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Mar 2020
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Rapid Genome Engineering of Pseudomonas Assisted by Fluorescent Markers and Tractable Curing of Plasmids
受荧光标记和易于固化质粒影响的假单胞菌快速基因组工程    

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Abstract

Precise genome engineering has become a commonplace technique for metabolic engineering. Also, insertion, deletion and alteration of genes and other functional DNA sequences are essential for understanding and engineering cells. Several techniques have been developed to this end (e.g., CRISPR/Cas-assisted methods, homologous recombination, or λ Red recombineering), yet most of them rely on the use of auxiliary plasmids, which have to be cured after the editing procedure. Temperature-sensitive replicons, counter-selectable markers or repeated passaging of plasmid-bearing cells have been traditionally employed to circumvent this hurdle. While these protocols work reasonably well in some bacteria, they are not applicable for other species or are time consuming and laborious. Here, we present a fast and versatile protocol of fluorescent marker-assisted genome editing in Pseudomonas putida, followed by clean curing of auxiliary plasmids through user-controlled plasmid replication. One fluorescent marker facilitates identification of genome-edited colonies, while the second reporter enables detection of plasmid-free bacterial clones. Not only is this protocol the fastest available for Pseudomonas species, but it can be easily adapted to any type of genome modifications, including sequence deletions, insertions, and replacements.


Graphical abstract:



Rapid genome engineering of Pseudomonas with curable plasmids



Keywords: Genome engineering (基因组工程), Synthetic biology (合成生物学), Pseudomonas (假单胞菌), Synthetic plasmid replication (合成质粒复制), Metabolic engineering (代谢工程), Gram-negative bacteria (革兰氏阴性菌)

Background

Targeted, precise genomic manipulation techniques have considerably advanced the field of microbial engineering. Such methods not only allow for assessing genotype-phenotype relationships, but also enable complex engineering of microbial cell factories. In recent years, CRISPR/Cas9 approaches have paved the way for precise genome engineering in eukaryotes. In bacteria, the use of CRISPR/Cas9 is mainly limited to its value as a counter-selection tool, as bacteria lack non-homologous end-joining to repair the double-strand breaks induced by the Cas9 nuclease. Therefore, engineering efforts in many bacteria rely on homologous recombination (HR) to alter the genome. The advantage of HR is that a broad range of alterations can be introduced in the target genome. Furthermore, it is applicable not only to the so-called model organisms, e.g., Escherichia coli and Saccharomyces cerevisiae, but also finds wide spread application in non-traditional hosts, e.g., Pseudomonas species. In this protocol, we provide a workflow for HR-based genome engineering of P. putida – paired with an advanced toolbox that includes several resistance markers – facilitated by the use of fluorescent markers that enable monitoring of every step (Wirth et al., 2020). The presented methodology relies on the co-integration of a suicide plasmid [controlled by the pir-dependent origin of replication ori(R6K)] at the locus of interest. The co-integration locus is determined by two homologous arms (HAs) on the suicide plasmid, which can be freely chosen by the user to mediate HR. A resolving step forces a second HR event that leads to removal of the plasmid backbone from the genome. This step is triggered by the action of the homing endonuclease I-SceI, acting on two recognition sequences that flank the homologous regions within the backbone of the suicide plasmid. The gene encoding I-SceI is supplied in trans from a helper plasmid, introduced into the cells after co-integration of the suicide plasmid. Our recently developed method facilitates rapid curing of this auxiliary plasmid through a synthetic, controllable replication mechanism (Volke et al., 2020) dependent on the presence of 3-methylbenzoic acid (3-mBz). Therefore, plasmid replication can be tightly regulated by the user by merely supplementing or omitting the inducer molecule in the culture medium. Plasmid curing is further aided by the expression of a fluorescent marker from the auxiliary vector, which is compatible with the reporter gene employed in the suicide plasmid. To broaden the use of this method, we developed different versions of the involved plasmids with several antibiotic resistance markers.


Materials and Reagents

Material

  1. Pipette tips (1,000 μl, 200 μl, 10 μl) (Sartorius, catalog numbers: 7902020, 790012, 791002)

  2. Sterile Petri dishes (Ø = 90 mm) (HiMedia Laboratories, catalog number: PW001)

  3. Eppendorf tubes (Tarsons Products, 1.5 ml, 2.0 ml)

  4. 50-ml conical tubes (Sarstedt, catalog number: 62.547.205)

  5. Electrocuvettes, 0.1-cm gap for E. coli (Bio-Rad, Gene Pulser, catalog number: 165-2089) and 0.2-cm gap for Pseudomonas (Bio-Rad, Gene Pulser, catalog number: 165-2086)

  6. Sterile 0.2-μm syringe filters (Sigma-Aldrich)


Reagents
  1. 3-Methylbenzoic acid (synonym m-toluic acid) (3-mBz; Sigma-Aldrich, ReagentPlus, catalog number: T36609)

  2. Sucrose (Sigma-Aldrich, Milipore, catalog number: 84100)

  3. Lysogeny broth (LB) (Sigma-Aldrich, catalog number: L3522); preparation according to the manufacturer’s instructions, storage for up to three weeks at room temperature

  4. Agarised LB (Sigma-Aldrich, catalog number: L3147); preparation according to the manufacturer’s instruction, storage for up to two months at 4 °C

  5. SOC medium (Sigma-Aldrich, catalog number: S1797)

  6. Kanamycin (TH-Geyer, catalog number: T832.3)

  7. Gentamicin (Sigma-Aldrich, catalog number: G1264)

  8. Streptomycin (Sigma-Aldrich, catalog number: S6501)

  9. Ampicillin (Mitolab, catalog number: K029)

  10. Oligonucleotides (Integrated DNA Technologies, Leuven, Belgium)

  11. Uracil-specific excision reagent (NEB Biolabs, USER enzyme, catalog number: M5505)

  12. DNA polymerase (Thermo Fisher Scientific, Phusion U Hot start, catalog number: F555)

  13. DNA polymerase reaction mix for colony PCRs including (NEB Biolabs, OneTaq® Hot Start Quick-Load® 2× master mix with standard buffer, catalog number: M0488L)

  14. Reagents to prepare chemically competent E. coli cells (Zymoresearch, Mix & Go!; catalog number: T3001)

  15. Escherichia coli DH5α λpir [endA1 hsdR17 glnV44 (supE44) thi-1 recA1 gyrA96 relA1 ϕ80dlacΔ(lacZ)M15 Δ(lacZYA-argF)U169 zdg-232::Tn10 uidA::pir+] (Platt et al., 2000)

