搜索

Transfer of Large Contiguous DNA Fragments onto a Low Copy Plasmid or into the Bacterial Chromosome
将大片段DNA克隆到低拷贝质粒或细菌染色体   

下载 PDF 引用 收藏 提问与回复 分享您的反馈

本文章节

Abstract

Bacterial pathogenicity islands and other contiguous operons can be difficult to clone using conventional methods due to their large size. Here we describe a robust 3-step method to transfer large defined fragments of DNA from virulence plasmids or cosmids onto smaller autonomously replicating plasmids or directly into defined sites in the bacterial chromosome that incorporates endogenous yeast and λ Red homologous recombination systems. This methodology has been successfully used to isolate and integrate at least 31 kb of contiguous DNA and can be readily adapted for the recombineering of E. coli and its close relatives.

Background

The ability to isolate and propagate large pieces of DNA has vastly expanded the study of gene networks and operons. However, the traditionally used engineered plasmids for this purpose, such as bacterial artificial chromosomes (BACs), while extremely useful, are limited by problems with DNA stability, copy number, and complex assembly requirements. Alternatively, incorporating constructs directly into the bacterial chromosome provides advantages by both reducing variations in gene expression arising from the presence of multiple gene copies and ensuring stable maintenance of genes, while also avoiding the need for antibiotic selection.
   The methodologies described here were originally designed to capture and transfer the 31 kb of DNA operons that encode the Shigella flexneri type 3 secretion system onto the Escherichia coli chromosome (Reeves et al., 2015). The procedure utilizes yeast homologous recombination to generate a capture vector, a plasmid that contains regions of DNA that flank the fragment to be transferred, followed by using the λ Red recombination system to transfer the region of DNA of interest from a large virulence plasmid or cosmid onto the capture vector. The introduction of unique ‘Landing Pad’ sequences flanking the target sequence can be used to transfer via site-specific recombination the region of DNA present on the capture vector to an experimentally defined location on the bacterial chromosome using a protocol previously established by Kuhlman and Cox (2010). The inclusion of flanking landing pad sequences does not preclude the propagation of the DNA of interest on an autonomously replicating plasmid, but rather affords the opportunity to subsequently introduce the captured DNA onto a defined site on the bacterial chromosome. While we favor the use of an engineered landing pad sequence, one could adapt the approach described below to target the insertion of the captured DNA to a specifically defined locus on the bacterial chromosome.

Materials and Reagents

  1. 1.7 ml microcentrifuge tubes
  2. Acid-washed, small glass beads, 425-600 μm (30-40 U.S. sieve) (Sigma-Aldrich, catalog number: G8772 )
  3. Large glass beads, 5 mm (Corning, catalog number: 7268-5 )
  4. Cell scrapers (Corning, Falcon®, catalog number: 353085 )
  5. Electroporation cuvettes (Thermo Fisher Scientific, Fisher Scientific, catalog number: FB101 )
  6. Petri dishes (100 x 15 mm) (VWR, catalog number: 89038-968 )
  7. ElectroMAXTM DH10β cells (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18290015 )
  8. Saccharomyces cerevisiae yeast strain (BY4741) (GE Healthcare Dharmacon, catalog number: YS1048 or other ura3 minus strain)
  9. E. coli DH10β atp/gidB::Landing pad cassette. Tetracycline resistant strain harboring an integrated landing pad cassette (for use if transferring captured DNA into the chromosome) (Addgene, catalog number: 83036 )
  10. pLLX13 plasmid, or another suitable yeast/bacteria shuttle vector (Addgene, catalog number: 79825 )
  11. pLLX8 plasmid, or another suitable vector that encodes the desired antibiotic resistance cassette (Addgene, catalog number: 79838 )
  12. pKD46, temperature sensitive, λ red recombinase expression plasmid, or similar (Datsenko and Wanner, 2000)
  13. pTKred plasmid, temperature sensitive, bacterial expression vector for λ Red recombinase and I-SceI (Addgene, catalog number: 41062 )
  14. Gene specific primers (see Table 1)
  15. Yeast nitrogen base with ammonium sulfate (MP Biomedicals, catalog number: 114027-532 )
  16. CSM-URA, uracil dropout supplement (MP Biomedicals, catalog number: 4511-222 )
  17. D-glucose (Thermo Fisher Scientific, Fisher Scientific, catalog number: D16-10 )
  18. Agar (Thermo Fisher Scientific, Fisher scientific, catalog number: BP1423-2 )
  19. L-(+)-arabinose (Sigma-Aldrich, catalog number: A3256-100 )
  20. Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518-25G )
  21. Kanamycin monosulfate (MP Biomedicals, catalog number: 02150029 )
  22. Tetracycline hydrochloride (Sigma-Aldrich, catalog number: T7660 )
  23. Spectinomycin dihydrochloride pentahydrate (Sigma-Aldrich, catalog number: S9007-5G )
  24. Distilled, deionized water
  25. Magnesium sulfate (MgSO4) (Sigma-Aldrich, catalog number: M2643-500G )
  26. KOD hot start DNA polymerase (EMD Millipore, catalog number: 71-086-3 )
  27. QIAquick Gel Extraction Kit (QIAGEN, catalog number: 28704 )
  28. NheI-HF® restriction enzyme (New England Biolabs, catalog number: R3131S )
  29. PmeI restriction enzyme (New England Biolabs, catalog number: R0560S )
  30. MluI-HF® restriction enzyme (New England Biolabs, catalog number: R3198S )
  31. Yeast extract (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP1422-2 )
  32. Peptone (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP1420-2 )
  33. Glycerol (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP229-4 )
  34. Polyethylene glycol 3350 (PEG 3350) (Sigma-Aldrich, catalog number: 202444 )
  35. Lithium acetate (LiOAc) (Sigma-Aldrich, catalog number: 517992 )
  36. DNA sodium salt from salmon testes (Sigma-Aldrich, catalog number: D1626 ), resuspended in TE buffer (2 mg/ml)
  37. Tris-HCl, pH 8.0 (Promega, catalog number: H5123 )
  38. EDTA (Sigma-Aldrich, catalog number: E5134-500G )
  39. QIAprep Spin Miniprep Kit (QIAGEN, catalog number: 27104 )
  40. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014-500G )
  41. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P5405-250G )
  42. Sodium acetate (NaOAc) (Sigma-Aldrich, catalog number: S2889 )
  43. Selective antibiotics
    Kanamycin monosulfate (1,000x stock) (see Recipes)
    Ampicillin sodium salt (1,000x stock) (see Recipes)
    Tetracycline hydrochloride (1,000x stock) (see Recipes)
    Spectinomycin dihydrochloride pentahydrate (1,000x stock) (see Recipes)
  44. CSM-Ura selective media (see Recipes)
  45. YEPD media (see Recipes)
  46. TE buffer (see Recipes)
  47. SOB broth (see Recipes)
  48. SOC broth (see Recipes)

Equipment

  1. 100-150 ml Erlenmeyer flask
  2. Vortex (Scientific Industries, catalog number: SI-0136 )
  3. Incubators, set to 37 °C and 30 °C
  4. Water baths or heat blocks, set to 42 °C, 37 °C and 30 °C
  5. Microcentrifuge (Eppendorf, model: 5424 )
  6. Large centrifuge, able to handle up to 50 ml (for making competent yeast and bacteria)
  7. Electrophoresis system
  8. UV transilluminator
  9. Thermocycler (Bio-Rad Laboratories, model: PTC 200 ) or any other conventional thermocycler
  10. GenePulser II electroporation system (Bio-Rad Laboratories) or other electroporation device
  11. Nanodrop Lite spectrophotometer (Thermo Fisher Scientific, model: NanoDrop Lite ), or any other equipment/method suitable for quantifying DNA concentration
  12. Roller drum or shaker

Procedure

Part I: Generation of a capture vector

Note: The objective of this section is to create a capture vector (Figure 1), a plasmid that can subsequently be used to capture large defined fragments of DNA (Part II). At this point in designing a protocol, a decision needs to be made regarding whether or not to engineer the resulting capture vector to include flanking unique ‘landing pad’ sequences, a procedure needed later if the ultimate goal is to introduce the DNA fragment into a defined site within the bacterial chromosome using Landing Pad technology previously described in detail by Kuhlman and Cox (2010) (Part III).

  1. Preparation of DNA to be recombined to generate the capture vector
    1. Overview of DNA fragments
      As outlined in Figure 1, the capture vector is assembled by combining the following 4 fragments of DNA via yeast endogenous homologous recombination: linearized vector, plus 3 PCR generated fragments of interest: targeting sequence 1 (TS1), targeting sequence 2 (TS2) and an ampicillin resistance (beta-lactamase) containing cassette.
      1. Vector backbone: pLLX13 is a 9.94 kb yeast/E. coli shuttle vector that carries origins of replication and selection markers to support plasmid maintenance in bacteria (oriV-incP and tetRA) and yeast (CEN and URA3) as well as an origin of transfer (oriT) sequence which facilitates transfer between bacteria via conjugation (Wolfgang et al., 2003) (Figure 1C).
      2. Targeting sequences 1 (TS1) and 2 (TS2): 1 kb regions of DNA that share homology with the up- and downstream borders of the region intended to be captured and cloned. TS1 and TS2 are generated via PCR using primers that add 40 base pairs of homology to linearized pLLX13 on one end, and a spacer region containing several restriction sites on the other end (Figure 1A, Table 1). This can be accomplished via one round of PCR. However, if chromosomal integration of the captured region of DNA is desired, landing pad sequences that flank the targeting sequences should be incorporated into the PCR product. Nested PCR is used to insert these landing pad sequences between the targeting sequences and the pLLX13 homology regions (Figure 1B, Table 1).
        Notes:
        1. As represented in Figure 1C, the I-SceI restriction sites present in the capture vector that flank the landing pad sequences can be used as described in Kuhlman and Cox (2010) to liberate and target the capture DNA to a previously engineered landing pad site. See Part III and Figure 3.
        2. The spacer regions incorporated into the 3’ end of the TS1 PCR and the 5’ end of the TS2 PCR provide homologous DNA sequence for recombination with the ampicillin resistance cassette (below) and assembly of the capture vector. Additionally, the spacer regions contain consecutive restriction digest sites that are used to linearize the capture vector prior to recombination with target DNA sequences (Figure 2). The TS1 spacer region includes PmeI and HpaI restriction sequences and the TS2 spacer region includes MluI and PacI restriction sites (Figure 1C, Table 1). These restriction sites can be replaced with other restriction sites if desired by altering the primer sequence, i.e., if sites are contained in TS1 and/or TS2.
      3. AmpR cassette: region of DNA inserted between TS1 and TS2 to facilitate selection of the capture vector plasmid using bacterial transformation following assembly of the capture vector plasmid using yeast recombination. The AmpR carrying fragment from pLLX8 is amplified with primers that provide homology to the Target Sequences 1 and 2 spacer region sequences. The flanking homology on these DNA sequences enables their assembly by homologous recombination when co-transformed into competent S. cerevisiae.


