, a vortexing adaptor that can house multiple tubes at once can be used).
  18. After completion of lysis by the glass bead breakage, proceed with the subsequent steps of your preferred commercial bacterial plasmid isolation protocol- for the Thermo Scientific system, add 250 μl of “Lysis buffer” and mix by inversion at least 10 times= and incubate at room temperature for 5 min. Add 350 μl of “Neutralization buffer” and mix by inversion at least 10 times immediately. Subject the resulting solution to centrifugation at maximum (13,200 rpm) in microfuge for 5 min.
  19. Transfer by pipetting the supernatant DNA-containing solution into a fresh, labeled tube and centrifuge at maximum speed for an additional 2 min (to remove any residual precipitate/cell debris). Transfer by pipetting the clarified supernatant solution into the plasmid-capturing spin column provided in the commercial bacterial plasmid isolation kit (see Materials and Reagents), centrifuge at maximum speed for 1 min and discard the flow-through.
  20. Wash the spin column with the commercial ethanol wash solution (500-750 μl), centrifuge at maximum speed for 1 min, and discard flow-through. Repeat (although this second wash step varies with the commercial plasmid prep kits and may be optional).
  21. To remove any residual wash solution, place the empty spin column in the microfuge and spin at maximum speed for 1 min, then place the spin column in a fresh Eppendorf tube and elute the bound DNA in the spin column by adding 25-30 μl of the “elution buffer” provided by the commercial supplier, incubating 1-2 min at room temperature, and then collecting the eluate by centrifugation at maximum speed for 2 min.
  22. To recover the desired constructed plasmid, a sample (10 μl) of the DNA-containing solution eluted from the spin column is gently mixed in a tube containing 100 μl of ultrachemically competent TOP10 bacterial cells (Materials and Reagents) that was taken from the -80 °C freezer and thawed on ice for ten minutes. After combining the DNA with the competent cells, the solution was left on ice for another ten min, then placed in a 42 °C water bath for 40 sec to subject the cells to a heat shock, returned to ice for two additional min, mixed with 500 μl of sterile SOC medium, and incubated on a rotator at 37 °C for 1 h.
  23. The resulting bacterial transformants (50-100 μl of the bacterial culture in SOC medium) are plated onto appropriate LB + drug (typically ampicillin or kanamycin) plates (prewarmed at 37 °C) and incubated at 37 °C overnight. Typically, 100-500 single colonies per plate grow out. Plasmid DNA from several representative single colonies are then isolated by standard molecular biology methods and tested using diagnostic PCR and DNA sequencing to ensure that the assembled gene fragments are all present and do not harbor any mutations or other alterations. This overall in vivo ligation process is so incredibly robust and efficient, it usually only requires 1-2 colonies to be analyzed to obtain the desired construct (see Notes).
  24. Important advice for avoiding pitfalls: Application of the above procedures for a multitude of different constructs (n > 1,000) over many years (a decade) indicates that the most common cause of any difficulty in successfully obtaining the desired constructs arises from the degree of sequence impurity in commercially purchased synthetic oligonucleotides. We have found that when mutations are observed, they reside in the junctions between gene fragments that correspond directly to the primer sequences used in the initial PCR amplifications. Thus, ensuring the high purity/quality/homogeneity of the initial oligonucleotide primers is critically important for maximizing the efficiency of this method of DNA assembly.

Notes

Our system includes a number of important differences from a previous description (Andersen, 2011) of this general approach for HR-driven fragment assembly and plasmid construction in yeast.