  16. P. putida KT2440 (strain ATCC 47054/DSM 6125/NCIMB 11950) (Belda et al., 2016)

  17. Sequencing kit (Eurofins, Mix2Seq Kit OVERNIGHT, catalog number: 3094-0ONMSK)

  18. Plasmid purification kit (Macherey-Nagel, NucleoSpin Plasmid, catalog number: 740588)

  19. Gel and PCR Clean-up Kit (Macherey-Nagel, NucleoSpin™ Gel and PCR Clean-up Kit, catalog number: 740588)

  20. Optional: DpnI (Thermo Fisher Scientific, FastDigest DpnI, catalog number: FD1703)

  21. Agarose (Bio-Rad, Certified Molecular Biology Agarose, catalog number: 1613102); prepare at 1% (w/v) in 1× TAE buffer for gel electrophoresis (use microwave heating to dissolve). Can be stored at 60 °C to keep molten for immediate use

  22. Fluorescent nucleic acid staining solution (Intronbio, Red safe, catalog number: 21141)

  23. DNA ladder (Thermo Fisher Scientific, GeneRuler 1 kB, catalog number: SM0314)

  24. 3-Methylbenzoic acid (3-mBz) solution (500 mM) (see Recipes)

  25. Sucrose solution (300 mM) (see Recipes)

  26. Polymerase chain reaction (PCR) reagents (see Recipes)

  27. Electro-competent Pseudomonas cells (see Recipes)

  28. Antibiotic stock solutions (see Recipes)

Equipment

  1. Electroporator (Bio-Rad, MicroPulser, catalog number: 1652100)

  2. Transilluminator (Thermo Fisher Scientific, Safe Imager 2.0 Blue Light, catalog number: G6600)

  3. Table centrifuge, used for 1.5- and 2-ml reaction tubes (VWR, model: Microstar 17R, catalog number: 521-1647)

  4. Table centrifuge, used for 50-ml reaction tubes (Thermo Fisher Scientific, model: Heraeus Multifuge X1R, catalog number: 75004250) with rotor (Thermo Fisher Scientific, model: TX-400, catalog number: 75003181) and buckets (Thermo Fisher Scientific, catalog number: 75003655) and adaptors (Thermo Fisher Scientific, catalog number: 75003683)

  5. Termoblock (Eppendorf, model: Thermo Mixer C, catalog number: 5382000015)

  6. pH-meter (Thermo Fisher Scientific, model: FE150pH, catalog number: S35924)

  7. Agarose chamber, gelcaster, combs and power supply (Bio-Rad, Mini-sub Cell GT system & Power pack, catalog number: 1645050)

  8. Gel visualization (Bio-Rad, Gel Doc XR+ Gel Documentation System, catalog number: 1708195EDU)

  9. PCR thermocycler (Eppendorf, Mastercycler Nexus X2 Thermocycler, catalog number: 6336000015)

Software

  1. AMUSER [http://www.cbs.dtu.dk/services/AMUSER (Genee et al., 2015)]

  2. DNA sequence design tool for example Benchling or Geneious (Biomatter Ltd.)

Procedure



Figure 1. Designing integration vectors for genome manipulations. In this example, we illustrate the construction of a plasmid for deleting gene(s) xyz on the chromosome of P. putida. First, the pSNW plasmid backbone as well as the two homology arms flanking xyz (HA1 and HA2) are amplified with Phusion U DNA polymerase, thereby introducing complementary, homologous overhangs containing a single deoxyuridine nucleoside (dU). The amplicons are combined and digested with the uracil excision reagent (USER), leading to the removal of dU to create single-stranded overhangs. After a subsequent annealing of the fragments, the plasmid is delivered into chemical-competent E. coli DH5α λpir cells.



  1. Cloning of integration vector

    A suicide plasmid for specific genomic manipulations is composed of a universal pSNW plasmid backbone (see Table 1), an upstream homology arm (HA1), if applicable: a DNA sequence to be inserted at the target site, and a downstream homology arm (HA2) (Figure 1). Each of these fragments is PCR-amplified. Thus, three to four pairs of primers are required per construct.


    Table 1. Suicide plasmids for co-integration

    1. Identify the sequence of the two regions flanking the target gene or locus on the chromosome of P. putida.

      The upstream homologous arm (HA1, see Figure 1) spans from 500-700 bp upstream of the chromosomal target to the first base of the sequence that is to be deleted or edited, or the integration site. The downstream homologous arm (HA2, see Figure 1) conversely starts after the last base of the chromosomal target and ends 500-700 bp further downstream.

      Tip: We recommend leaving the START and STOP codons of the target gene intact and deleting only the interjacent sequence to minimize the chance of polar effects due to gene deletions and to avoid the potential creation of toxic, truncated polypeptides.

    2. Design primers for the construction of the application-specific pSNW plasmid

      Plasmids from the pSNW series can be linearized for USER cloning using the same pair of primers for every procedure (pSNW-USER_F: 5’-AGT CGA CCU GCA GGC ATG CAA GCT TCT-3’, and pSNW-USER_R: 5’-AGG ATC UAG AGG ATC CCC GGG TAC CG-3’; dU residues highlighted in red in the primer sequences), so that only insert-specific primers need to be designed for every genomic manipulation.

      Open the AMUSER online software (http://www.cbs.dtu.dk/services/AMUSER/) and enter the sequence of each fragment comprising the pSNW plasmid insert identified in STEP 1 (HA1, HA2, and, if applicable: integration fragment) in FASTA format (including a header preceded by “>” and a DNA sequence). At STEP 2: Output construct, select linear and click on Submit query. The primer sequences from the AMUSER report can directly be used for the amplification of the fragments. Add the motif 5’-AGA TCC U-3’ as the primer overhang to the forward primer of HA1, and 5’-AGG TCG ACU-3’ as overhang to the reverse primer of HR2. These two overhangs match the ones that have been used to linearize vector pSNW.

      Tip: Standard cloning procedures can be also established for Gibson Assembly or Golden Gate cloning to streamline the process.

      One standard set of primers can be used to check for the correct insert size after construction of the pSNW derivative in colony PCR, as well as to sequence the insert region. The two primers bind within the pSNW backbone immediately upstream (pSNW_seq_F: 5’-TGT AAA ACG ACG GCC AGT-3’) and downstream (pSNW_seq_R: 5’-CTT TAC ACT TTA TGC TTC CGG-3’) of the insert region, respectively.

    3. Design primers for genotyping after the genome manipulation

      Design one pair of primers binding within a range of 50 bp upstream of HA1 and 50 bp downstream of HA2 in the genome, respectively. These primers are used to test for the cells’ genotype after the recombination step via integration of vector pSNW. For small insertions or modifications that do not alter the total length of the sequence comprising both HAs, an additional primer that specifically binds within the insertion/modification is helpful to identify the engineered genotype.