        Figure 1. Schematic illustration of yeast recombination-based strategy utilized to generate a capture vector. A. Fragments with small overlapping regions of homology are generated via PCR using the depicted primers. The overlapping homologous sequences facilitate recombination and assembly in yeast. These fragments include those that contain homology to the 5’ and 3’ targeting sequences (TS1 and TS2) spacer regions (gray) plus an ampicillin resistance cassette (AmpR) (green). B. Landing pad (LP) sequences (blue) can be introduced into the capture vector by including a round of nested PCRs. The initial PCR amplification introduces homology to the spacer regions and flanking landing pad sequences, while a second round of PCR introduces homology to the pLLX13 vector backbone. C. When introduced into yeast, the 3 PCR generated fragments that contain overlapping homologous sequences and linearized (NheI-digested) pLLX13 vector backbone, undergo homologous recombination to generate a capture vector that can be selected for by growth on CSM-URA media. The resulting capture vector plasmid can be harvested and selected for in E. coli by plating on media containing ampicillin and tetracycline. The spacer regions (gray) that separate the target sequences and ampicillin resistance cassette introduce PmeI and MluI restriction sites into the capture vector which can be used to confirm proper capture vector assembly. Alternatively, SceI restriction sites can be used to liberate the entire assembled 5 kb fragment. Additionally, oligos pLLX F and pLLX R (Table 1), denoted by arrows, can be used to sequence the assembled capture vector to confirm that the proper recombination-mediated events occurred.

    2. Preparation of PCR generated fragments (TS1, TS2, AMPR)
      1. General PCR reaction mixture using high fidelity polymerase:
        16 μl distilled water
        2.5 μl KOD 10x reaction buffer
        2.5 μl dNTPs (2 mM each)
        1.5 μl 25 mM MgSO4
        0.5 μl forward oligo 20 μM (see oligo descriptions below and in Table 1)
        0.5 μl reverse oligo 20 μM
        1 μl template DNA (50-250 ng for genomic DNA and 1-10 ng for plasmid DNA)
        0.5 μl KOD hot start polymerase (1 U/μl)
      2. Oligos and template DNA
        Note: Choice of forward oligo (+/-LP) depends on whether downstream chromosomal integration of target DNA is desired.
        1. TS1 PCR (+/-LP):
          Forward oligo (+LP): 5_LP1_TS1 (Oligos are described in Table 1)
          Forward oligo (-LP): 5_pLLX_TS1
          Reverse oligo: 3_TS1
          Template: Gene specific template such as bacterial chromosomal or virulence plasmid DNA
        2. TS2 PCR (+/-LP):
          Forward oligo: 5_TS2
          Reverse oligo (+LP): 3_LP2_TS2
          Reverse oligo (-LP): 3_pLLX_TS2
          Template: Gene specific template such as bacterial chromosomal or virulence plasmid DNA
        3. Ampicillin resistance cassette PCR:
          Forward oligo: 5_Amp
          Reverse oligo: 3_Amp
          Template: pLLX8 or other plasmid containing desired antibiotic resistance cassette for selection
        4. PCR profile:
          Note: These conditions can be modified for the use of a different polymerase or for gene specific primers as needed.
          1 cycle, 94 °C, 5 min
          30 cycles, 94 °C 30 sec; 55 °C 30 sec; 72 °C 2-3 min
          1 cycle, 72 °C 10 min
      3. Expected fragments
        The TS1 and TS2 PCR products should be ~1 kb and the AmpR cassette ~3 kb. 
      4. Nested PCR (if adding flanking landing pad [LP] sequences)
        Prepare the same KOD hot start polymerase PCR mixture as above with following primer and template modifications: 
        1. Nested TS1 PCR oligos:
          Forward oligo: 5_pLLX_LP1
          Reverse oligo: 3_TS1
          Template: 1 μl of purified, confirmed TS1 PCR
        2. Nested TS2 PCR oligos:
          Forward oligo: 5_TS2
          Reverse oligo: 3_pLLX_LP2
          Template: 1 μl of purified, confirmed TS2 PCR
      5. Nested PCR analysis
        The TS1 and TS2 nested PCR products are still approximately 1 kb, but now contain a region of homology to the pLLX13 vector backbone to be used in the downstream recombination reaction.
      6. Clean the PCR products using the QIAquick Gel Extraction Kit or other equivalent methodology.
    3. Preparation of linearized vector DNA
      1. Digest 1 μg of the pLLX13 vector with NheI-HF restriction enzyme.
      2. Gel purify the digested plasmid DNA and quantify concentration on a Nanodrop Lite spectrophotometer or other equipment/method suitable for DNA quantification.

        Table 1. Primer sequences (5’-3’ sequence)

        aThese sequences should be added to the 5’ end (TS1) or 3’ end (TS2) of gene specific sequence to yield PCR products that can easily recombine to form a capture vector in yeast.
        bThese oligos contain PmeI and HpaI restriction sites as part of a spacer region.
        cThese oligos contain PacI and MluI restriction sites as part of a spacer region.
        NNN, denotes where target DNA specific sequence should be added.
        Bold, Landing Pad sequence
        Underlined, homology to pLLX13 vector
        Italics, homology to pLLX8 ampicillin resistance cassette encoding vector. Sequences in italics can be substituted for vector of choice containing desired antibiotic resistance cassette.

  2. Use of yeast recombination to generate capture vector
    1. Preparation of competent yeast (Gietz and Woods, 2002)
      1. Day 1: Inoculate 5 ml YEPD broth with a large colony of the host S. cerevisiae yeast strain and incubate on a roller drum overnight at 30 °C.
      2. Day 2: Measure the culture OD600 on a spectrophotometer, and back dilute (subculture) into 50 ml fresh YEPD broth in a 100-150 ml Erlenmeyer flask to an OD600 of 0.3.
      3. Incubate yeast on a shaker at 30 °C for 3-5 h until yeast cells undergo two doublings to reach an OD600 of ~1.2.
      4. Harvest yeast by centrifuging in a large centrifuge at 850 x g for 3 min. Decant supernatant.
        Note: Centrifuging at a higher x g makes the yeast pellet more difficult to resuspend.
      5. Resuspend yeast in 25 ml sterile water.
      6. Centrifuge at 850 x g for 3 min in a large centrifuge to pellet yeast. Decant supernatant.
      7. Resuspend yeast in 1 ml sterile water.
        Note: Remaining yeast can be resuspended in 10% glycerol and frozen at -80 °C for future use.
    2. Yeast transformation
      1. Centrifuge 100 μl of competent yeast in microcentrifuge tube using tabletop centrifuge at 850 x g for 1 min and remove supernatant.
      2. Overlay the yeast with 240 μl filter sterilized 50% PEG 3350 (w/v).
        Note: PEG 3350 is viscous, use plastic based materials to pipet.
      3. Assemble transformation mixture composed of:
        1. 36 μl 1 M LiOAc
        2. 10 μl DNA sodium salt (ssDNA) from salmon testes (2 mg/ml), resuspended in TE buffer.
          Note: Before first use, boil ssDNA for 5 min and cool on ice.
        3. DNA transformation mixture:
          100 ng linearized, NheI-digested, pLLX13 vector
          200 ng targeting sequence 1 (TS1) amplified fragment
          200 ng targeting sequence 2 (TS2) amplified fragment
          600 ng amplified fragment that contains ampicillin resistance gene
          Distilled, deionized water to a final volume of 65 μl
          Note: A second DNA mixture can also be assembled as a control, which contains only 100 ng linearized, NheI-digested, pLLX13 vector.
      4. Overlay the DNA transformation mixture upon the PEG and pipet or vortex until yeast pellet is fully resuspended.
      5. Incubate yeast at 30 °C for 30 min in a water bath or heat block.
      6. Transfer tube to a 42 °C water bath or heat block and incubate for an additional 15 min.
      7. Harvest cells by centrifuging at 2,400 x g for 1 min.
      8. Resuspend pellet in 100 μl sterile TE buffer or water and plate all onto a CSM-URA plate. Spread across plate using sterile large (5 mm) glass beads.
      9. Incubate in 30 °C incubator for up to 5 days. Colonies usually appear within 2-3 days, but growth times may vary.
    3. Isolate recombined capture vector
      1. Use a plate scraper or sterile pipet tip to resuspend all yeast colonies present on the transformation plate in 500 μl of YEPD media. Transfer mixture to a 1.7 ml microcentrifuge tube and harvest cells by centrifuging at 2,400 x g for 1 min.
      2. Decant supernatant and lyse yeast to extract assembled capture vectors using a QIAprep Spin MiniPrep Kit protocol modified as follows:
      3. Resuspend yeast in 250 μl of P1 buffer.
      4. Add 250 μl of small (425-600 μm) acid-washed glass beads to the tube and vortex on maximal speed setting for 5 min to lyse the yeast.
      5. Allow ~1 min for the glass beads to settle before transferring the yeast lysate into a new 1.7 ml microcentrifuge tube.
      6. Add 250 μl P2 buffer, mix by inversion 4-6 times and incubate at room temperature for 5 min.
      7. Add 350 μl N3 buffer, invert 4-6 times to mix, and spin at 16,500 x g for 10 min.
      8. Transfer supernatant onto a QIAprep column and collection tube and spin at 16,500 x g for 30 sec.
      9. Decant flow through and wash column with 750 μl PE wash buffer. Spin at 16,500 x g for 30 sec.
      10. Discard the flow through solution and reassemble spin column and collection tube. Spin at 16,500 x g for 1 min to dry the column.
      11. Transfer spin column to a fresh, labeled 1.7 ml microcentrifuge tube.
      12. Add 40 μl EB buffer (elution buffer), incubate at room temperature for 1 min, and perform a final spin at 16,500 x g for 1 min.
      13. Mix 3 μl of the eluted DNA with E. coli ElectroMAX DH10β cells (or any suitable competent E. coli cells) in a 1.7 ml microcentrifuge tube.
      14. Transfer DNA and bacteria mixture into a 0.1 mm electroporation cuvette.
      15. Electroporate using a GenePulser II (Bio-Rad Laboratories) or equivalent electroporation device using manufacturer’s settings.
        Note: For the GenePulser II, use 1.8 kV and 25 μF settings with E. coli.
      16. Add 450 μl of SOC and incubate for 1 h in a 37 °C heat block or water bath.
      17. Plate transformed bacteria on LB containing tetracycline and ampicillin to select for recombinant plasmids that have combined all 4 DNA fragments. Colonies will likely appear one day post-transformation, but can sometimes take an additional day to appear.
      18. To screen candidate capture vector colonies, inoculate at least 4 single colonies individually into 10 ml LB broth containing ampicillin and tetracycline and grow overnight at 37 °C.
        Note: We recommend using a larger volume for the miniprep at this step (up to 10 ml), because the low copy of the pLLX13 vector backbone typically results in a lower DNA yield. The bacterial pellet from all 10 ml of culture can then be pooled into a single miniprep.
      19. The next morning, harvest plasmid DNA using a QIAprep Spin Miniprep Kit following manufacturer’s instructions.