  1. Our methodology for plasmid creation takes advantage of the incorporation of a PCR fragment that contains a selectable marker (auxotrophic or drug-resistance cassette) that is distinct from any which may reside on any of the template DNAs used or on the parent vector into which the construct will be inserted (Figure 1B). In this way, even if the vector recircularizes, it will not generate any false positives when the selection is imposed to isolate clones carrying the desired construct.
  2. Whenever it is unavoidable that the template and the vector have the same selectable marker, in our procedure false positives can still be efficiently eliminated by treatment of the PCR reaction products with DpnI restriction enzyme before their incorporation into the vector.
  3. Our method eliminates the use of phenol: chloroform in the yeast plasmid isolation step.
  4. Use of a unique lineage of S. cerevisiae strains (SF838-1Dα-based) allows for more efficient plasmid rescue from yeast.
  5. Our approach allows for the insertion at any location of any desired sequence wherever it may have utility for meeting any experimental goal. Such sequences could include, but are not limited to, at the protein level, internal epitope tags, protease cleavage sites, aptamers for protein recognition domains, flexible linkers, and subcellular localization signals and, at the nucleic level, restriction endonuclease cut sites, stem-loop sequences for RNA-binding proteins, loxP cassettes, etc. (Figure 1C).
  6. Our approach exploits and highlights the power of in vivo HR to yield complex multi-part gene fusions, domain deletions, or tagged constructs in a single assembly step. Prior to our methods, this approach had only been used as a means to simply clone by reassembly a large contiguous stretch of the yeast genome (Andersen, 2011).
  7. Routinely, even for plasmids constructed from 5 or more separate PCR fragments (with a final plasmid size of 7-15 kb), only 1-2 bacterial clones need to be examined to obtain the construct of interest. Overall, the process of in vivo ligation and plasmid recovery described here is an efficient, cost-effective means for accurately generating complex DNA assemblies and can be applied in a massively parallel manner, when needed, to generate hundreds of plasmid constructs simultaneously.
  8. Finally, because a selectable marker is incorporated into each construct, it can be amplified (or excised) from the vector containing it and used for one-step gene replacement (Rothstein, 1991) in any desired recipient organism, such as the yeast KanR-marked haploid or diploid genome deletion collections (Brachmann et al., 1998) (Figure 1D).


    Figure 1. Complex DNA assembly using in vivo ligation by homologous recombination in yeast. A. A diagram illustrating the workflow of the protocol to construct plasmids using homologous recombination in yeast. B. An example construct is illustrated using homologous overlapping primer tails (identical colors show homology and the dotted lines illustrate the resulting recombination event across the homologous segments). Digested CEN-based yeast vector, such as LEU2-based pRS315 (Sikorski and Hieter, 1989). This illustration represents a single example of a series of gene fragments assembled into a single construct in one step using in vivo ligation. Modifications to this arrangement can be used to assemble multiple domains within a single gene or genes, include multiple epitope or fluorescent tags at either the N- or C-termini, and/or include flanking UTR sequences for subsequent integration at a non-native chromosomal locus. C. This example specifically addresses a useful property of this process. Specifically, one can encode in the homologous overlapping tails between two adjacent gene fragments to be assembled any desired non-native sequence information to introduce additional sequences of interest (such as, but not limited to, an epitope tag, a Gly-Ser rich linker sequence, restriction enzyme cut sites, protein cleavage motifs, signal sequences, etc.). D. Following construction, the entire construct can be PCR-amplified and integrated into the genome in a single step using either designed homology (5’- and 3’-UTR) within the system or, in the case of the MX-4-based drug-resistance cassette system devised by Goldstein and McCusker (2001), homology between the common sequence (blue) installed downstream of each type of drug-resistance gene, in which ADH1(t) is the terminator sequence from the ADH1 locus. Replacement of the gene deletion cassette with the modified gene of interest also allows for a swap in the growth selection markers (either drug resistance or nutrient). This system can also be modified to accommodate essential genes, as long as the recipient cell also harbors a URA3-based vector expressing the WT essential gene from a second plasmid, which can be subsequently eliminated by counter-selection on 5-FOA medium (Boeke et al., 1984).

Recipes

  1. 1 M sorbitol, 0.1 M Na2EDTA
    Adjusted to pH 7.5, filter sterilized
  2. YPD liquid media
    1% yeast extract
    2% peptone
    2% dextrose
    Autoclaved
  3. SOB medium
    2% tryptone
    0.5% yeast extract
    10 mM NaCl
    2.5 mM KCl
    1 mM MgCl2
    1 mM MgSO4
  4. SOC medium
    SOB medium
    20 mM glucose final concentration
  5. LB plates (with appropriate drug included)
    15 g/L agar
    1% tryptone
    0.5% yeast extract
    10 g/L NaCl
    4 mM NaOH

Acknowledgments

This work was supported by a Miller Research Fellowship from the Miller Institute for Basic Research in Science at the Univ. of California, Berkeley (to G.C.F.) and by NIH Research Grants GM21841 (to J.T.) and GM101314 (to Eva Nogales and J.T.). This protocol was adapted and modified from Finnigan et al. (2011).