    4. Amplification of regions by PCR

      We recommend using a ‘touchdown’ temperature protocol (Don et al., 1991) for each PCR amplification (see Table 2), as it circumvents the needs for optimizing the annealing temperature and leads to higher yields and specificity (Korbie and Mattick, 2008). Amplify vector pSNW using 5 ng of plasmid, mini-prep isolated to increase template concentration and purity, and primers pSNW-USER_F and pSNW-USER_R using the Phusion U Hot Start DNA polymerase (see Recipe 3). Use the temperature protocol illustrated in Table 2, with an elongation time of 3 min. Adopt the same temperature protocol to amplify each of the fragments constituting the pSNW insert, only adjusting the duration of the extension steps according to the amplicon lengths. Utilise purified genomic Pseudomonas DNA with the appropriate primer pairs to generate the HR1 and HR2 fragments. Perform, if needed, additional PCRs to generate the DNA fragments required for insertions.

      Tip: We recommend to gel-purify the linearized pSNW fragment using a gel and PCR clean-up kit according to the manufacturer’s instructions. By using the purified product as template for future PCRs, digestion with DpnI to remove circular plasmids (i.e., template) can be omitted. We further recommend generating a large amount of linearized pSNW vector in several parallel PCRs for repeated usage in USER cloning.


      Table 2. Touchdown temperature protocol for Phusion U PCR


    5. Check PCRs on agarose gel

      Analyse a 3-μl aliquot of each PCR by agarose gel electrophoresis [1% (w/v) agarose and 1× fluorescent nucleic acid gel stain in 1× TAE buffer] to verify the correct amplification of the fragments. The relative amplicon concentrations can be semi-quantitatively estimated from the intensities of their bands. If agarose gel electrophoresis reveals the presence of non-specific by-products, the desired bands have to be excised from the gel and purified prior to cloning using a gel and PCR clean-up kit according to the manufacturer’s instructions. If the product appears clean, the PCR reaction can be directly used in the assembly reaction.

    6. USER reaction

      In a PCR tube, combine equimolar amounts of all fragments (HR regions, insert and backbone) in 10 µl with a total amount of around 50 ng. Add 1 µl of 1 U µl–1 USER enzyme. Set up a thermocycler and run a reaction program as follows: deoxyuracil excision: 30 min at 37 °C; annealing 1: decrease from 28 °C to 18 °C with -2 °C per step of each 3 min; annealing 2: 10 min and hold at 10 °C.

      !!! If a plasmid was used as template for the amplification of one of the fragments that contains the same antibiotic resistance as the employed pSNW vector, add 0.5 µl of FastDigest DpnI to the reaction mix prior to incubation at 37 °C !!!

    7. Transformation of E. coli DH5α λpir cells

      Transform 50 µl (or 100 µl) aliquots of chemically-competent E. coli DH5α λpir cells with 5 µl (or 10 µl) of the assembly reaction from the previous step. To prepare competent cells and transform them with assembled plasmids, we recommend using the Mix & Go! E. coli Transformation Buffer Set (Zymo Research) and the associated protocol. After delivery of the plasmid into the cells (via heat-shock or incubation, see Mix & Go! Instruction manual), add 1 ml of SOC medium and incubate for 1 h at 37 °C with gentle shaking at 200-300 rpm. Pellet the cells at 10,000 × g for 1 min, resuspend in 50-100 µl of SOC medium and plate the suspension onto LB agar plates supplemented with the respective antibiotic for the pSNW plasmid.

      !!! Critical: It is essential to use an E. coli strain harboring the λ phage-derived pir gene for replication the of ori(RK6) !!!

      Tip: If a circular plasmid was used as template in the PCR for linearization of vector pGNW and if the reaction was directly employed for the assembly reaction (rather than using a gel-purified plasmid), spread the transformed E. coli DH5α λpir cells on agar plates containing 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) at 40 µg ml–1. The disruption of the pSNW-borne lacZα sequence then allows for the identification of E. coli colonies harbouring “empty” template plasmids via their blue colour in contrast to white colonies, carrying a pSNW with insert.

    8. Check for correct insert size

      Perform a colony PCR (using OneTaq® 2× Master Mix) on eight to ten colonies that show green fluorescence under blue-light exposure (inspect plate with transformed E. coli DH5α λpir on a blue-light transilluminator) with primers pSNW_seq_F and pSNW_seq_R. To this end, prepare 50 µl of a 1× master mix by mixing 25 µl of OneTaq® 2× Master Mix with 23 µl DNAse-free water and 1 µl of each primer (scale up if necessary). Transfer 6 µl of the 1× master mix into PCR tubes and add a small amount of biomass from the E. coli colonies grown on the culture plate. Run a PCR with the following temperature protocol (Table 3, adjust the elongation time according to the expected insert size).

      !!! For E. coli, P. putida and many other bacteria, small amounts of biomass from colonies can be directly transferred with a pipette tip or inoculation loop to the reaction mix (avoid transferring agar from the plate to the reaction mixture, as it will inhibit the amplification). For some bacteria, it might be necessary to boil biomass in water and dilute before colony PCR for good amplification results !!!


      Table 3. Touchdown temperature protocol for OneTaq colony PCR


    9. Check colony PCRs on agarose gel

      Analyse a 3-μl aliquot of each PCR by agarose gel electrophoresis [1% (w/v) agarose in 1× TAE buffer] to verify the correct insert size.

    10. Verify insert sequence integrity by sequencing

      If no band other than that of the expected size is visible in the agarose gel, the reaction sample can directly be sent out for sequencing. For this, mix 0.5 μl of the PCR reaction with 14.5 μl of DNase-free water and 2 μl of the primer pSNW_seq_F or pSNW_seq_R (one sequencing sample for each primer and each E. coli clone) in a barcoded tube from a Mix2Seq kit and send the tubes for sequencing. If non-specific bands appeared in the agarose gel, inoculate 3-5 ml of LB medium (add the corresponding antibiotic) in 50-ml centrifuge tubes with three individual clones that were tested for a correct insert size. Incubate the cultures at 37 °C for 12-18 h in a shaking incubator at 180-250 rpm (depending on the type of incubator). Purify plasmid DNA from the E. coli cultures and send the purified plasmid DNA for sequencing. Use a sequence-verified plasmid for the subsequent procedure in P. putida.


  2. Integration of suicide plasmid into desired genome locus

    1. Transformation of P. putida

      Follow the recipe for preparing electro-competent Pseudomonas cells (Recipe 4). Electroporate the cells with 300-500 ng of the previously constructed pSNW plasmid (see Procedure A). Figure 2 presents an overview of the procedure described in this section.

      Tip: If the strain has a low competence or the plasmid has a low integration efficiency, triparental conjugation can be used instead of electroporation. See protocols in Martínez-García and de Lorenzo (2011), Sánchez-Pascuala et al. (2017) and Durante-Rodríguez et al. (2018) for further details.