  3. Data analysis Part I: Confirm correct assembly of capture vector
    The plasmids isolated from individual bacterial transformants can be analyzed for the presence of an intact, recombined capture vector by:
    1. Digesting with SceI to liberate the inserted (~5 kb) TS1-AmpR-TS2 fragment (Figure 1C).
    2. Digesting with PmeI and MluI to liberate the (~3 kb) AmpR cassette.
    3. Using PCR to confirm the presence of both TS1 and TS2.
    4. Sequencing the recombined junctions with oligos pLLX F and pLLX R (Table 1 and Figure 1).

Part II: Capture the target sequence using λ Red homologous recombination

  1. Prepare capture vector DNA for transformation
    Linearize the capture vector to generate a fragment of DNA such that TS1 and TS2 are accessible as free ends to promote recombination when introduced into bacteria. This will greatly improve the efficiency of λ Red recombination and acts to remove the ampicillin resistance cassette (AmpR) prior to recombination (Figure 2A).
    1. Digest 1 μg pLLX13-TS1-amp-TS2 capture vector DNA with PmeI plus MluI.
      Note: HpaI can be used as a substitute for PmeI, and PacI can be used as a substitute for MluI.
    2. Gel purify the ~10 kb linearized capture vector DNA using a QIAquick Gel Extraction Kit purification kit according to manufacturer’s instructions.
    3. Quantify recovered linearized DNA using a Nanodrop Lite spectrophotometer or other DNA quantification method.

  2. Introduce source of target DNA to be captured into E. coli ElectroMAX DH10β cells
    For the following protocol, the target DNA must first be engineered to contain a selectable marker to facilitate selection of recombination based capture events. For example, the Shigella virulence plasmid contained a KanR cassette in place of ipaJ (Reeves et al., 2015).
    Notes:
    1. If removal of the antibiotic resistance cassette is desired for downstream applications, FRT (FLP recognition target) sites can be included that flank the antibiotic resistance cassette as described in Datsenko and Wanner (2000). It may be possible to capture an unmarked region of DNA via recombination, however this possibility was not investigated here.
    2. It is not absolutely necessary to transfer the DNA source into E. coli, if the DNA source is present in a bacterial species that can efficiently perform λ Red recombination. However, in our experience, performing this step greatly improved the efficiency and success of DNA capture.
    1. Transform marked cosmid or virulence plasmid into ElectroMAX DH10β cells via electroporation and select on LB plates containing kanamycin (or suitable antibiotic resistance marker).
      Note: Only with these commercially available cells have we successfully introduced the large 220 kb Shigella virulence plasmid into a laboratory strain of E. coli.

  3. Use λ Red recombination system to capture DNA sequence of interest (Figure 2B).
    1. To enable the expression of a homologous recombination system in the target bacterial strain, the plasmid pKD46 (Datsenko and Wanner, 2000) is first introduced into the strain by standard electroporation and selection on solid media containing ampicillin and 0.2% glucose (to suppress λ Red expression) at 30 °C.
      Note: pKD46 is a temperature sensitive, ampicillin resistant plasmid that carries an allele of λ red recombinase under the control of an arabinose-inducible promoter. Other plasmids encoding λ Red recombinase can also be used (Murphy and Campellone, 2003).


      Figure 2. Schematic illustrating how to use the capture vector to isolate a target DNA sequence onto the yeast-generated capture vector. A. The capture vector is linearized by digestion with PmeI and MluI to generate free DNA ends corresponding to TS1 and TS2 DNA to enhance recombination efficiency and to remove the ampicillin resistance cassette. B. The linearized DNA is introduced into an E. coli strain that contains a virulence plasmid or cosmid which encodes the target DNA sequence engineered to contain a selectable marker, i.e., a kanamycin resistance cassette, plus a plasmid that conditionally expresses the λ Red recombinase,i.e., pKD46 (not shown). The previous introduction of an antibiotic selection marker into the target DNA facilitates the selection of the DNA fragment following its integration into the capture vector. Expression of λ Red recombinase results in the transfer of the target DNA onto the linearized capture vector via homologous recombination (dashed lines).

    2. Day 1: Inoculate a single kanamycin, ampicillin resistant colony of bacteria that carries both the target DNA and pKD46 into LB broth containing kanamycin, ampicillin, and 0.2% glucose and grow overnight on a roller drum at 30 °C.
    3. Day 2: In the morning, back dilute the pKD46 containing bacteria 1:100 into 50 ml of SOB broth that contains kanamycin, ampicillin, and 0.2% arabinose (to induce λ Red recombinase expression). Incubate at 30 °C on a shaker for approximately 3-4 h until the culture reaches an OD600 of 0.6.
    4. Generate electrocompetent cells
      1. Transfer the cells into a sterile bottle and centrifuge at 6,000 x g for 10 min at 4 °C.
      2. Decant the supernatant and resuspend bacteria in 25 ml ice cold 10% glycerol.
        Note: Keep the bacterial pellets cold through entire procedure by placing tubes on ice in between wash steps.
      3. Repeat centrifugation at 6,000 x g for 10 min.
      4. Decant supernatant and repeat the 10% glycerol wash 3 more times, for a total of 4 washes.
        Note: The 4 washes may be performed with ice-cold sterile water instead of 10% glycerol, if desired (steps C6-C8).
      5. Resuspend the final pellet in 500 μl of cold 10% glycerol.
    5. Transfer 100 μl of electrocompetent, recombinase-expressing cells into a new microcentrifuge tube.
    6. Add 100 ng of linearized, gel purified, capture vector (Figure 2) and transfer mixture of bacterial cells and DNA into a 0.1 mm cuvette.
    7. Electroporate using a GenePulser II (Bio-Rad Laboratories) or equivalent electroporation device following manufacturer’s settings.
    8. Add 450 μl SOC broth to the electroporated cells and transfer to a microcentrifuge tube.
    9. Incubate cells at 37 °C for 1 h on a heat block or in a water bath.
    10. Plate entire transformation on LB plates containing tetracycline (to select for the capture vector backbone), and kanamycin or an appropriate antibiotic (to select for the region of target DNA to be captured) and incubate at 37 °C overnight.
      Notes:
      1. If no colonies arise after overnight incubation, continue incubating for an additional 24 h, as sometimes the recombination process can slow growth of the colonies.
      2. Incubation at 37 °C should eliminate the temperature sensitive pKD46 λ Red recombinase expressing plasmid, resulting in transformants that are ampicillin susceptible. The loss of pKD46 can be confirmed by patching individual colonies on LB containing ampicillin, if desired.
    11. Inoculate several candidate tetracycline/kanamycin resistant colonies into 10 ml of LB broth containing kanamycin and tetracycline and grow overnight at 37 °C.
    12. The next day, harvest DNA using a QIAprep Spin Miniprep Kit or other suitable miniprep kit.
      Note: This step provides size exclusion of the recombined ‘captured’ DNA-containing plasmids away from the virulence plasmid and genomic DNA.
    13. Transform harvested plasmids into ElectroMAX DH10β cells or other suitable electrocompetent bacterial host strain and plate on LB plates containing tetracycline and kanamycin.
      Notes:
      1. We recommend ElectroMAX DH10β cells for this purpose, as these cells are efficient at transformation and propagation of large DNA constructs.
      2. Importantly, if pursuing integration of the captured target DNA into the landing pad site of a bacterial chromosome, the harvested plasmids can be directly transformed into a landing pad-containing host strain (see Part III). However, if the user is unable to successfully transform the landing pad host strain directly, (i.e., because the plasmid containing captured target DNA is very large), we recommend transforming ElectroMAX DH10β cells first, and then using conjugation to transfer the plasmid into the landing pad-containing host strain.
      3. Once transformed into a host strain, the confirmed large plasmid can be transferred between bacterial strains via DNA conjugation, if desired.

  4. Data analysis Part II: Confirm transfer of the target DNA segment onto the capture vector
    1. We recommend using several complementary approaches to confirm that the correct target DNA segment has been transferred onto the capture vector. In our experience, nearly 100% of plasmids recovered from E. coli that carry both antibiotic markers contain the correct target DNA fragment.
    2. Candidate plasmids should be screened for the correct recombination event by:
      1. PCR analysis of junction regions (i.e., PCR using the pLLXF or pLLXR oligo [Table 1 and Figure 1]) and a suitable target DNA oligo.
      2. PCR analysis using gene specific primers for sequences that should be included in the captured region of DNA. Positive and negative controls should be included, for example, using the empty capture vector as PCR template for the negative control, and the parent bacterial strain that contains the target DNA segment as a positive control.
      3. PCR reactions using primers that specifically bind to regions unique to the starting virulence plasmid or cosmid, but not present in the captured DNA region, can be performed to confirm loss of the parental virulence plasmid or cosmid.
      4. Sequence analysis of the captured target DNA using target gene specific oligos, pLLXF, or pLLXR.

Part III: Integration of target DNA into a landing pad site on the bacterial chromosome

Notes:

  1. This section describes how to transfer a large piece of target DNA from the capture vector, assembled in Part II, into the chromosome of a bacterial strain that harbors a landing pad site. The introduction of a landing pad site into the bacterial chromosome is described in detail in the methods paper by Kuhlman and Cox (2010), and is therefore not discussed here. Thus, to continue with Part III of this protocol, a bacterial strain, such as E. coli DH10β, that harbors an integrated landing pad site is required.
  2. E. coli DH10β, that harbors an integrated landing pad site at the atp/gidB locus, is available at Addgene, catalog number: 83036. The landing pad site also introduces a tetracycline resistance cassette into the chromosome (Figure 3).
  1. Preparation of the bacterial strain for integration of captured, target DNA into the chromosome at a landing pad site.
    Notes:
    1. In order to transfer target DNA into the landing pad site of a bacterial chromosome, the captured target DNA plasmid must contain flanking landing pad sequences introduced by nested PCR (see Part I and Figure 1B).
    2. The landing pad host strain must contain two plasmids:(1) the pLLX13 capture vector containing the captured target DNA sequences (generated and confirmed in Part II) and (2) pTKred, a spectinomycin resistant, temperature sensitive plasmid that expresses an IPTG-driven λ red recombinase and an arabinose driven SceI restriction endonuclease. This protocol uses E. coli DH10β harboring a landing pad site at the atpI/gidB locus. If the captured target DNA plasmid (from Part II) was already introduced into E. coli DH10β-landing pad in Part II, begin this protocol by introducing pTKred into the strain by going directly to step B (Introduce the pTKred plasmid into E. coli DH10β-landing pad bacteria containing the captured target DNA).
    1. Introduce the capture vector plasmid containing target DNA into E. coli DH10β-landing pad cells
      1. Day 1: Inoculate a single tetracycline resistant E. coli DH10β-landing pad colony into LB broth containing tetracycline and grow overnight on a roller drum at 37 °C.
      2. Day 2: In the morning, back-dilute the E. coli DH10β-landing pad bacteria 1:100 into 10 ml of LB broth that contains tetracycline. Incubate at 37 °C on a roller drum or shaker for approximately 1-2 h until the culture reaches an OD600 of 0.6.
      3. Generate electrocompetent E. coli DH10β-landing pad cells:
        1. Incubate the cells on ice for 1 h.
        2. Transfer the cells into a sterile bottle and centrifuge at 6,000 x g for 10 min at 4 °C.
        3. Decant the supernatant and resuspend bacteria in 5 ml ice cold 10% glycerol.
          Note: Keep the bacterial pellets cold through entire procedure by placing tubes on ice in between wash steps.
        4. Repeat centrifugation at 6,000 x g for 10 min.
        5. Decant supernatant and repeat the 10% glycerol wash 3 more times, for a total of 4 washes.
          Note: The 4 washes may be performed with ice-cold sterile water instead of 10% glycerol, if desired.
        6. Resuspend the final pellet in 200 μl of cold 10% glycerol.
      4. Transfer 100 μl of electrocompetent cells into a new microcentrifuge tube.
      5. Add 200 ng of plasmid containing captured target DNA (Figure 2B) and transfer mixture of bacterial cells and DNA into a 0.1 mm cuvette.
      6. Electroporate using a GenePulser II (Bio-Rad Laboratories) or equivalent electroporation device following manufacturer’s settings.
      7. Add 450 μl SOC broth to the electroporated cells and transfer to a microcentrifuge tube.
      8. Incubate cells at 37 °C for 1 h on a heat block or in a water bath.
      9. Plate entire transformation on LB plates containing tetracycline (to select for the landing pad site on the chromosome), and kanamycin or an appropriate antibiotic (to select for the region of captured target DNA) and incubate at 37 °C overnight.