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简介

这里提出的方案允许使用发芽酵母(酿酒酵母)中的同源重组和体内连接,容易地生成各种复杂的多部分DNA构建体(标记的基因产物,基因融合体,嵌合蛋白和其他变体)。该方法是直接,有效和成本有效的,并且可以用于载体创建和用于后续的一步,高频整合到酵母中的染色体位点。该方法利用PCR扩增多个片段之间的同源性的寡核苷酸“尾”,以允许在单次转化中重组酵母,随后用酵母高效质粒提取(用于转化为细菌)的方法。后者是对现有的酵母质粒提取方法的改进,其历史上已经是恢复所需构建体的限制步骤。我们描述了我们的技术的实用性和便利性,并提供了几个例子。
【背景】酿酒酵母中的同源重组(HR)早已被公认为在体内组装DNA片段的非常方便的方法(Szostak等,1983; Ma等,1987; Oldenburg等,1997)。鉴于酵母中人力资源的效率,它已经被利用了增加其效用,增强其多功能性并允许其应用于广泛的实验目标的方式。这种一般方法的改进包括使用体内连接作为定向诱变的平台(Muhlrad等人,1992),引入反选择以帮助质粒产生(Gunyuzlu等人,2001; Anderson和Haj-Ahmad,2003)和使体内组装适应于不能在酵母中繁殖的载体(Iizasa和Nagano,2006; Joska等人,2014)。然而,以前的研究并没有充分利用人力资源的力量和效用。我们已经发现HR在体内允许在单个转化步骤中非常有效地装配和回收含有大量(> 5个独立片段)DNA片段的质粒。因此,我们已经能够构建各种各样的基因破坏盒和整合盒,克隆异常大的基因,组装多部分嵌合基因,并产生多重标记的基因产物。我们的方法的主要用途和功能是能够在单个步骤中正确有效地组装转化成酵母的多个DNA片段。我们的方法(图1A)已成功使用:(i)在两个或多个不同基因之间组装框内嵌合体; (ii)将它们的N-和/或C-末端或两者的基因产物融合到荧光探针和/或表位标签; (iii)产生具有大量非翻译侧翼序列的基因缺失盒; (iv)在组装的基因或基因片段之间引入一个或多个短接头序列或表位标签; (v)利用各种转录启动子和终止子;并且重要的是,(vi)在一个步骤中,产生标记有药物抗性基因盒或可选营养基因盒的构建体,其在所需基因座处整合到基因组中。因为酵母细胞中的HR进行体内构建过程(如果需要,则进行后续整合),不需要试剂盒或专有系统,并且可以大量平行的方式进行质粒收集装配。在这方面,我们的系统比体外酶驱动的“Gibson克隆”(Gibson,2011)程序便宜得多,但仍然非常有效。此外,我们的系统(不同于需要限制酶消化插入基因片段的系统)不会导致任何核苷酸的插入(或丢失),这有时会在经典的限制性位点克隆中发生。在我们的方法中,可以实现对编码序列和侧翼非翻译区(UTR)的精确控制。最后,使用该系统产生的构建体可以与单倍体酵母基因组缺失收集(Winzeler等人,1999; Giaever等人,2002)相结合,以允许在任何设计的基因座处简单且有效的一步整合。在我们的方法中唯一的速率限制因素是需要转化的酵母细胞在构建体(或遗传改变的细胞)被回收之前几天生长。

尽管可能存在类似的整体方法(Andersen,2011),但在我们的方案中,我们开发了几个重要的改进,大大提高了酵母细胞中DNA构建体的有效回收,包括:(i)易于溶解的特定酵母基因型比标准实验室菌株,如S288C(及其衍生物,如BY4741); (ii)造球步骤(破坏酵母细胞壁); (iii)玻璃珠打浆以获得更好的核酸提取;和(iv)化学处理用于超高效DNA转化的细菌。