      Figure 2. Overview of the genome editing procedure. 1a) P. putida is transformed with a suicide plasmid from the pSNW series. 1b) Genomic integration of the vector is enforced by plating on selective medium. 1c) The genome integration is confirmed via green fluorescence of antibiotic-resistant colonies. 2a) Confirmed co-integrants are propagated in selective medium and electroporated with an auxiliary plasmid from the pQURE series. 2b) The co-integration is resolved via the expression of I-SceI from pQURE. 2c) Single colonies from suspension are obtained by dilution-streaking and 2d) the resolution of co-integration is confirmed by the absence of GFP-fluorescence. 2e) The genotype of selected colonies is confirmed by colony PCR; colonies can be either revertants or carry the desired genome alteration. 3a) A modified clone is propagated in non-selective medium. 3b) Single colonies are obtained by dilution-streaking and clones that have lost pQURE are identified by the absence of red fluorescence.


    2. Plating

      Plate on LB agar with either kanamycin for pSNW2; gentamicin for pSNW6 or streptomycin for pSNW4 and incubate the plates at 30 °C for ~16 h. The growth conditions may have to be adjusted depending on the strain to be edited. Grown colonies should be big enough to identify fluorescence on a blue-light transilluminator and to be easily picked with an inoculation loop.

    3. Fluorescence check on transilluminator

      Colonies with the suicide plasmid integrated should display green fluorescence (Figure 3A). Mark several fluorescent colonies for the further steps.

      Tip: Fluorescence increases significantly over time after plating due to maturation of the fluorophore. Therefore, plates can be incubated for an extended time at room temperature or at a lower temperature (4 °C) to increase the signal. Usually, all antibiotic-resistant colonies should display green fluorescence. The (unlikely) absence of fluorescence may indicate the presence of contaminations.


  3. Resolving of co-integration

    1. Propagation of confirmed co-integrant and transformation with pQURE plasmid

      Pick up to six co-integrant colonies, verified by fluorescence, and transfer their biomass together into 10 ml of LB medium in a 50-ml centrifuge tube with the appropriate antibiotic. Incubate at 30 °C for ~16 h with shaking. Prepare the culture for electroporation, and use ~10 ng of pQURE plasmid (Table 4). Add 3-mBz at 2 mM directly to the recovery medium.

      !!! Choose a pQURE variant whose antibiotic resistance is compatible with the employed suicide plasmid and organism (i.e., no natural resistance and not the same antibiotic resistance marker as used for pSNW) !!!

      !!! Critical: Omit the antibiotic used to select for pSNW co-integration from now on, otherwise mutations within pSNW are selected which rules out its successful resolution !!!

      Table 4. Plasmids for resolving of co-integration

    2. Induce double strand break and homologous recombination event

      Continue culturing the cells for 2 h before adding the appropriate antibiotic to select for pQURE. After 1-3 additional hours, either (i) plate a volume of 70 μl onto LB agar supplemented with 2 mM 3-mBz and the appropriate antibiotic to select for pQURE and incubate the plate overnight at 30 °C, and/or (ii) pass the suspension on to 10 ml of selective LB medium with 2 mM 3-mBz and continue incubation at 30 °C with shaking for about 16 h.

      Tip: The longer the growth in selective liquid medium, the higher the resolving efficiency and the lower the chance to obtain genetically heterogeneous colonies containing revertants and cells with the desired genetic alteration. The resolving efficiency can vary strongly between different loci.

    3. Isolating single mutants

      If (ii) was chosen in the previous step, perform a dilution streak of the culture on selective (for pQURE) solid medium with 2 mM 3-mBz.

      Tip: You want dozens of isolated colonies. You can perform multiple dilution streaks from once culture on a single plate to increase the numbers of isolated colonies.

    4. Check fluorescence on a blue-light transilluminator

      Resolved colonies do not display green fluorescence and should appear red under blue-light exposure (from the RFP encoded on pQURE, Figure 3B and 3C). In general, the majority of colonies will be resolved. Mark several colonies without green fluorescence for further analysis.

      !!! Critical: 3-mBz in the plates is slowly degraded by some microbes, e.g., P. putida, leading to a brown coloration of the colonies and the surrounding medium. The brown pigment produced conceals the colonies’ fluorescence after prolonged storage (several days to weeks) and therefore, the colonies should be marked immediately !!!



      Figure 3. Exemplary plates for different steps of the genome engineering process. A. Colonies of P. putida after co-integration of the suicide plasmid display green fluorescence (marked with green circles) after ~24 h of incubation. B. Resolved culture after ~18 h of incubation. Most colonies display a slightly red colour (marked with green circles). Only a few show green fluorescence (marked with a red circle). The latter still contain the co-integrated plasmid. C. Culture plate with cured colonies after ~24 h of incubation. Several resolved colonies without red fluorescence (highlighted with green circles) are kept for further analysis.


    5. Check genome editing in resolved colonies

      Check the locus of interest with the genotyping primers designed in A3 via colony PCR (see A8 and Table 3) for at least eight colonies. For genomic deletions or insertions, the amplicon size differs for the two genotypes and thus allows for their identification. For small insertions or sequence replacements, a primer specific for the modified sequence together with the external primer binding on the opposite strand will yield a product only for modified genotypes. The ratio of revertants, i.e., unmodified genotype, and modified genotype can vary greatly, depending on either a fitness advantage for one of the two genotypes or the sequence context of the modified locus.

      Tip: An aliquot of the amplified region can be directly used for sequencing. For this, add 0.5 μl of the PCR product to 14.5 μl of DNAse-free water and 2 μl of the respective primer used for its amplification. For genomic manipulations that cause a severe detriment to the bacterium’s growth, the use of CRISPR/Cas9 counter selection can facilitate in obtaining the desired mutant genotype [please refer to Wirth et al. (2020) and Volke et al. (2020) for details].

  4. Curing of auxiliary plasmid

    1. Propagate a colony with the desired genotype

      Inoculate 10 ml of LB medium without any additives (neither antibiotics nor 3-mBz) with biomass from a single colony. Incubate the culture at 30 °C until it reaches stationary phase (typically overnight).

      Tip: It is advantageous to use as little biomass as possible to allow for a maximum number of replications of the cells in liquid LB medium.

    2. Selection of the cured strain

      Make a dilution streak of the culture on an LB plate without antibiotic or 3-mBz to obtain isolated colonies. Incubate the plate at 30 °C for ~16 h. Plasmid-cured strains can be identified on a blue-light transilluminator by the absence of red fluorescence.

      Tips:

      1. The wavelengths created by the blue-light transilluminator are not optimal for RFP visualization. Thus, high amounts of matured RFP are needed. Since the maturation time of RFP is long, incubation of the plate at 4-8 °C for several hours is helpful to increase the fluorescence intensity. The signal matures during the course of several days and can even be seen with the naked eye after incubating the plate for 2 days at 4 °C.