  2. Introduce the pTKred plasmid into E. coli DH10β-landing pad bacteria containing the captured target DNA
    1. Day 1: Inoculate a single colony of tetracycline/kanamycin resistant E. coli DH10β-landing pad bacteria containing the captured target DNA plasmid into LB broth containing tetracycline and kanamycin and grow overnight on a roller drum at 37 °C.
    2. Day 2: In the morning, back dilute the E. coli DH10β-landing pad bacteria containing the captured target DNA plasmid 1:100 into 10 ml of LB broth that contains tetracycline and kanamycin. Incubate at 37 °C on a roller drum or shaker for approximately 1-2 h until the culture reaches an OD600 of 0.6.
    3. Generate electrocompetent cells
      1. Incubate the cells on ice for 1 h.
      2. Transfer the cells into a sterile bottle and centrifuge at 6,000 x g for 10 min at 4 °C.
      3. Decant the supernatant and resuspend bacteria in 5 ml ice cold 10% glycerol.
        Note: Keep the bacterial pellets cold through entire procedure by placing tubes on ice in between wash steps.
      4. Repeat centrifugation at 6,000 x g for 10 min at 4 °C.
      5. Decant supernatant and repeat the 10% glycerol wash 3 more times, for a total of 4 washes.
        Note: The 4 washes may be performed with ice-cold sterile water instead of 10% glycerol, if desired.
    4. Add 100 ng of pTKred plasmid and transfer mixture of bacterial cells and DNA into a 0.1 mm cuvette.
    5. Electroporate using a GenePulser II (Bio-Rad Laboratories) of equivalent electroportation device following manufacturer’s settings.
    6. Add 450 μl SOC broth to the electroporated cells and transfer to a microcentrifuge tube.
    7. Incubate cells at 30 °C for 1 h.
      Note: pTKred is temperature sensitive so the outgrowth and plating must be performed at 30 °C.
    8. Plate entire transformation on LB plates containing tetracycline (to select for landing pad on the chromosome), kanamycin (to select for the captured target DNA plasmid), spectinomycin (to select for pTKred), and 0.2% D-glucose (to inhibit expression of λ Red recombinase) and incubate overnight at 30 °C.

  3. Insertion of target DNA into the landing pad on the chromosome
    Notes:
    1. This part of the protocol is modified from Kuhlman and Cox (2010). During this step, arabinose is used to induce expression of the SceI restriction enzyme while IPTG induces expression of λ Red recombinase. SceI introduces double stranded DNA breaks into the E. coli DH10β chromosome at the site of the landing pad integration. Simultaneously, SceI introduces double stranded DNA breaks on the captured target DNA plasmid. Expression of λ Red recombinase leads to the homologous recombination between landing sites on the chromosome and those present at the free ends of the target DNA. Successful SceI digestion and homologous recombination leads to double stranded break repair of the chromosome, excision of the tetracycline cassette, and integration of the target DNA sequence in its place (Figure 3). The introduction of double stranded breaks generates free ends of DNA that greatly increases the efficiency of homologous recombination.
    2. The 18 bp SceI restriction recognition site does not naturally exist in the E. coli chromosome.
    1. Day 1: In the morning, inoculate 5 ml of SOB broth containing 0.5% glycerol, 2 mM IPTG, and 0.2% arabinose with a single colony of the E. coli DH10β-landing pad strain containing the captured target DNA plasmid and pTKred. Grow for 1 h at 37 °C on a roller drum or shaker.
    2. Add 5 μl of spectinomycin (100 mg/ml) to the broth and incubate on a roller drum or shaker at 30 °C for 4 h.
    3. Add 5 μl of kanamycin (100 mg/ml) to the broth and incubate on a roller drum or shaker at 30 °C overnight.
    4. Day 2: Make serial dilutions of the overnight culture in LB broth from 10-1 to 10-5 and plate 100 μl of cells onto LB plates containing kanamycin.
    5. Incubate plates overnight at 37 °C.
    6. Day 3: Patch kanamycin resistant colonies onto LB plates containing tetracycline and LB plates containing kanamycin to screen for colonies that have become tetracycline susceptible.
    7. Incubate plates overnight at 37 °C.
    8. Colonies that are kanamycin resistant and tetracycline susceptible are candidates for successful target DNA integration at the landing pad site. (i.e., the tetracycline resistance cassette present on the chromosome has been replaced by the target DNA sequence).
      Note: Incubation at 37 °C should also eliminate the temperature sensitive pTKred plasmid. Loss of pTKred can be confirmed by patching colonies onto LB plates containing spectinomycin. If growth at 37 °C is not sufficient to drop the pTKred plasmid, individual colonies can be grown for 4 h or overnight at 42 °C and then replated onto LB plates containing kanamycin at 37 °C, then retested for spectinomycin sensitivity. Passaging at the higher temperature should ensure loss of pTKred.

  4. Data analysis Part III: Confirm integration of target DNA into the chromosomal landing pad site
    Individual colonies can be analyzed for the presence of integrated target DNA by:
    1. PCR analysis using primers to assess integration at junction regions in the chromosome. If the E. coli DH10β-landing pad strain was used, the atpI/gidB F and atpI/gidB R oligos (Figure 3 and Table 1) can be used with target gene specific primers to assess junctions.
    2. PCR analysis using primers to assess presence or absence of expected target DNA genes in the integrant strains.
    3. Functional analysis of integrated target DNA genes can be performed, if appropriate.


      Figure 3. Schematic illustrating the transfer of target DNA from the capture vector into a bacterial chromosome at a landing pad site. Captured, target DNA sequences can be integrated into the chromosome of a bacterial host strain that harbors an integrated landing pad site. Landing pad site 1 (LP1) and 2 (LP2) are shown as blue boxes. Two plasmids must be introduced into the host strain: (1) the capture vector containing target DNA flanked by landing pad sites and SceI restriction sites (see Part I and Table 1 for design) and (2) pTKred, which encodes IPTG inducible λ Red recombinase and arabinose inducible SceI restriction endonuclease (not shown). Induction of SceI with arabinose introduces double stranded DNA breaks into the host strain chromosome and on the captured target DNA adjacent to the landing pad sites. The free ends of DNA are then joined by homologous recombination through the IPTG-induced expression of λ Red recombinase (dashed lines). Bacterial isolates containing the correct insertion of target DNA can be identified by screening for kanamycin resistance and tetracycline susceptibility, which indicates a loss of both the landing pad intervening sequence from the chromosome and the original capture vector containing target DNA. The original 1 kb targeting sequence 1 (TS1) is denoted by a purple box and targeting sequence 2 (TS2) is denoted by a red box. The original pLLX13 homology sequences are denoted as black boxes. PCR analysis with the atpI/gidB F or atpI/gidB R oligos, denoted by arrows, (Table 1) and target DNA specific oligos can be used to confirm integration at the landing pad site.

Notes

  1. Additional application: Capturing fragments of chromosomal DNA can be accomplished by modifying this protocol as described in Wolfgang et al. (2003). For example, a capture vector can be designed to include targeting sequences homologous to regions on the bacterial chromosome.
  2. Repetitive elements in DNA. An effort should be made when designing the initial capture vector targeting sequences (TS1 and TS2) to ensure the 1 kb of target sequence is unique and does not include a repetitive element or transposon sequence, as this can affect the specificity of the λ Red recombination reaction.

Recipes

  1. Kanamycin monosulfate (1,000x stock)
    30 mg/ml in dH2O, filter sterilize
  2. Ampicillin sodium salt (1,000x stock)
    100 mg/ml in dH2O, filter sterilize
  3. Tetracycline hydrochloride (1,000x stock)
    12.5 mg/ml in 100% methanol, filter sterilize
  4. Spectinomycin dihydrochloride pentahydrate (1,000x stock)
    100 mg/ml in dH2O, filter sterilize
  5. CSM-URA selective media (per liter 900 ddH2O)
    6.7 g yeast nitrogen base with ammonium sulfate
    0.77 g CSM-URA, uracil dropout supplement
    ~20 g agar
    Before pouring plates add 100 ml sterile 20% glucose
  6. YEPD (per liter ddH2O)
    10 g yeast extract
    20 g peptone)
    20 g D-glucose
    ~15 g agar
  7. TE buffer
    10 mM Tris-HCl, pH 8.0
    1.0 mM EDTA
  8. SOB broth (per liter ddH2O)
    5 g yeast extract
    20 g tryptone
    0.584 g NaCl
    0.186 g KCl
    2.4 g MgSO4
    Adjust to pH 7.5 before use
  9. SOC broth (per liter ddH2O)
    Add 10 ml sterile 20% D-glucose solution to 990 ml SOB broth

Acknowledgments

This protocol was adapted from Reeves et al. (2015). Work was supported by R01AI064285, R21AI103882, and the Massachusetts General Hospital Research Scholar Award 2016 to CFL. AZR is supported by an MGH ECOR Fund for Medical Discovery Fellowship.