关键字:同源重组, 质粒组装, 体内结扎, 酵母, 质粒拯救

材料和试剂

  1. 酵母菌株:SF838-1Dα(MATαura3-52 leu2-3,122 his4-519 ade6 pep4-3gal2; Rothman和Stevens,1986)和THS4218(SF838-1Dα; HIS4his3Δ:: HygR )
  2. KOD热启动DNA聚合酶(EMD Millipore,由VWR International分销,目录号:80511-384)
  3. DNA寡核苷酸引物(Integrated DNA Technologies,Inc。)
  4. dpn I限制酶(New England Biolabs,目录号:R0176S)
  5. 适用于消化的限制酶(New England Biolabs)
  6. PEG:聚(乙二醇),BioXtra avg。分子量3,350(Sigma-Aldrich,目录号:P4388-1KG)
  7. 乙酸锂二水合物(Sigma-Aldrich,目录号:L6883-1KG)
  8. ssDNA:来自鲑鱼睾丸的脱氧核糖核酸钠盐(Sigma-Aldrich,目录号:D1626-1G)
  9. GeneJET质粒小量制备试剂盒(Thermo Fisher Scientific,目录号:K0503)
  10. 从Arthrobacter leuteus (Amsbio LLC,目录号:120493-1)获得的Zymolyase ® 100T。
  11. One Shot TOP10化学感受态大肠杆菌(Life Technologies,Invitrogen TM ,目录号:C4040-03)
    注意:这些用作使用CCMB80缓冲液的进一步化学感受性程序的种子培养物。我们的感受态细胞通过用One Shot TOP10细胞接种1L SOB培养基培养物(Hanahan等,1991)在23℃下将它们生长至A600nm≈0.3。收获后,TOP10细胞通过如下所述(Hanahan等人,1991)处理而被制成化学感受态,其中"CCMB80 缓冲液"(10mM KOAc,pH 7.0,80mM CaCl 2
  12. 氨苄青霉素(终浓度为100μg/ml; Research Products International Corp.,目录号:A40040-100.0)和卡那霉素(终浓度为50μg/ml; Life Technologies,目录号:11815-024)
  13. 1M山梨醇,0.1M Na 2 EDTA(参见Recipes)
  14. YPD液体介质(参见配方)
  15. SOB培养基(参见配方)
  16. SOC介质(参见配方)
  17. LB板(含适当药物)(见配方)

设备

  1. 0.5mm玻璃珠(BioSpec Products,目录号:11079105)
  2. 离心机(Eppendorf微型离心机型号:5415D,目录号:022621408; Eppendorf转子,型号:F-45-24-11,用于24×1.5/2ml,目录号:022636502)
  3. 培养皿(100×15mm尺寸; VWR International,目录号:25384-088)
  4. 管(Axygen Microtubes 1.5ml透明,均聚物,防沸腾,目录号:MCT-150-C)
  5. 涡旋适配器(Microtube foam insert for Fisher Vortex Genie 2 mixer,Scientific Industries,Inc。;目录号:504-0234-00)
  6. PCR机(MJ Research PTC-200Peltier Thermo Cycler,双30孔α块)