      2. Use of a light source with a wavelength closer to the excitation maximum of RFP (i.e., 558 nm) increases the fluorescence signal significantly, enabling the selection of colonies with relatively low RFP levels.

    3. Storage of cured, engineered strain

      Inoculate 10 ml of LB medium with the cured colony. After incubation at 30 °C for ~16 h, you can either store the strain or proceed working with it.

Recipes

  1. 3-Methylbenzoic acid (3-mBz) solution (500 mM)

    Ingredients                  Per 250 ml

    3-Methylbenzoic acid 17 g

    1. Add 3-mBz into 200 ml of distilled water

    2. Stir vigorously with a magnetic stirrer and insert pH-probe

    3. Add drop-wise 5 M NaOH solution until all 3-mBz is dissolved and the pH reaches 7.0

    4. Fill up to 250 ml with distilled water

    5. Sterile-filter the solution with a 0.2-μm filter

    Note: Solution can be stored at room temperature for several months. 3-mBz is added in a 1:500-ratio to culture media immediately before use. Let molten agar cool down to ~50 °C before adding 3-mBz and immediately cast plates.

  2. Sucrose solution (300 mM)

    Ingredients Per 500 ml

    Sucrose        52.3 g

    1. Add sucrose into 400 ml of distilled water

    2. Stir vigorously with magnetic stirrer until dissolved

    3. Fill up with distilled water to 500 ml

    4. Sterile-filter with a 0.2-μm filter

    Note: Solution can be stored at room temperature for several months. Aliquoting (50 ml) is recommended to prevent contamination.

  3. Polymerase chain reaction (PCR)

    Ingredients Per 50 μl [μl]

    Phusion U              0.5

    dNTPs                      1

    Buffer HF                10

    DMSO                     1.5

    Oligonucleotides 2.5 each

    Template              10 ng plasmid (or biomass)

    H2O                        Fill up to 50 μl

  4. Electro-competent Pseudomonas cells

    1. Inoculate 10 ml of LB medium with a single colony in a 50-ml tube.

    2. Grow the culture for ~16 h at 30 °C in a shaking incubator (200-250 rpm).

  5. Note: All of the following steps are performed at room temperature, if not otherwise stated.

    1. Harvest the cells by centrifugation (4,000 × g; 10 min, room temperature), resuspend in 1 ml of 300 mM sucrose and transfer into a microreaction tube (1.5 ml or 2 ml).

    2. Pellet the cells by centrifugation (10,000 × g, 1 min, room temperature) and resuspend in 1 ml of the sucrose solution.

    3. Repeat the washing step (resuspension and centrifugation) two times. Finally, resuspend the pellet in 400 μl of 300 mM sucrose. The cell suspension can be stored for several hours on ice before use or transformed directly.

    4. Mix 100 μl of cell suspension with 10 ng or >200 ng DNA for replicated or suicide plasmids, respectively. Electroporate (2.5 kV, 25 μF capacitance, 200 Ω resistance), immediately add 1 ml of LB medium and transfer the suspension into a test tube (recommended: 14-ml round bottom test tube). Recover the cells for 2 h at 30 °C with shaking at 200-250 rpm.

    Note: In the following steps, you can either plate the culture to obtain single transformants or transfer the suspension into selective liquid medium, if isolated clones are not needed.

  6. Antibiotic stock solutions

    Kanamycin: Dissolve 0.5 g of kanamycin in 10 ml of MilliQ water.

    Gentamicin: Dissolve 0.1 g of gentamicin in 10 ml of MilliQ water.

    Streptomycin: Dissolve 0.5 g of streptomycin in 10 ml of MilliQ water.

    Note: Sterile-filter the solution and aliquot at volumes of 1 ml. Solutions can be stored at -20 °C for several months. Add antibiotic stock solution to liquid and solid media immediately before use in a 1:1,000 ratio. Let molten agar cool down to ~50 °C before adding antibiotics and immediately cast plates.

Acknowledgments

The authors thank all members of the laboratory for testing the protocol and for useful suggestions for improvement, especially to Laura Friis who was involved in developing the prototype on which this protocol is based. This work was financially supported by the Novo Nordisk Foundation (NNF10CC1016517 and NNF18CC0033664), the Danish Council for Independent Research (SWEET, DFF-Research Project 8021-00039B), and the European Union’s Horizon2020 Research and Innovation Program under grant agreement No. 814418 (SinFonia). This protocol has been adapted from Volke et al. (2020).

Competing interests

The authors declare there are no potential conflicts of interest.

References

  1. Belda, E., van Heck, R. G. A., López-Sánchez, M. J., Cruveiller, S., Barbe, V., Fraser, C., Klenk, H. P., Petersen, J., Morgat, A., Nikel, P. I., Vallenet, D., Rouy, Z., Sekowska, A., Martins dos Santos, V. A. P., de Lorenzo, V., Danchin, A. and Médigue, C. (2016). The revisited genome of Pseudomonas putida KT2440 enlightens its value as a robust metabolic chassis. Environ Microbiol 18(10): 3403-3424.
  2. Don, R. H., Cox, P. T., Wainwright, B. J., Baker, K. and Mattick, J. S. (1991). Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res 19(14): 4008-4008.
  3. Durante-Rodríguez, G., de Lorenzo, V. and Nikel, P. I. (2018). A post-translational metabolic switch enables complete decoupling of bacterial growth from biopolymer production in engineered Escherichia coli. ACS Synth Biol 7(11): 2686-2697.
  4. Genee, H. J., Bonde, M. T., Bagger, F. O., Jespersen, J. B., Sommer, M. O., Wernersson, R. and Olsen, L. R. (2015). Software-supported USER cloning strategies for site-directed mutagenesis and DNA assembly. ACS Synthetic Biology 4(3): 342-349.
  5. Korbie, D. J. and Mattick, J. S. (2008). Touchdown PCR for increased specificity and sensitivity in PCR amplification. Nat Protoc 3(9): 1452-1456.
  6. Martínez-García, E. and de Lorenzo, V. (2011). Engineering multiple genomic deletions in Gram-negative bacteria: analysis of the multi-resistant antibiotic profile of Pseudomonas putida KT2440. Environ Microbiol 13(10): 2702-2716.
  7. Platt, R., Drescher, C., Park, S. K. and Phillips, G. J. (2000). Genetic system for reversible integration of DNA constructs and lacZ gene fusions into the Escherichia coli chromosome. Plasmid 43(1): 12-23.
  8. Sánchez-Pascuala, A., de Lorenzo, V. and Nikel, P. I. (2017). Refactoring the Embden-Meyerhof-Parnas pathway as a whole of portable GlucoBricks for implantation of glycolytic modules in Gram-negative bacteria. ACS Synthetic Biology 6(5): 793-805.
  9. Volke, D. C., Friis, L., Wirth, N. T., Turlin, J. and Nikel, P. I. (2020). Synthetic control of plasmid replication enables target- and self-curing of vectors and expedites genome engineering of Pseudomonas putida. Metabolic Engineering Communications 10: e00126.
  10. Wirth, N. T., Kozaeva, E. and Nikel, P. I. (2020). Accelerated genome engineering of Pseudomonas putida by I-SceI-mediated recombination and CRISPR-Cas9 counterselection. Microb Biotechnol 13(1): 233-249.