References

  1. Datsenko, K. A. and Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97(12): 6640-6645.
  2. Gietz, R. D. and Woods, R. A. (2002). Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350: 87-96.
  3. Kuhlman, T. E. and Cox, E. C. (2010). Site-specific chromosomal integration of large synthetic constructs. Nucleic Acids Res 38(6): e92.
  4. Murphy, K. C. and Campellone, K. G. (2003). Lambda red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli. BMC Mol Biol 4: 11.
  5. Reeves, A. Z., Spears, W. E., Du, J., Tan, K. Y., Wagers, A. J. and Lesser, C. F. (2015). Engineering Escherichia coli into a protein delivery system for mammalian cells. ACS Synth Biol 4(5): 644-654.
  6. Wolfgang, M. C., Kulasekara, B. R., Liang, X., Boyd, D., Wu, K., Yang, Q., Miyada, C. G. and Lory, S. (2003). Conservation of genome content and virulence determinants among clinical and environmental isolates of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 100(14): 8484-8489.

简介

由于其大尺寸,细菌致病性岛和其它连续操纵子可能难以使用常规方法克隆。在这里我们描述了一个强大的3步法从毒力质粒或粘粒转移大型定义的片段到更小的自主复制质粒或直接到细胞染色体,并入内源性酵母和λ红色同源重组系统的定义网站。该方法已经成功地用于分离和整合至少31kb的连续DNA,并且可以容易地适应于E的重组。大肠杆菌及其近亲。

[背景] 分离和繁殖大片DNA的能力大大扩展了基因网络和操纵子的研究。然而,用于该目的的传统使用的工程质粒,例如细菌人工染色体(BAC),虽然极其有用,但是受到DNA稳定性,拷贝数和复杂装配要求的问题的限制。或者,将构建体直接并入细菌染色体中通过减少由于存在多个基因拷贝引起的基因表达的变化以及确保基因的稳定维持而提供了优点,同时还避免了对抗生素选择的需要。这里描述的方法最初被设计为捕获和转移编码Shigella flexneri 3型分泌系统的31kb DNA操纵子到大肠杆菌染色体上(Reeves em>等人,2015)。该方法利用酵母同源重组产生捕获载体,质粒包含位于待转移片段两侧的DNA区域,然后使用λRed重组系统从大的病毒质粒或粘粒转移感兴趣的DNA区域到捕获载体上。在靶序列侧翼引入独特的"着陆垫"序列可以用于通过位点特异性重组将存在于捕获载体上的DNA区域转移到细菌染色体上的实验定义的位置,使用先前由Kuhlman和Cox建立的方案(2010)。包含侧翼着陆区序列并不排除目标DNA在自主复制质粒上的繁殖,而是提供随后将捕获的DNA引入细菌染色体上的确定位点的机会。虽然我们赞成使用工程着陆垫序列,但是可以采用下面描述的方法来靶向将捕获的DNA插入到细菌染色体上特定定义的基因座。

材料和试剂

  1. 1.7 ml微量离心管
  2. 酸洗涤的小玻璃珠,425-600μm(30-40 U.S.筛)(Sigma-Aldrich,目录号:G8772)
  3. 大玻璃珠,5mm(Corning,目录号:7268-5)
  4. 细胞刮刀(Corning,Falcon ,目录号:353085)
  5. 电穿孔杯(Thermo Fisher Scientific,Fisher Scientific,目录号:FB101)
  6. 培养皿(100×15mm)(VWR,目录号:89038-968)
  7. ElectroMAX TMDH10β细胞(Thermo Fisher Scientific,Invitrogen TM ,目录号:18290015)
  8. 酿酒酵母菌株(BY4741)(GE Healthcare Dharmacon,目录号:YS1048或其它ura3阴性菌株)。
  9. E。大肠杆菌DH10β atp/gidB ::着陆垫盒。含有整合着陆垫盒的四环素抗性菌株(用于将捕获的DNA转移到染色体中)(Addgene,目录号:83036)
  10. pLLX13质粒或另一种合适的酵母/细菌穿梭载体(Addgene,目录号:79825)
  11. pLLX8质粒或编码所需抗生素抗性盒的另一合适载体(Addgene,目录号:79838)
  12. pKD46,温度敏感的λ红色重组酶表达质粒或类似物(Datsenko和Wanner,2000)
  13. 用于λRed重组酶和I-SceI的pTKred质粒,温度敏感的细菌表达载体(Addgene,目录号:41062)
  14. 基因特异性引物(参见表1)
  15. 酵母氮源与硫酸铵(MP Biomedicals,目录号:114027-532)
  16. CSM-URA,尿嘧啶滴出补充剂(MP Biomedicals,目录号:4511-222)
  17. D-葡萄糖(Thermo Fisher Scientific,Fisher Scientific,目录号:D16-10)
  18. 琼脂(Thermo Fisher Scientific,Fisher scientific,目录号:BP1423-2)
  19. L - (+) - 阿拉伯糖(Sigma-Aldrich,目录号:A3256-100)
  20. 氨苄青霉素钠盐(Sigma-Aldrich,目录号:A9518-25G)
  21. 卡那霉素一硫酸盐(MP Biomedicals,目录号:02150029)
  22. 盐酸四环素(Sigma-Aldrich,目录号:T7660)
  23. 壮观霉素二盐酸盐五水合物(Sigma-Aldrich,目录号:S9007-5G)
  24. 蒸馏水,去离子水
  25. 硫酸镁(MgSO 4)(Sigma-Aldrich,目录号:M2643-500G)
  26. KOD热启动DNA聚合酶(EMD Millipore,目录号:71-086-3)
  27. QIAquick凝胶提取试剂盒(QIAGEN,目录号:28704)
  28. NheI-HF限制酶(New England Biolabs,目录号:R3131S)
  29. PmeI限制酶(New England Biolabs,目录号:R0560S)
  30. MluI-HF限制酶(New England Biolabs,目录号:R3198S)
  31. 酵母提取物(Thermo Fisher Scientific,Fisher Scientific,目录号:BP1422-2)
  32. 蛋白胨(Thermo Fisher Scientific,Fisher Scientific,目录号:BP1420-2)
  33. 甘油(Thermo Fisher Scientific,Fisher Scientific,目录号:BP229-4)
  34. 聚乙二醇3350(PEG 3350)(Sigma-Aldrich,目录号:202444)
  35. 乙酸锂(LiOAc)(Sigma-Aldrich,目录号:517992)
  36. 来自鲑鱼睾丸的DNA钠盐(Sigma-Aldrich,目录号:D1626)重悬于TE缓冲液(2mg/ml)中
  37. Tris-HCl,pH8.0(Promega,目录号:H5123)
  38. EDTA(Sigma-Aldrich,目录号:E5134-500G)
  39. QIAprep Spin Miniprep Kit(QIAGEN,目录号:27104)
  40. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S3014-500G)
  41. 氯化钾(KCl)(Sigma-Aldrich,目录号:P5405-250G)
  42. 乙酸钠(NaOAc)(Sigma-Aldrich,目录号:S2889)
  43. 选择性抗生素
    卡那霉素一硫酸盐(1,000x储备液)(见配方)
    氨苄青霉素钠盐(1,000x原液)(见配方)
    盐酸四环素(1,000x原液)(参见配方)
    壮观霉素二氢氯化物五水合物(1,000x储备液)(见配方)
  44. CSM-Ura选择性培养基(参见配方)
  45. YEPD媒体(见配方)
  46. TE缓冲区(参见配方)
  47. SOB肉汤(见配方)
  48. SOC肉汤(见配方)

设备

  1. 100-150ml锥形瓶
  2. Vortex(Scientific Industries,目录号:SI-0136)
  3. 孵育器,设置为37°C和30°C
  4. 水浴或加热块,设置为42°C,37°C和30°C
  5. 微量离心机(Eppendorf,型号:5424)
  6. 大型离心机,能够处理高达50毫升(用于制造胜任的酵母和细菌)
  7. 电泳系统
  8. 紫外透射仪
  9. 热循环仪(Bio-Rad Laboratories,型号:PTC 200)或任何其他常规热循环仪
  10. GenePulser II电穿孔系统(Bio-Rad Laboratories)或其他电穿孔装置
  11. Nanodrop Lite分光光度计(Thermo Fisher Scientific,型号:NanoDrop Lite)或适用于定量DNA浓度的任何其他设备/方法
  12. 滚筒或振动器

程序

第I部分:生成捕获向量

注意:本节的目的是创建一个捕获载体(图1),一种随后可用于捕获DNA的大片段的质粒(第二部分)。在设计方案的这一点上,需要做出关于是否设计所得捕获载体以包括侧翼独特的"着陆垫"序列的决定,如果最终目标是将DNA片段引入使用先前由Kuhlman和Cox(2010)(第III部分)详细描述的Landing Pad技术在细菌染色体内定义位点。

  1. 待重组的DNA的制备以产生捕获载体
    1. DNA片段概述
      如图1所示,通过经由酵母内源性同源重组结合DNA的以下4个片段来装配捕获载体:线性化载体,加上3个PCR产生的感兴趣的片段:靶向序列1(TS1),靶向序列2(TS2)和含有氨苄青霉素抗性(β-内酰胺酶)的盒
      1. 载体骨架:pLLX13是9.94kb酵母/em。大肠杆菌穿梭载体,其携带复制起点和选择标记以支持在细菌(oriV-incP 和 tetRA )和酵母(CEN和 URA3)以及促进细菌通过共轭转移的转移起始( oriT )序列(Wolfgang等人,2003)(图1C) 。
      2. 靶向序列1(TS1)和2(TS2):与欲捕获和克隆的区域的上游和下游边缘具有同源性的DNA的1kb区域。使用在一端添加40个碱基对与线性化pLLX13的同源性的引物,以及在另一端包含几个限制性位点的间隔区,通过PCR产生TS1和TS2(图1A,表1)。这可以通过一轮PCR实现。然而,如果期望DNA的捕获区域的染色体整合,则应该将靶向序列侧翼的着陆区序列并入PCR产物中。嵌套PCR用于将这些着陆垫序列插入靶向序列和pLLX13同源区之间(图1B,表1)。
        注意:
        1. 如图1C所示,可以如Kuhlman和Cox(2010)中所述使用存在于接头焊盘序列侧翼的捕获载体中的I-SceI限制性位点,以释放捕获DNA并将其靶向先前设计的接头垫位点。请参阅第III部分和图3。
        2. 结合到TS1 PCR的3'末端和TS2 PCR的5'末端的间隔区提供了用于与氨苄青霉素抗性盒重组(以下)和捕获载体的装配的同源DNA序列。另外,间隔区含有连续的限制性消化位点,用于在与靶DNA序列重组之前线性化捕获载体(图2)。 TS1间隔区包括PmeI和HpaI限制性序列,TS2间隔区包括MluI和PacI限制性位点(图1C,表1)。如果需要,可以通过改变引物序列,即如果位点包含在TS1和/或TS2中,则可以用其它限制性位点替换这些限制性位点。
      3. Amp 盒:插入在TS1和TS2之间的DNA区域,以便利用酵母重组装配捕获载体质粒后使用细菌转化来选择捕获载体质粒。使用提供与靶序列1和2间隔区序列的同源性的引物扩增来自pLLX8的Amp 携带片段。当共转化到感受态细胞中时,这些DNA序列上的侧翼同源性使其能够通过同源重组装配。酿酒厂。