程序

  1. 开始时,设计寡核苷酸以允许以期望的顺序扩增每个必需的基因片段,表位或荧光蛋白标签,营养标记和/或耐药性盒及其重组装配(图1B) 。后一个目的通过允许每个扩增片段通过HR连接到其邻居的每个引物上的同源性"[尾部] [通常30个核苷酸(Baudin等人,1993)]实现。重叠在质粒DNA中具有相应侧翼序列的C-末端最多片段的N-末端最多片段和3'-尾部中的5'-尾中的30个核苷酸的序列也足以重组插入载体。
  2. 可以使用基因组或质粒DNA(参见步骤3)作为模板作为模板扩增所需的片段序列。对于来自大多数商业试剂盒(参见材料和试剂)的纯化的质粒DNA,需要小于0.25μl作为模板DNA;对于染色体DNA,使用来自酵母的标准基因组DNA制备物(Amberg等人,2006),每50μLPCR反应推荐大约2-3μl。我们已经发现使用两相PCR方法实现具有突出尾部的PCR片段的最佳产生:在较低退火温度下5个循环,随后在较高退火温度下25个循环。为了确定初始温度,确定两个引物中的每一个的非突出部分的T m(以℃计)(为此目的,我们使用可在线获得的算法 http://www.basic.northwestern.edu/biotools/oligocalc.html ),并且推荐的初始退火温度由公式[(T sub) forward + T sub reverse /2] -5。为了确定第二温度,确定每个全引物的T m并应用相同的公式。根据DNA聚合酶,扩增的序列,热循环仪,甚至使用的管,PCR反应可能需要优化。关键点是需要产生预期长度的单个干净的PCR产物,因此,所得PCR产物的样品应该在DNA琼脂糖凝胶,以确保(i)产生正确大小的产物,和(ii)没有多个对应于引导错误的DNA条带。
  3. 如果任何序列扩增用作在E中繁殖的基于酵母的质粒的模板。大肠杆菌,随后用Dpn I(New England Biolabs)消化允许甲基化模板DNA的破坏,并且实际上消除了在选择标记物的情况下潜在的假阳性的恢复。构建的感兴趣的载体与模板DNA中包含的载体相同。用PCR管混合物(不需要缓冲液)在37℃下消化过夜(不需要热灭活)。
  4. 所需的目标酵母载体(图1B)应用单一独特的限制性内切核酸酶消化;此网站应该是已经存在于载体中的任何插入片段的下游。一种策略包括预先将启动子序列(5'-UTR)克隆到所需的基于酵母的质粒中(我们优选CEN载体)。
  5. 所得载体在启动子3'末端的消化应使用适当的限制酶进行并温育过夜。
    注意:该消化的产物不必提取或凝胶纯化,因为即使未切割的载体将不会产生任何假阳性,只要用于选择所需构建体的标记(例如潮霉素抗性)不同于亲本质粒(例如,URA3)(然而,可能谨慎的是,通过在琼脂糖凝胶上运行小样品确保质粒已经线性化)。
  6. 导致所需质粒DNA的有效装配和随后的高产量的酵母菌株是SF838-1Dα(MATαura3-52 leu2-3,122 his4-519 ade6 pep4-3 gal2 )(Rothman和Stevens, 1986)。然而,为了构建其中HIS3将是所选标记(而不是潮霉素抗性)的质粒,衍生的THS4218(SF838-1DαHIS4his3Δ:: Hyg R )。我们已经发现,从该背景的菌株中分离质粒可重复产生更高的质粒产量,并且比其他实验室菌株更可靠,包括BY4741(MATaleu2Δura3Δmet15Δhis3Δ)及其衍生物(Brachmann&特别是当与超化学性质的TOP10细菌转化(见下文)组合时。
  7. 酵母应在30℃下在富含(YPD)培养基中生长过夜至饱和,在10ml YPD中回稀释至A 600nm 0.25,并在30℃振荡下生长大致4-5小时直到A 600nm -1。对于通过这种体内连接程序的每种质粒构建,需要10ml培养物的酵母。因此,如果多个质粒构建同时进行,则需要相应的更多的培养物
  8. 通过在临床离心机中以6,000rpm离心5分钟收集酵母,并倒出YPD培养基。将所得沉淀重悬在500μl无菌水中,轻轻吹打,并在微量离心机中以6,000rpm再次沉淀1分钟。
  9. 对于酵母转化,应当使用修饰的基于乙酸锂的高效方案(Eckert-Boulet等人,2012)。为此,用移液管除去水,并将酵母沉淀重悬在500μl无菌的100mM乙酸锂溶液中,并以6,000rpm再收集细胞1分钟,除去上清液和保持酵母颗粒。
  10. 向每个酵母沉淀物中加入以下管:240μl50%聚乙二醇(PEG;高压灭菌并灭菌),36μl无菌1M乙酸锂,50μl10mg/ml单链鲑精DNA ssDNA在水中)。包含大量ssDNA以(i)用作转化的DNA的有效载体,和(ii)ssDNA在10mg/ml的浓度下是极粘的(并且可以聚集成阻碍通过微量移液管转移的球状物),因此,在将溶液吸移到每个管(或主混合物溶液)中时,显着量的损失。因此,为了确保包括足够的ssDNA,我们建议大约50μl,理解一些量将在溶液制备中损失。成分应按此顺序添加,首先将PEG溶液加入酵母中。或者,如果同时进行多次转化,可以将PEG,乙酸锂和ssDNA的"主混合物"混合在一起,并剧烈涡旋30秒,然后加入每个酵母颗粒。这些试剂的两个方面是重要的。首先,鲑鱼精子DNA的储备应煮沸10分钟,并在冰上冷却10分钟,每次使用。第二,如果使用"主混合",对于每个实验必须是新的(它不能被存储和重复使用)。