简介

[摘要]精确的基因组工程已成为代谢工程的一种普遍技术。同样,基因和其他功能性DNA序列的插入,缺失和改变对于理解和改造细胞也是必不可少的。几种技术已经发展到该端部(例如,CRISPR / CAS-辅助方法,同源重组,或 λ 红色重组),但其中大多数依赖于辅助质粒的使用,必须在编辑程序后将其固化。传统上已采用对温度敏感的复制子,反向选择标记或带有质粒的细胞的重复传代来规避这一障碍。尽管这些协议在某些细菌中可以很好地发挥作用,但它们不适用于其他物种,或者既费时又费力。在这里,我们提出了快速和通用的荧光假单胞菌荧光标记辅助基因组编辑协议,然后通过用户控制的质粒复制干净固化辅助质粒。一种荧光标记有助于鉴定基因组编辑的菌落,而第二种报道分子能够检测无质粒的细菌克隆。该协议不仅是用于假单胞菌物种的最快方法,而且可以轻松地适应任何类型的基因组修饰,包括序列删除,插入和替换。



图形概要:

带有可治愈质粒的假单胞菌的快速基因组工程


[背景]靶向,精确的基因组操纵技术已经大大推进了微生物工程领域。这样的方法不仅允许评估基因型与表型的关系,而且使微生物细胞工厂的复杂工程化成为可能。近年来,CRISPR / Cas9方法为真核生物的精确基因组工程铺平了道路。在细菌中,CRISPR / Cas9的用途主要限于其作为反选择工具的价值,因为细菌缺乏非同源的末端连接来修复由Cas9核酸酶诱导的双链断裂。因此,许多细菌的工程研究都依赖于同源重组(HR)来改变基因组。HR的优点是可以在靶基因组中引入广泛的变化。此外,它不仅适用于所谓的模式生物,例如,大肠杆菌和酿酒酵母,但也发现在非传统的主机,广泛推广应用例如,假单胞菌属的物种。在此协议中,我们为P的基于HR的基因组工程提供了工作流程。putida –与高级工具箱配对,该工具箱包含多个电阻标记 –通过使用荧光标记物,可以监测每个步骤而促进了这种情况(Wirth等,2020)。所提出的方法依赖于自杀质粒[受pir依赖性复制起点ori (R6K )控制]在目标基因座的共整合。共整合位点由自杀质粒上的两个同源臂(HA)确定,使用者可以自由选择这些介导臂来介导HR。解决步骤迫使发生第二次HR事件,从而导致从基因组中去除质粒骨架。该步骤由归巢核酸内切酶I- Sce I的作用触发,作用于自杀质粒主链内同源区域两侧的两个识别序列。从辅助质粒反式提供编码I- Sce I的基因,在自杀质粒共整合后将其引入细胞。我们最近开发的方法通过取决于3-甲基苯甲酸(3- m Bz)的存在的合成,可控制的复制机制(Volke等人,2020)促进该辅助质粒的快速固化。因此,使用者仅通过补充或省略培养基中的诱导剂分子就可以严格地调控质粒的复制。来自辅助载体的荧光标记物的表达进一步辅助了质粒的固化,该辅助标记物与自杀质粒中采用的报告基因相容。为了拓宽此方法的使用范围,我们开发了具有多个抗生素抗性标记物的涉及质粒的不同版本。

关键字:基因组工程, 合成生物学, 假单胞菌, 合成质粒复制, 代谢工程, 革兰氏阴性菌

材料和试剂
材料
移液器吸头(1,000 μ升,200 μ升,10 μ升)(赛多利斯,目录号:7902020,790012,791002)
STE RILE培养皿(直径= 90 MM)(HIMEDIA实验室,目录号:PW001)
Eppendorf管(Tarsons Products,1.5 ml,2.0 ml)
50 ml锥形管(Sarstedt ,目录号:62.547.205)
Electrocuvettes ,0.1厘米间隙为大肠杆菌(BIO - Rad公司,基因脉冲,目录号:165-2089)和0.2厘米间隙为假单胞菌(BIO - RAD,基因脉冲,目录号:165-2086)
无菌0.2 μ米注射器过滤器(Sigma-Aldrich公司)

试剂种类
1. 3-甲基苯甲酸(同义词米-toluic酸)(3-米的Bz ; Sigma-Aldrich公司,ReagentPlus ,目录号:T36609)     
2.蔗糖(Sigma-Aldrich,Milipore ,目录号:84100)     
3.溶源性肉汤(LB)(Sigma-Aldrich,目录号:L3522 );根据制造商的说明进行准备,在室温下最多可保存三周     
4.琼脂LB(Sigma-Aldrich,目录号:L3147 );根据制造商的说明进行准备,在4下最多可存放两个月       °C
5. SOC介质(Sigma-Aldrich,目录号:S1797)     
6.卡那霉素(TH-Geyer,目录号:T832.3)     
7.庆大霉素(Sigma-Aldrich,目录号:G1264)     
8.链霉素(西格玛奥德里奇,目录号:S6501)     
9.氨苄西林(Mitolab ,目录号:K029)     
10.寡核苷酸(整合DNA技术,比利时鲁汶) 
11.尿嘧啶特异性切除试剂(NEB Biolabs,用户酶,目录号:M5505) 
12. DNA聚合酶(Thermo Fisher Scientific ,Phusion U热启动,目录号:F555) 
13.用于菌落PCR中包括DNA聚合酶反应混合物(NEB Biolabs公司,一个的Taq ®热启动快速负载® 2 ×主混合物与标准缓冲液,产品目录号:M0488L) 
14.制备化学感受态大肠杆菌细胞的试剂(Zymoresearch ,Mix&Go ! ;目录号:T3001) 
15.大肠杆菌DH5    α λ PIR [ endA1 hsdR17 glnV44 (supE44 )THI-1 recA1 gyrA96 relA1 φ80d紫胶 (lacZ启动)M15  (lacZYA-ARGF )U169 ZDG-232 :: Tn的10的uidA :: PIR + ] (普拉特等人,2000 )