        图1.用于产生捕获载体的基于酵母重组的策略的示意图。A.使用所描述的引物通过PCR产生具有小重叠同源区域的片段。重叠的同源序列促进酵母中的重组和装配。这些片段包括与5'和3'靶向序列(TS1和TS2)间隔区(灰色)加上氨苄青霉素抗性盒(Amp )(绿色)同源的那些。可以通过包括一轮嵌套PCR将接头垫(LP)序列(蓝色)引入捕获载体。初始PCR扩增向间隔区和侧翼着陆区序列引入同源性,而第二轮PCR向pLLX13载体主链引入同源性。当引入酵母中时,3个PCR产生的包含重叠同源序列和线性化(NheI消化的)pLLX13载体骨架的片段经历同源重组以产生可通过在CSM-URA培养基上生长而选择的捕获载体。可以收获所得的捕获载体质粒,并在E中进行选择。通过在含有氨苄青霉素和四环素的培养基上电镀大肠杆菌。分离靶序列和氨苄青霉素抗性盒的间隔区(灰色)将PmeI和MluI限制性位点引入捕获载体,其可用于确认正确的捕获载体装配。或者,SceI限制性位点可用于释放整个组装的5kb片段。此外,用箭头表示的寡核苷酸pLLX F和pLLX R(表1)可用于对装配的捕获载体进行测序,以确认发生了适当的重组介导的事件。

    2. PCR产生的片段(TS1,TS2,AMP R )的制备
      1. 使用高保真聚合酶的通用PCR反应混合物:
        16μl蒸馏水
        2.5μlKOD 10x反应缓冲液
        2.5μldNTP(各2mM) 1.5μl25mM MgSO 4 0.5μl正向寡核苷酸20μM(见下文和表1中的寡核苷酸描述) 0.5μl反向寡核苷酸20μM
        1μl模板DNA(50-250ng基因组DNA和1-10ng质粒DNA)
        0.5μlKOD热启动聚合酶(1 U /μl)
      2. 寡核苷酸和模板DNA
        注意:选择正向寡核苷酸(+/- LP)取决于是否需要靶DNA的下游染色体整合。
        1. TS1 PCR(+/- LP):
          正向寡核苷酸(+ LP):5_LP1_TS1(寡核苷酸在表1中描述)
          正向寡核苷酸(-LP):5_pLLX_TS1
          反向寡核苷酸:3_TS1
          模板:基因特异性模板如细菌染色体或毒力质粒DNA
        2. TS2 PCR(+/- LP):
          正向寡核苷酸:5_TS2
          反向寡核苷酸(+ LP):3_LP2_TS2
          反向寡核苷酸(-LP):3_pLLX_TS2
          模板:基因特异性模板如细菌染色体或毒力质粒DNA
        3. 氨苄青霉素抗性盒PCR:
          前向寡核苷酸:5_Amp
          反向寡核苷酸:3_Amp
          模板:pLLX8或含有用于选择的所需抗生素抗性盒的其他质粒
        4. PCR谱:
          注意:根据需要,可以修改这些条件以使用不同的聚合酶或基因特异性引物。
          1个循环,94℃,5分钟
          30个循环,94℃30秒; 55℃30秒; 72℃2-3分钟
          1个循环,72℃10分钟
      3. 预期分段
        TS1和TS2 PCR产物应为〜1kb,Amp R 盒〜3kb。
      4. 嵌套PCR(如果添加侧翼着陆点[LP]序列)
        准备与上述相同的KOD热启动聚合酶PCR混合物,并进行以下引物和模板修饰:
        1. 嵌套的TS1 PCR寡核苷酸:
          正向寡核苷酸:5_pLLX_LP1
          反向寡核苷酸:3_TS1
          模板:1μl纯化,确认TS1 PCR
        2. 嵌套的TS2 PCR寡核苷酸:
          正向寡核苷酸:5_TS2
          反向寡核苷酸:3_pLLX_LP2
          模板:1μl纯化的,证实的TS2 PCR
      5. 嵌套PCR分析
        TS1和TS2巢式PCR产物仍然约为1kb,但是现在含有与用于下游重组反应的pLLX13载体骨架同源的区域。
      6. 使用QIAquick凝胶提取试剂盒或其他等效方法清洁PCR产物
    3. 线性化载体DNA的制备
      1. 用NheI-HF限制酶消化1μg的pLLX13载体
      2. 凝胶纯化消化的质粒DNA,并在Nanodrop Lite分光光度计或适用于DNA定量的其他设备/方法上定量浓度。

        表1.引物序列(5'-3'序列)

        这些序列应该添加到基因特异性序列的5'末端(TS1)或3'末端(TS2),以产生可以容易地重组以在酵母中形成捕获载体的PCR产物。
        b

        NNN,表示应添加目标DNA特异性序列。
        ,着陆垫顺序
        下划线
        ,与pLLX8氨苄青霉素抗性盒编码载体的同源性。斜体序列可以替代含有所需抗生素抗性盒的选择载体。

  2. 使用酵母重组产生捕获载体
    1. 感受态酵母的制备(Gietz和Woods,2002)
      1. 第1天:接种具有大宿主宿主的5ml YEPD肉汤。酿酒酵母菌株,并在滚筒上于30℃温育过夜
      2. 第2天:在分光光度计上测量培养物OD 600,并在100-150ml锥形瓶中将其稀释(传代培养)至50ml新鲜YEPD肉汤中至OD 600,为0.3。
      3. 在30℃下在振荡器上孵育酵母3-5小时,直到酵母细胞经历两次倍增,达到〜1.2的OD 600.
      4. 通过在大离心机中以850×g离心3分钟收获酵母。倾析上清液。
        注意:以更高的x g离心会使酵母颗粒更难以重悬。
      5. 将酵母悬浮于25ml无菌水中
      6. 在大离心机中以850×g离心3分钟以沉淀酵母。倾析上清液。
      7. 重悬在1毫升无菌水中的酵母。
        注意:剩余的酵母可以重悬于10%甘油中,并在-80℃冷冻备用。
    2. 酵母转化
      1. 使用台式离心机在850×g离心1分钟,离心100微升感受态酵母在微量离心管中,并除去上清液。
      2. 用240μl过滤灭菌的50%PEG 3350(w/v)覆盖酵母 注意:PEG 3350是粘稠的,使用塑料基材料移液。
      3. 组装转化混合物组成:
        1. 36微升1 M LiOAc
        2. 10μl来自鲑鱼睾丸(2mg/ml)的DNA钠盐(ssDNA),重悬于TE缓冲液中。
          注意:第一次使用前,煮ssDNA 5分钟,在冰上冷却。
        3. DNA转化混合物:
          100ng线性化,NheI消化的pLLX13载体
          200ng靶向序列1(TS1)扩增片段
          200ng靶向序列2(TS2)扩增片段
          600ng含有氨苄青霉素抗性基因的扩增片段 蒸馏去离子水至最终体积为65μl
          注意:第二种DNA混合物也可以作为对照组装,其仅含有100ng线性化,NheI消化的pLLX13载体。
      4. 将DNA转化混合物覆盖在PEG和移液管或涡旋上,直到酵母团块完全重悬
      5. 在30℃下在水浴或加热块中孵育酵母30分钟
      6. 将管转移到42℃水浴或加热块中,再孵育15分钟
      7. 通过在2,400×g离心1分钟收获细胞。
      8. 重悬在100微升无菌TE缓冲液或水和板的沉淀在CSM-URA板上。使用无菌大(5mm)玻璃珠在平板上铺展。
      9. 在30°C孵育器孵育长达5天。菌落通常在2-3天内出现,但生长时间可能不同。
    3. 分离重组捕获载体
      1. 使用板刮板或无菌移液管尖重新悬浮存在于转化板上的500微升YEPD培养基中的所有酵母菌落。将混合物转移到1.7ml微量离心管中,并通过在2,400×g离心1分钟收获细胞。
      2. 倾析上清液和裂解酵母以使用如下修改的QIAprep Spin MiniPrep Kit方案提取组装的捕获载体:
      3. 将酵母悬浮于250μlP1缓冲液中
      4. 添加250微升的小(425-600微米)酸洗玻璃珠到管和涡流在最大速度设置5分钟,以裂解酵母。
      5. 允许〜1分钟的玻璃珠沉降,然后将酵母裂解液转移到新的1.7毫升微量离心管中。
      6. 加入250μlP2缓冲液,通过颠倒混合4-6次,并在室温下孵育5分钟
      7. 加入350μlN3缓冲液,反转4-6次混合,并在16,500×g下旋转10分钟。
      8. 将上清液转移到QIAprep柱和收集管上,并在16,500×g下旋转30秒。
      9. 倾析流过和用750μlPE洗涤缓冲液洗涤柱。旋转16,500英寸x g 30秒。
      10. 丢弃通过溶液的流,并重新组装离心柱和收集管。旋转16,500英寸x g 1分钟以干燥色谱柱。
      11. 将转移柱转移到新鲜的,标记的1.7ml微量离心管中
      12. 加入40μlEB缓冲液(洗脱缓冲液),在室温下孵育1分钟,并以16,500×g进行最终旋转1分钟。
      13. 混合3微升洗脱的DNA用E。大肠杆菌 ElectroMAXDH10β细胞(或任何合适的感受态大肠杆菌细胞)。
      14. 转移DNA和细菌混合物到0.1毫米电穿孔小杯。
      15. 使用GenePulser II(Bio-Rad Laboratories)或等效电穿孔装置使用制造商的设置进行电穿孔。
        注意:对于GenePulser II,使用1.8 kV和25μF设置与大肠杆菌。
      16. 加入450微升的SOC并孵育1小时在37℃加热块或水浴。
      17. 在含有四环素和氨苄青霉素的LB平板上转化细菌,以选择组合了所有4个DNA片段的重组质粒。转化后一天可能出现菌落,但有时可能需要额外一天才能出现
      18. 为了筛选候选捕获载体集落,将至少4个单菌落分别接种到含有氨苄青霉素和四环素的10ml LB肉汤中,并在37℃下生长过夜。
        注意:我们建议在此步骤使用更大体积的小量制备(最多10 ml),因为pLLX13载体骨架的低拷贝通常导致较低的DNA产量。然后可以将来自所有10ml培养物的细菌沉淀物合并成单个小量制备物。
      19. 第二天早上,使用QIAprep Spin Miniprep Kit按照制造商的说明书收获质粒DNA
  3. 数据分析 >:确认捕获矢量
    的正确组装 从单个细菌转化体中分离的质粒可以通过以下方式分析完整的重组捕获载体的存在:
    1. 用SceI消化以释放插入的(〜5kb)TS1-Amp R -TS2片段(图1C)。
    2. 用PmeI和MluI消化以释放(〜3kb)Amp R 盒。
    3. 使用PCR确认TS1和TS2的存在。
    4. 用寡核苷酸pLLX F和pLLX R测序重组的接头(表1和图1)。