  11. 加入适当的DNA片段(15-20μl来自每个PCR反应;浓度将取决于PCR反应的效率,片段的长度,但通常这对应于大约3-5μg的每个PCR反应)和切割载体(来自限制酶消化的15-20μl;以及约2μg总DNA含量,取决于所使用的亲本载体的大小)至乙酸锂和PEG处理的酵母的管中。以最大速度涡旋管30秒,将酵母重悬于溶液中,并在42℃下孵育45分钟。
  12. 收集酵母在微型离心机中以6,000rpm离心1分钟,去除上清液,轻轻地将酵母沉淀物重悬在550μl无菌YPD培养基中,并在30℃的培养箱中在架子中静置过夜。这种过夜孵育步骤对于使用药物抗性盒的任何构建体是绝对必需的(Kan等人,Nat Immunol或Hyg Immunol);酵母需要足够的时间来表达药物 - 并且在它们被存在于选择培养基中的药物攻击之前变得抗性。与通过摇动或旋转通气相反,静置过夜孵育允许缓慢且大部分厌氧生长,从而降低CO 2和其它挥发物的产生速率,在我们的经验中,这些挥发物可以使盖子流出的标准Eppendorf管
  13. 将全管酵母涂布在合适的琼脂培养基(YPD +药物或合成的丢失培养基)上,并在30℃下孵育平板至少两天。任选的对照包括用(i)仅消化的载体和/或(ii)仅仅每个PCR片段转化相同的酵母菌株 - 两者都不应导致在选择平板上生成质粒构建体和不生长。
  14. 为了回收质粒DNA,将酵母从平板上刮下(避免琼脂) - 使用整个培养皿(直径150mm的培养皿;假设存在完整的覆盖草坪)的表面积的至少25%。理想地,这种高效率的体内连接技术与高效率酵母转化结合应该在选择性培养基上产生完全的草坪,尽管其它因素(例如,组装的复杂性)可以降低最终菌落计数。建议只使用在选择平板上产生充分覆盖的构建体进行方案。如果在初始选择平板上只有单个菌落,建议收集所有的菌落,并重新放在新鲜(选择性)平板上长出一个足够密实的酵母菌草坪,以便在本协议中使用。
  15. 将从平板上刮下的酵母重悬浮于500μl的1M山梨醇,0.1M Na 2 EDTA(pH 7.5)(Amberg等人,2006)中,短暂涡旋(最大速度)以确保完全再悬浮(预期溶液将非常稠和浓缩),然后将5-7.5μl储备液(终浓度为25mg/ml,在50%甘油中)的Zymolyase 100T(储存在4 ℃)。在37℃下在旋转器上孵育至少1小时(但不超过2小时)后,在微量离心机中以12,000rpm收集所得的原生质球。
  16. 去除上清液后,将原生质球沉淀物重悬浮于250μl专有细菌质粒分离溶液(参见材料和试剂)中。通过将溶液吸入和移出移液管来研磨;在这一点上,溶解的原生质球的溶液是非常粘的,并且需要大量的努力来确保非常彻底的重悬。
  17. 加入大致相等体积(大约100-250μl)的玻璃珠(直径0.5mm)。在微型离心机中以2,000rpm短暂旋转5秒(以确保所有玻璃珠与酵母溶液混合,而不是粘在管壁上)。最大涡旋5分钟(如果您平行做多个结构,可以使用可同时容纳多个管的涡旋适配器)。
  18. 玻璃珠裂解完成裂解后,继续您的首选商业细菌质粒分离方案的后续步骤 - 为Thermo Scientific系统,添加250μl"裂解缓冲液",通过反转混合至少10次=和孵育在室温下5分钟。加入350微升的"中和缓冲液",通过倒置至少10次立即混合。使所得溶液在微量离心机中最大(13,200rpm)离心5分钟
  19. 通过吸取含有上清液的溶液到新鲜的,标记的管中,并以最大速度离心另外2分钟(以去除任何残留的沉淀物/细胞碎片)转移。通过将澄清的上清液吸移到商业细菌质粒分离试剂盒(参见材料和试剂)中提供的质粒捕获旋转柱中来转移,以最大速度离心1分钟并丢弃流出物。
  20. 用商业乙醇洗涤溶液(500-750μl)洗涤离心柱,以最大速度离心1分钟,弃去流出液。重复(尽管此第二次洗涤步骤随商业质粒制备试剂盒而变化,并且可以是可选的)
  21. 为了除去任何残留的洗涤溶液,将空离心柱置于微量离心机中并以最大速度旋转1分钟,然后将离心柱置于新鲜的Eppendorf管中,通过加入25-30μl的由商业供应商提供的"洗脱缓冲液",在室温下孵育1-2分钟,然后通过以最大速度离心2分钟收集洗脱液。
  22. 为了回收所需构建的质粒,将从旋转柱洗脱的含有DNA的溶液的样品(10μl)在含有100μl超化学感受态TOP10细菌细胞(材料和试剂)的管中温和混合, 80℃冰箱中,并在冰上解冻10分钟。将DNA与感受态细胞组合后,将溶液在冰上放置另外10分钟,然后置于42℃水浴中40秒以使细胞经受热休克,再次加入冰另外两分钟,混合用500μl无菌SOC培养基,并在旋转器上在37℃孵育1小时
  23. 将所得细菌转化体(50-100μl的SOC培养基中的细菌培养物)接种在合适的LB +药物(通常为氨苄青霉素或卡那霉素)平板(在37℃预热)中,并在37℃下孵育过夜。通常,每板100-500个单菌落生长。然后通过标准分子生物学方法分离来自几个代表性单个菌落的质粒DNA,并使用诊断PCR和DNA测序来测试,以确保组装的基因片段全部存在并且不具有任何突变或其他改变。这种整体的体内连接过程是非常强大和有效的,它通常仅需要分析1-2个菌落以获得所需的构建体(参见注释)。
  24. 避免陷阱的重要建议:在多年(十年)中对多种不同构建体(n> 1,000)的上述程序的应用表明,成功获得所需构建体的任何困难的最常见原因来自于序列杂交。我们已经发现,当观察到突变时,它们存在于基因之间的连接中 片段,其直接对应于在初始PCR扩增中使用的引物序列。因此,确保初始寡核苷酸引物的高纯度/质量/均一性对于最大化该DNA装配方法的效率至关重要。