16. P 。putida KT2440 (株ATCC 47054 / DSM 6125 / NCIMB 11950)(Belda等人,2016) 
17.测序试剂盒(Eurofins,Mix2Seq Kit OVERNIGHT,目录号:3094-0ONMSK) 
18.质粒纯化试剂盒(Macherey -Nagel,NucleoSpin质粒,目录号:740588) 
19.凝胶和PCR纯化试剂盒(Macherey -Nagel,NucleoSpin ™凝胶和PCR纯化试剂盒,目录号:740588) 
20.可选:Dpn I (Thermo Fisher Scientific ,FastDigest DpnI ,目录号:FD1703) 
21.琼脂糖(Bio-Rad,认证分子生物学琼脂糖,目录号:1613102);在1 × TAE缓冲液中以1%(w / v)的比例制备凝胶电泳(使用微波加热溶解)。可以在60 °C下保存以保持熔融状态,以便立即使用 
22.荧光核酸染色溶液(Intronbio ,Red safe,目录号:21141) 
23. DNA阶梯(Thermo Fisher Scientific ,GeneRuler 1 kB,目录号:SM0314) 
24. 3-甲基苯甲酸(3- m Bz)溶液(500 mM)(请参阅食谱) 
25.蔗糖溶液(300 mM)(请参阅食谱) 
26.聚合酶链反应(PCR)试剂(请参阅食谱) 
27.具有电感受态的假单胞菌细胞(请参阅食谱) 
28.抗生素储备溶液(请参阅食谱) 

设备
电穿孔仪(BIO - Rad公司,MicroPulser ,目录号:1 652100)
透照仪(Thermo Fisher Scientific,Safe Imager 2.0 Blue Li ght,目录号:G6600)
台式离心机,用于1.5和2毫升ř反应的影响管(VWR,型号:MICROSTAR 17R,目录号:521-1647)
台式离心机,用于带有转子(Thermo Fisher Scientific ,型号:TX-400,目录号:75003181 )和水桶(Thermo Fisher Scientific )的50 ml反应管(Thermo Fisher Scientific ,型号:Heraeus Multifuge X1R,目录号:75004250 ),目录号:75003655 )和适配器(Thermo Fisher Scientific ,目录号:75003683)
Termoblock (Eppendorf,型号:Thermo Mixer C ,目录号:5382000015)
pH计(Thermo Fisher Scientific,型号:FE150pH,目录号:S35924)
琼脂糖腔,凝胶浇铸机,梳子和电源(Bio - Rad,Mini-sub Cell GT系统和电源组,目录号:1645050)
凝胶可视化(Bio - Rad,Gel Doc XR +凝胶文献系统,目录号:1708195EDU)
PCR热循环仪(Eppendorf,Mastercycler Nexus X2热循环仪,货号:6336000015)

软件
AMUSER [ http://www.cbs.dtu.dk/services/AMUSER(Genee等人,2015)]
DNA序列设计工具,例如Benching或Geneious (Biomatter Ltd.)

程序

图1.设计用于基因组操作的整合载体。在这个例子中,我们说明了用于删除恶臭假单胞菌染色体上的基因xyz的质粒的构建。首先,用Phusion U DNA聚合酶扩增pSNW质粒主链以及xyz侧翼的两个同源臂(HA1和HA2),从而引入包含单个脱氧尿苷核苷(dU )的互补同源突出端。将扩增子合并,并用尿嘧啶切除试剂(USER )消化,从而去除dU ,形成单链突出端。片段的随后的退火后,质粒被输送到化学感受态大肠杆菌DH5 α λ PIR细胞。


整合载体的克隆
用于特定基因组操作的自杀质粒由通用pSNW质粒主链(请参见表1 ),上游同源臂(HA1)(如果适用)组成:要插入目标位点的DNA序列和下游同源臂(HA2) )(图1 )。这些片段中的每一个都是PCR扩增的。因此,每个构建体需要三到四对引物。


表1。用于共整合的自杀质粒


确定恶性疟原虫染色体上目标基因或基因座两侧的两个区域的序列。
上游同源臂(HA1,参见图1 )跨度从染色体靶标的上游500-700bp到要删除或编辑的序列的第一碱基或整合位点。相反,下游同源臂(HA2,参见图1 )在染色体靶标的最后一个碱基之后开始,并在下游进一步终止500-700 bp。

提示:我们建议保留目标基因的START和STOP密码子完整无缺,并仅删除中间序列,以最大程度地减少由于基因缺失而产生极性效应的机会,并避免潜在地产生有毒的截短的多肽。

设计用于构建特定应用pSNW质粒的引物
可以将pSNW系列的质粒线性化以用于USER克隆,每个步骤使用相同的引物对(pSNW -USER_F:5'- AGT CGA CC U GCA GGC ATG CAA GCT TCT -3',而pSNW -USER_R:5'- AGG ATC U AG AGG ATC CCC GGG TAC CG -3';dU残基在引物序列中以红色突出显示),因此对于每种基因组操作,只需设计插入特异性引物。

打开AMUSER在线软件(http://www.cbs.dtu.dk/services/AMUSER/),然后输入每个片段的序列,这些片段包含在步骤1中识别的pSNW质粒插入片段(HA1,HA2,如果适用:整合)片段)以FASTA格式显示(包括带有“>”和DNA序列的标头)。在步骤2:输出构造中,选择线性,然后单击提交查询。AMUSER报告中的引物序列可直接用于片段扩增。将基序5'-AGA TCC U -3'作为引物突出到HA1的正向引物上,将5'- AGG TCG AC U -3'作为引物突出到HR2的反向引物。这两个突出端与用于线性化向量pSNW的突出端匹配。

提示:还可以为Gibson Assembly或Golden Gate克隆建立标准的克隆程序,以简化流程。

一组标准的引物可用于在菌落PCR中构建pSNW衍生物后检查正确的插入片段大小,以及对插入片段进行测序。两条引物在紧接插入区上游(pSNW_seq_F :5'- TGT AAA ACG ACG GCC AGT -3')和下游(pSNW_seq_R :5'- CTT TAC ACT TTA TGC TTC CGG -3')的pSNW主链内结合,分别。

设计基因组操作后进行基因分型的引物
设计一对分别在基因组中HA1上游50 bp和HA2下游50 bp范围内结合的引物。这些引物用于重组步骤后通过整合载体pSNW来测试细胞的基因型。对于不改变包含两个HA的序列的总长度的小插入或修饰,在插入/修饰内特异性结合的另外的引物有助于鉴定工程基因型。