第二部分:使用λRed同源重组捕获靶序列

  1. 准备转移的捕获载体DNA
    使捕获载体线性化以产生DNA片段,使得TS1和TS2作为游离末端可接近,以在引入细菌时促进重组。这将极大地提高λRed重组的效率,并且在重组之前起到去除氨苄青霉素抗性盒(Amp R )的作用(图2A)。
    1. 用PmeI和MluI消化1μgpLLX13-TS1-amp-TS2捕获载体DNA。 注意:HpaI可以用作PmeI的替代品,PacI可以用作MluI的替代品。
    2. 凝胶使用QIAquick凝胶提取试剂盒纯化试剂盒根据制造商的说明书纯化〜10kb线性化的捕获载体DNA。
    3. 使用Nanodrop Lite分光光度计或其他DNA定量方法定量回收的线性化DNA
  2. 引入要捕获到的靶DNA的来源。大肠杆菌 ElectroMAXDH10β细胞
    对于以下方案,必须首先将靶DNA工程化为含有选择性标记以促进基于重组的捕获事件的选择。例如,Shigella 毒力质粒含有Kan R 盒代替ipaJ (Reeves等人 2015)。
    注意:
    1. 如果需要去除抗生素抗性盒以用于下游应用,可以包括侧接抗生素抗性盒的FRT(FLP识别靶)位点,如Datsenko和Wanner(2000)中所述。可能通过重组捕获DNA的未标记区域,然而这里没有调查这种可能性。
    2. 不是绝对必要的将DNA来源转移到 E。如果DNA来源存在于可以有效地进行λRed重组的细菌物种中,则可以使用大肠杆菌。然而,根据我们的经验,执行这一步骤大大提高了DNA捕获的效率和成功。
    1. 通过电穿孔将标记的粘粒或毒力质粒转化到ElectroMAXDH10β细胞中,并在含有卡那霉素(或合适的抗生素抗性标记)的LB平板上选择。 注意:只有使用这些商业上可获得的细胞,我们才成功地将大的220kb志贺氏菌属毒力质粒引入到实验室菌株E/E中。大肠杆菌 。

  3. 使用λ红色重组系统捕获感兴趣的DNA序列(图2B)
    1. 为了能够在靶细菌菌株中表达同源重组系统,首先通过标准电穿孔将质粒pKD46(Datsenko和Wanner,2000)引入菌株,并在含有氨苄青霉素和0.2%葡萄糖的固体培养基上进行选择(以抑制λRed表达)。
      注意:pKD46是温度敏感的氨苄青霉素抗性质粒,其在阿拉伯糖 - 诱导型启动子的控制下携带λ红色重组酶的等位基因。也可以使用编码λRed重组酶的其他质粒(Murphy和Campellone,2003)
      2.示意图说明如何使用捕获载体将目标DNA序列分离到酵母产生的捕获载体上。   A。捕获载体通过用PmeI和MluI消化而线性化,以产生对应于TS1和TS2 DNA的游离DNA末端,以增强重组效率并除去氨苄青霉素抗性盒。 B.线性化的DNA被引入 E。大肠杆菌 ="" 菌株,其含有编码含有可选择标记的靶dna序列的毒力质粒或粘粒;<=""> ie ie ,pKD46(未示出)。先前将抗生素选择标记引入靶DNA中有助于在DNA片段整合到捕获载体中之后选择DNA片段。 λRed重组酶的表达导致目标DNA通过同源重组(虚线)转移到线性化捕获载体上。

    2. 第1天:将携带靶DNA和pKD46的单个卡那霉素氨苄青霉素抗性菌落接种到含有卡那霉素,氨苄青霉素和0.2%葡萄糖的LB肉汤中,并在30℃的滚筒上生长过夜。
    3. 第2天:在早上,将含有细菌1:100的pKD46稀释到含有卡那霉素,氨苄青霉素和0.2%阿拉伯糖的50ml SOB肉汤(以诱导λRed重组酶表达)中。在振荡器上在30℃下孵育约3-4小时,直到培养物达到OD 600的0.6。
    4. 生成电感受态细胞
      1. 将细胞转移到无菌瓶中,并在6,000×g离心10分钟,在4℃。
      2. 滗析上清液和重悬细菌在25毫升冰冷的10%甘油。
        注意:在整个过程中,通过将管子放在冰上的洗涤步骤之间,让细菌沉淀冷。
      3. 在6,000xg下重复离心10分钟。
      4. 倾析上清液并重复10%甘油洗涤3次以上,共4次洗涤 注意:如果需要,4次洗涤可以用冰冷的无菌水代替10%甘油进行(步骤C6-C8)。
      5. 将最终的沉淀重悬于500μl冷的10%甘油中
    5. 转移100微升电感受态重组酶表达细胞到新的微量离心管中
    6. 加入100 ng线性化,凝胶纯化的捕获载体(图2),并将细菌细胞和DNA的混合物转移到0.1 mm比色杯中。
    7. 使用GenePulser II(Bio-Rad Laboratories)或等效电穿孔装置根据制造商的设置进行电穿孔。
    8. 添加450微升SOC肉汤到电穿孔的细胞,并转移到微量离心管。
    9. 在37℃下在加热块或水浴中孵育细胞1小时
    10. 在含有四环素(以选择捕获载体骨架)和卡那霉素或合适的抗生素(以选择待捕获的靶DNA的区域)的LB平板上铺板整个转化,并在37℃孵育过夜。
      注意:
      1. 如果过夜孵育后没有菌落产生,继续孵育另外24小时,因为有时重组过程可以减缓菌落的生长。
      2. 在37℃下孵育应消除温度敏感的pKD46λRed重组酶表达质粒,产生氨苄青霉素易感的转化体。如果需要,可以通过修补含有氨苄青霉素的LB上的单个菌落来确认pKD46的缺失。
    11. 将几个候选四环素/卡那霉素抗性菌落接种到含有卡那霉素和四环素的10ml LB肉汤中,并在37℃下生长过夜。
    12. 第二天,使用QIAprep Spin Miniprep试剂盒或其他合适的小量制备试剂盒收集DNA。
      注意:该步骤提供了从毒力质粒和基因组DNA中排除重组的"捕获的"含DNA质粒的大小排阻。
    13. 将收获的质粒转化到ElectroMAXDH10β细胞或其他合适的电感受态细菌宿主菌株中,并在含有四环素和卡那霉素的LB平板上平板。
      注意:
      1. 我们推荐ElectroMAXDH10β细胞用于此目的,因为这些细胞在大DNA构建体的转化和增殖中是有效的。
      2. 重要的是,如果追踪将捕获的靶DNA整合到细菌染色体的着陆点位置,可以将收获的质粒直接转化到含着陆垫的宿主菌株中(参见第III部分)。然而,如果用户不能直接成功转化着陆垫宿主菌株(即,因为含有捕获的靶DNA的质粒非常大),我们建议首先转化ElectroMAXDH10β细胞,然后使用接合将质粒转移到含着床垫的宿主菌株。
      3. 一旦转化到宿主菌株中,如果需要,确认的大质粒可以通过DNA缀合在细菌菌株之间转移。

  4. 数据分析 >:确认将目标DNA片段转移到捕获载体上
    1. 我们建议使用几种补充方法来确认正确的靶DNA片段已转移到捕获载体上。根据我们的经验,接近100%的质粒从E中回收。携带两种抗生素标记的大肠杆菌含有正确的目标DNA片段
    2. 候选质粒应通过以下筛选正确的重组事件:
      1. (即使用pLLXF或pLLXR寡核苷酸[表1和图1]的PCR)和合适的靶DNA寡核苷酸的PCR分析。
      2. 使用基因特异性引物对应当包括在DNA捕获区域中的序列进行PCR分析。应该包括阳性和阴性对照,例如,使用空捕获载体作为阴性对照的PCR模板,并且包含靶DNA区段的亲本细菌菌株作为阳性对照。
      3. 可以进行使用特异性结合起始毒力质粒或粘粒特异性区域但不存在于捕获的DNA区域中的引物的PCR反应,以证实亲本毒力质粒或粘粒的丧失。
      4. 使用靶基因特异性寡核苷酸,pLLXF或pLLXR对捕获的靶DNA进行序列分析。

第三部分:将目标DNA整合到细菌染色体上的着陆点位置

注意:

  1. 本部分描述了如何将来自捕获载体的大片靶DNA转移到含有着陆垫位点的细菌菌株的染色体中。在Kuhlman和Cox(2010)的方法论文中详细描述了将着陆点位点引入细菌染色体中,因此这里不讨论。因此,为了继续本方案的第III部分,需要含有整合着陆点位点的细菌菌株,例如大肠杆菌DH10β。
  2. E。在atp/gidB基因座处具有整合着陆点位点的大肠杆菌DH10β可在Addgene,目录号:83036获得。着陆点位点还将四环素抗性盒引入染色体中(图3)。
  1. 制备用于将捕获的靶DNA整合入染色体中的着陆区位点的细菌菌株 注意:
    1. 为了将靶DNA转移到细菌染色体的着陆点位点,捕获的靶DNA质粒必须含有通过巢式PCR引入的侧翼着陆序列(参见第I部分和图1B)。
    2. 着陆垫宿主菌株必须含有两个质粒:(1)含有捕获的靶DNA序列(在第II部分中产生和证实)的pLLX13捕获载体和(2)pTKred,一种壮观霉素抗性的温度敏感性质粒, IPTG驱动的λ红色重组酶和阿拉伯糖驱动的SceI限制性内切核酸酶。该方案使用在atpI/gidB基因座具有着陆点位点的大肠杆菌DH10β。如果捕获的靶DNA质粒(来自部分II)已经被引入到部分II中的大肠杆菌DH10β-着陆垫中,则通过直接进入步骤B(将pTKred质粒引入大肠杆菌中)将pTKred引入菌株中开始该方案含有捕获的靶DNA的DH10β-着陆垫细菌)。
    1. 将含有靶DNA的捕获载体质粒导入EM。大肠杆菌DH10β-着陆垫细胞
      1. 第1天:接种单个四环素抗性的E。大肠杆菌DH10β-接合垫菌落到含有四环素的LB肉汤中,并在37℃的滚筒上生长过夜。
      2. 第2天:早上,向后稀释 E。大肠杆菌DH10β-着陆垫细菌1:100加入含有四环素的10ml LB肉汤中。在滚筒或摇动器上在37℃下孵育约1-2小时,直到培养物达到0.6的OD 600。
      3. 生成电子能力。大肠杆菌DH10β-着陆垫细胞:
        1. 在冰上孵育细胞1小时。
        2. 将细胞转移到无菌瓶中,并在6,000×g离心10分钟,在4℃。
        3. 滗析上清液和重悬细菌在5毫升冰冷的10%甘油。
          注意:在整个过程中,通过将管子放在冰上的洗涤步骤之间,让细菌沉淀冷。
        4. 在6,000xg下重复离心10分钟。
        5. 倾析上清液并重复10%甘油洗涤3次以上,共4次洗涤 注意:如果需要,4次洗涤可以用冰冷的无菌水代替10%甘油进行。
        6. 将最终沉淀重悬在200μl冷的10%甘油中。
      4. 将100μl电感受态细胞转移到新的微量离心管中
      5. 加入200ng含有捕获的靶DNA的质粒(图2B),并将细菌细胞和DNA的混合物转移到0.1mm比色杯中。
      6. 使用GenePulser II(Bio-Rad Laboratories)或等效电穿孔装置根据制造商的设置进行电穿孔。
      7. 添加450微升SOC肉汤到电穿孔的细胞,并转移到微量离心管
      8. 在37℃下在加热块或水浴中孵育细胞1小时
      9. 在含有四环素(以选择染色体上的着陆点位点)和卡那霉素或合适的抗生素(以选择捕获的靶DNA的区域)的LB平板上铺板整个转化,并在37℃下孵育过夜。