笔记

我们的系统包括与先前描述(Andersen,2011)的这种用于驱动酵母中的HR驱动的片段装配和质粒构建的一般方法的一些重要差异。

  1. 我们用于质粒制备的方法利用了含有选择性标记(营养缺陷型或耐药性盒)的PCR片段的掺入,其不同于可以位于使用的任何模板DNA上的任何模板DNA,或者其中构建体将被插入(图1B)。以这种方式,即使载体再循环,当选择用于分离携带所需构建体的克隆时,它不会产生任何假阳性。
  2. 当模板和载体不可避免地具有相同的选择标记时,在我们的程序中,通过用Dpn I限制性内切酶处理PCR反应产物,可以有效地消除假阳性,然后将它们掺入矢量
  3. 我们的方法在酵母质粒分离步骤中不使用苯酚:氯仿
  4. 使用 S的独特谱系。酿酒酵母菌株(基于SF838-1Dα)允许从酵母更有效的质粒拯救。
  5. 我们的方法允许在任何期望的序列的任何位置插入,无论它可以用于满足任何实验目标。这样的序列可以包括但不限于在蛋白质水平,内部表位标签,蛋白酶切割位点,蛋白识别结构域的适体,柔性接头和亚细胞定位信号,并且在核酸水平上包括限制性内切核酸酶切割位点,茎RNA结合蛋白的循环序列,loxP盒等。(图1C)。
  6. 我们的方法利用并强调体内的HR在单个组装步骤中产生复杂的多部分基因融合,结构域缺失或标记的构建体的能力。在我们的方法之前,这种方法只是作为一种手段简单克隆通过重组装大连续酵母基因组(Andersen,2011)。
  7. 通常,即使对于由5个或更多个单独的PCR片段(具有7-15kb的最终质粒大小)构建的质粒,仅需检查1-2个细菌克隆以获得感兴趣的构建体。总的来说,这里描述的体内连接和质粒回收的过程是用于精确产生复杂DNA组装体的有效的,成本有效的方法,并且当需要时可以以大规模并行的方式应用以产生 数百个质粒构建体
  8. 最后,因为选择性标记物被掺入每个构建体中,所以其可以从包含它的载体扩增(或切除)并用于任何期望的受体生物体中的一步基因置换(Rothstein,1991),例如酵母KanR-标记的单倍体或二倍体基因组删除集合(Brachmann等人,1998)(图1D)。