  2. 将pTKred质粒导入em。大肠杆菌DH10β-着陆垫细菌含有捕获的目标DNA
    1. 第1天:接种单个菌落的四环素/卡那霉素抗性E。将含有捕获的靶DNA质粒的大肠杆菌DH10β-着陆垫细菌加入含有四环素和卡那霉素的LB肉汤中,并在37℃的滚筒上生长过夜。
    2. 第2天:早上,背部稀释 E。将含有捕获的靶DNA质粒1:100的大肠杆菌DH10β-着陆垫细菌加入到10ml含有四环素和卡那霉素的LB肉汤中。在滚筒或摇动器上在37℃下孵育约1-2小时,直到培养物达到0.6的OD 600。
    3. 生成电感受态细胞
      1. 在冰上孵育细胞1小时。
      2. 将细胞转移到无菌瓶中,并在6,000×g离心10分钟,在4℃。
      3. 滗析上清液和重悬细菌在5毫升冰冷的10%甘油。
        注意:在整个程序中,通过将管置于冰之间的洗涤步骤之间保持细菌沉淀冷。
      4. 在4℃下,以6000xg重复离心10分钟。
      5. 倾析上清液并重复10%甘油洗涤3次以上,总共4次洗涤。
        注意:如果需要,4次洗涤可以用冰冷的无菌水代替10%甘油进行。
    4. 加入100 ng pTKred质粒,并将细菌细胞和DNA的混合物转移到0.1 mm比色皿中。
    5. 使用GenePulser II(Bio-Rad Laboratories)根据制造商的设置的等效电端口装置进行电穿孔。
    6. 添加450微升SOC肉汤到电穿孔的细胞,并转移到微量离心管
    7. 孵育细胞在30℃下1小时。
      注意:pTKred对温度敏感,因此必须在30℃下进行生长和电镀。
    8. 在含有四环素(以选择染色体上的着陆点),卡那霉素(以选择捕获的靶DNA质粒),壮观霉素(选择pTKred)和0.2%D-葡萄糖(抑制表达λRed重组酶)并在30℃下孵育过夜。

  3. 将目标DNA插入染色体上的着陆垫
    注意:
    1. 协议的这部分修改从Kuhlman和Cox(2010)。在该步骤中,使用阿拉伯糖诱导SceI限制酶的表达,而IPTG诱导λRed重组酶的表达。 SceI在着陆垫整合的位点处将双链DNA断裂引入大肠杆菌DH10β染色体中。同时,SceI在捕获的靶DNA质粒上引入双链DNA断裂。 λRed重组酶的表达导致染色体上的着陆位点和存在于靶DNA的游离末端的那些之间的同源重组。成功的SceI消化和同源重组导致染色体的双链断裂修复,四环素盒的切除和靶DNA序列在其位置的整合(图3)。双链断裂的引入产生DNA的游离末端,大大增加同源重组的效率。
    2. 18bp SceI限制性识别位点不自然存在于大肠杆菌染色体中。
    1. 第1天:早上,用单一集落的E接种5ml含有0.5%甘油,2mM IPTG和0.2%阿拉伯糖的SOB肉汤。含有捕获的靶DNA质粒和pTKred的大肠杆菌DH10β-着陆垫菌株。在滚筒或摇床上在37℃下生长1小时
    2. 向肉汤中加入5μl壮观霉素(100mg/ml),并在滚筒或振荡器上在30℃下孵育4小时。
    3. 向肉汤中加入5μl卡那霉素(100mg/ml),并在滚筒或振荡器上在30℃下孵育过夜。
    4. 第2天:在10℃-1至10℃下在LB肉汤中进行系列稀释的过夜培养物,并将100μl细胞铺板于含有卡那霉素的LB平板上。 >
    5. 在37℃下孵育平板过夜
    6. 第3天:将含卡那霉素抗性菌落铺在含有四环素和含有卡那霉素的LB平板的LB平板上,筛选已成为四环素易感的菌落。
    7. 在37℃下孵育平板过夜。
    8. 具有卡那霉素抗性和四环素敏感性的菌落是在着陆点位点成功靶DNA整合的候选物。 (即存在于染色体上的四环素抗性盒已被靶DNA序列替代)。
      注意:在37℃下孵育也应该消除温度敏感的pTKred质粒。 pTKred的缺失可以通过将菌落接种到含有壮观霉素的LB平板上来确认。如果在37℃下生长不足以使pTKred质粒下降,则单个菌落可以在42℃下生长4小时或过夜,然后在37℃下再次接种到含有卡那霉素的LB平板上,然后重新测试壮观霉素敏感性。在较高温度下传代应当确保pTKred的损失。

  4. 数据分析 >:确认靶DNA整合到染色体着陆垫位点
    可以通过以下方式分析个体集落中是否存在整合的靶DNA:
    1. 使用引物进行PCR分析以评估染色体中连接区域的整合。如果 E。使用大肠杆菌DH10β-着陆垫菌株,atpI/gidB F和atpI/gidB R寡核苷酸(图3和表1)可与靶基因特异性引物一起使用以评估连接。
    2. 使用引物进行PCR分析以评估整合菌株中预期靶DNA基因的存在或不存在。
    3. 如果合适,可以对整合的靶DNA基因进行功能分析

      图3.说明靶DNA从捕获载体转移到在着陆点位点的细菌染色体中的示意图。捕获的靶DNA序列可以整合到细菌宿主菌株的染色体中,所述菌株含有集成着陆垫站点。着陆点位置1(LP1)和2(LP2)被示出为蓝色框。必须将两种质粒引入宿主菌株:(1)含有侧翼为着陆垫位点和SceI限制位点的靶DNA的捕获载体(见设计的第I部分和表1)和(2)pTKred,其编码IPTG诱导型λRed重组酶和阿拉伯糖可诱导的SceI限制性内切核酸酶(未示出)。用阿拉伯糖诱导SceI导致双链DNA断裂进入宿主菌株染色体和邻近着陆点位点的捕获的靶DNA上。然后通过IPTG诱导的λRed重组酶表达(虚线)通过同源重组连接DNA的游离末端。可以通过筛选卡那霉素抗性和四环素敏感性来鉴定含有靶DNA正确插入的细菌分离物,这表明来自染色体的着陆点间插序列和含有靶DNA的原始捕获载体都失去了。原始的1kb靶向序列1(TS1)由紫色框表示,靶向序列2(TS2)由红色框表示。原始的pLLX13同源序列表示为黑框。用atpI/gidB F或atpI/gidB R寡核苷酸(由箭头表示)(表1)和靶DNA特异性寡核苷酸的PCR分析可用于确认着陆点位点处的整合。

笔记

  1. 额外的应用:捕获染色体DNA的片段可以通过修改如Wolfgang等人所述的该方案来实现。 (2003)。例如,捕获载体可以设计为包括与细菌染色体上的区域同源的靶向序列
  2. DNA中的重复元件。在设计初始捕获载体靶向序列(TS1和TS2)时应该努力以确保1kb的靶序列是独特的,并且不包括重复元件或转座子序列,因为这可以影响λRed重组的特异性反应。

食谱

  1. 卡那霉素一硫酸盐(1,000x储备液)
    30mg/ml在dH 2 O中,过滤除菌
  2. 氨苄青霉素钠盐(1,000x原液)
    100mg/ml在dH 2 O中,过滤除菌
  3. 盐酸四环素(1,000x原液)
    12.5mg/ml在100%甲醇中,过滤灭菌
  4. 壮观霉素二氢氯化物五水合物(1,000x原液)
    100mg/ml在dH 2 O中,过滤除菌
  5. CSM-URA选择性培养基(每升900 ddH 2 O)
    6.7克酵母氮源与硫酸铵
    0.77克CSM-URA,尿嘧啶缺失补充剂
    〜20g琼脂
    在倒板前加入100ml无菌20%葡萄糖
  6. YEPD(每升ddH 2 O)
    10g酵母提取物
    20 g蛋白胨)
    20克D-葡萄糖
    〜15g琼脂
  7. TE缓冲区
    10mM Tris-HCl,pH8.0 1.0 mM EDTA
  8. SOB肉汤(每升ddH 2 O)
    5g酵母提取物
    20克胰蛋白酶
    0.584g NaCl
    0.186克KCl
    2.4g MgSO 4 使用前调整pH值为7.5
  9. SOC肉汤(每升ddH 2 O)
    向990ml SOB肉汤中加入10ml无菌20%D-葡萄糖溶液

致谢

该协议改编自Reeves等人。 (2015)。工作由R01AI064285,R21AI103882和马萨诸塞州总医院研究学者奖2016支持到CFL。 AZR由MGH ECOR医学发现奖学金基金支持。

参考文献

  1. Datsenko,KA和Wanner,BL(2000)。  One使用PCR产物在大肠杆菌K-12中染色体基因的步骤失活。 Proc Natl Acad Sci USA 97(12):6640-6645。
  2. Gietz,RD和Woods,RA(2002)。  转型酵母通过乙酸锂/单链载体DNA /聚乙二醇法。方法Enzymol 350:87-96。
  3. Kuhlman,TE和Cox,EC(2010)。  网站 - 特异性染色体整合大合成构建体。核酸研究38(6):e92。
  4. Murphy,KC and Campellone,KG(2003)。  Lambda红介导的肠创伤和肠致病性E的重组工程化。大肠杆菌。 BMC Mol Biol 4:11.
  5. Reeves,AZ,Spears,WE,Du,J.,Tan,KY,Wagers,AJand Lesser,CF(2015)。  将大肠杆菌工程化为哺乳动物细胞的蛋白质递送系统。


    ACS Synth Biol 4 (5):644-654
  6. Wolfgang,MC,Kulasekara,BR,Liang,X.,Boyd,D.,Wu,K.,Yang,Q.,Miyada,CG和Lory,S。(2003)。  绿脓假单胞菌的临床和环境分离物中的基因组内容和毒力决定簇的保存 。 Proc Natl Acad Sci USA 100(14):8484-8489。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Reeves, A. Z. and Lesser, C. F. (2016). Transfer of Large Contiguous DNA Fragments onto a Low Copy Plasmid or into the Bacterial Chromosome. Bio-protocol 6(22): e2002. DOI: 10.21769/BioProtoc.2002.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。