    图1.在酵母中通过同源重组在体内连接的复杂DNA装配。 A.说明在酵母中使用同源重组构建质粒的方案的工作流程图。 B.使用同源重叠引物尾(相同的颜色显示同源性,虚线说明跨越同源区段产生的重组事件)举例说明实施例构建体。基于EST的基于酵母的载体,例如基于LEU2的pRS315(Sikorski和Hieter,1989)。该图表示使用体内连接在一个步骤中装配成单个构建体的一系列基因片段的单个实例。对这种排列的修饰可以用于在单个基因内组装多个结构域,在N-或C-末端包括多个表位或荧光标签,和/或包括侧翼UTR序列,用于随后在非天然染色体上整合轨迹。 C.该实施例特别涉及该方法的有用性质。具体地,可以在待组装的两个相邻基因片段之间的同源重叠尾部中编码任何期望的非天然序列信息,以引入另外的目的序列(例如但不限于表位标签,富含Gly-Ser的接头序列,限制性酶切位点,蛋白质切割基序,信号序列等)。 D.构建后,整个构建体可以PCR扩增并在单个步骤中使用系统内设计的同源性(5'-和3'-UTR)整合到基因组中,或者在MX-4- Goldstein和McCusker(2001)设计的基于抗原的药物抗性盒系统,安装在每种类型的耐药性基因下游的共同序列(蓝色)之间的同源性,其中ADH1(t)是终止子来自 ADH1 基因座的序列。用修饰的感兴趣的基因替换基因缺失盒还允许在生长选择标记(药物抗性或营养物)中的交换。该系统也可以被修改以适应必需基因,只要受体细胞还含有表达来自第二质粒的WT必需基因的基于URA3基的载体,其可以随后通过反选择而消除在5-FOA培养基上(Boeke等人,1984)

食谱

  1. 1M山梨醇,0.1M Na 2 EDTA 调节至pH 7.5,过滤灭菌
  2. YPD液体培养基
    1%酵母提取物
    2%蛋白胨
    2%葡萄糖 高压灭菌
  3. SOB介质
    2%胰蛋白酶
    0.5%酵母提取物
    10mM NaCl 2.5mM KCl
    1mM MgCl 2
    1mM MgSO 4
  4. SOC介质
    SOB介质
    20mM葡萄糖终浓度
  5. LB板(含适当药物)
    15g/L琼脂 1%胰蛋白酶
    0.5%酵母提取物
    10g/L NaCl
    4 mM NaOH

致谢

这项工作得到米勒研究基金会的支持,米勒研究所的基础研究在科学大学。 加利福尼亚州伯克利(到G.C.F.)和NIH Research Grants GM21841(到J.T.)和GM101314(到Eva Nogales和J.T.)。 该协议从Finnigan等人(2011)修改和修改。

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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Finnigan, G. C. and Thorner, J. (2015). Complex in vivo Ligation Using Homologous Recombination and High-efficiency Plasmid Rescue from Saccharomyces cerevisiae. Bio-protocol 5(13): e1521. DOI: 10.21769/BioProtoc.1521.
  2. Finnigan, G. C., Hanson-Smith, V., Houser, B. D., Park, H. J. and Stevens, T. H. (2011). The reconstructed ancestral subunit a functions as both V-ATPase isoforms Vph1p and Stv1p in Saccharomyces cerevisiae. Mol Biol Cell 22(17): 3176-3191.
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