通过PCR扩增区域
我们建议使用“T ouchdown”温度协议(唐等人对每个PCR扩增,1991)(见表2 ),因为它绕过了退火温度和引线优化以更高的产率和特异性(需求Korbie和Mattick , 2008)。使用5 ng质粒扩增载体pSNW,进行小量制备以提高模板浓度和纯度,并使用Phusion U热启动DNA聚合酶引物pSNW -USER_F和pSNW -USER_R (请参见食谱3 )。使用表2中所示的温度规程,延长时间为3分钟。采用相同的温度方案扩增构成pSNW插入片段的每个片段,仅根据扩增子的长度调整延伸步骤的持续时间。利用纯化的基因组假单胞菌DNA与适当的引物对生成HR1和HR2片段。如果需要,执行其他PCR,以生成插入所需的DNA片段。

提示:我们建议根据制造商的说明,使用凝胶和PCR纯化试剂盒对线性化的pSNW片段进行凝胶纯化。通过将纯化的产物用作未来PCR的模板,可以省略DpnI的消化以去除环状质粒(即模板)。我们进一步建议在多个并行PCR中生成大量线性化的pSNW载体,以便在USER克隆中重复使用。


表2 。Phusion U PCR的着陆温度协议


检查琼脂糖凝胶上的PCR
              分析3- μ通过琼脂糖凝胶电泳每个PCR的等分试样升[1%(W / V)琼脂糖和1 ×荧光核酸凝胶染料在1 × TAE缓冲液],以确认所述片段的正确放大。相对扩增子浓度s可以根据其条带的强度进行半定量估计。如果琼脂糖凝胶电泳显示存在非特异性副产物,则必须按照生产商的说明,使用凝胶和PCR纯化试剂盒从凝胶上切下所需的条带并纯化,然后再进行克隆。如果产物看起来干净,则PCR反应可直接用于组装反应。

用户反应
在PCR管中,将等摩尔量的所有片段(HR区域,插入片段和骨架)混合在10 µl中,总量约为50 ng。加入1 µl 1 U µl –1 USER酶。设置一个热循环仪并运行以下反应程序:脱氧尿嘧啶切除:在37°C下30分钟;退火1 :从28℃至18℃下用降低- 2℃每的每3分钟步骤; 退火2:10分钟,并保持在10°C。

!!! 如果使用质粒作为模板扩增包含与所用pSNW载体相同的抗生素抗性的片段之一,请在反应混合物中加入0.5 µl FastDigest DpnI ,然后在37° C下孵育!

转化大肠杆菌DH5 α λ PIR细胞
              变换50微升(或100微升)化学感受的等分试样大肠杆菌DH5 α λ PIR细胞与来自前一步骤的装配反应的5微升(或10微升)。要准备感受态细胞并用组装的质粒转化它们,我们建议使用Mix&Go!大肠杆菌转化缓冲液集(Zymo研究)和相关协议。将质粒递送到细胞中后(通过热休克或温育,请参见Mix&Go!说明手册),添加1 ml SOC培养基,并在37°C下温育200到300 rpm并孵育1 h。将细胞以10,000 × g的速度沉淀1分钟,重悬于50-100 µl SOC培养基中,并将悬浮液铺在补充有pSNW质粒相应抗生素的LB琼脂平板上。

!!! 关键:必须使用带有λ噬菌体衍生的pir基因的大肠杆菌菌株复制ori (RK6)!

提示:如果环状质粒是在PCR作为模板用于向量的线性化pGNW并且如果将反应混合物直接用于装配反应(而不是使用凝胶纯化的质粒),传播转化大肠杆菌我DH5 α λ琼脂平板上的pir细胞含有40 µg ml –1的5-溴-4-氯-3-吲哚基-β -D-吡喃半乳糖苷(X-Gal)。的破坏的pSNW -borne的lacZ α序列然后允许的大肠杆菌菌落的鉴定窝藏经由他们的蓝色“空”的模板质粒颜色对比的白色菌落,背着pSNW与插入件。

检查正确的插入尺寸
执行菌落PCR(使用一个的Taq ® 2 ×上八至十个菌落,显示下蓝光曝光(与转化的检查板的绿色荧光主混合物)大肠杆菌DH5 α λ PIR上的蓝色光透用引物)pSNW_seq_F和pSNW_seq_R 。为此,制备50微升1 ×通过混合25微升主混合物之一的Taq ® 2 ×主混合物与23微升DNA酶-free水和1μl的每种引物(必要时扩展)。将6 µl 1 ×预混液转移到PCR管中,并从培养板上生长的大肠杆菌菌落中添加少量生物质。使用以下温度规程运行PCR(表3 ,根据预期的插入片段大小调整延长时间)。

!!! 对于大肠杆菌,恶臭假单胞菌和许多其他细菌,可使用移液器吸头或接种环将菌落中的少量生物质直接转移至反应混合物中(避免将琼脂从平板转移至反应混合物中,因为这会抑制琼脂放大)。对于某些细菌,为了获得良好的扩增结果,可能有必要先将水中的生物质煮沸并稀释后再进行菌落PCR。




表3. OneTaq菌落PCR的着陆温度协议


检查琼脂糖凝胶上的菌落PCR
分析3- μ通过琼脂糖凝胶电泳每个PCR的等分试样升[1%(W / V)琼脂糖中1 × TAE缓冲液],以确认正确的插入物大小。

通过测序验证插入序列的完整性
如果在琼脂糖凝胶上看不到除预期大小以外的其他条带,则可将反应样品直接送出进行测序。为此,混合0.5 μ升的PCR反应物与14.5 μ升DNA酶自由水和2的μ升引物pSNW_seq_F或pSNW_seq_R (对于每个引物和每一个样品测序的大肠杆菌克隆)在从一个条形码管Mix2Seq试剂盒,然后将试管发送进行测序。如果琼脂糖凝胶中出现非特异性条带,则在50 ml离心管中接种3-5 ml LB培养基(添加相应的抗生素),并用三个单独的克隆进行测试,以检测插入片段的正确大小。将培养物在37°C下于180-250 rpm的振荡培养箱中孵育12-18 h(取决于培养箱的类型)。从大肠杆菌培养物中纯化质粒DNA,然后将纯化的质粒DNA发送进行测序。在恶臭假单胞菌的后续步骤中使用经过序列验证的质粒。           

自杀质粒整合到所需的基因组座位
恶臭假单胞菌的转化
遵循制备电感受态假单胞菌细胞的配方(配方4 )。用300-500 ng先前构建的pSNW质粒电穿孔细胞(请参阅步骤A )。图2概述了本节中描述的过程。

提示:如果菌株的能力低或质粒的整合效率低,则可以使用三亲结合来代替电穿孔。参见Martínez-García和de Lorenzo(2011),Sánchez-Pascuala等人的协议。(2017)和Durante-Rodríguez等人。(2018)了解更多详情。



图2.基因组编辑程序概述。1a)用来自pSNW系列的自杀质粒转化恶臭假单胞菌。1b)通过在选择性培养基上铺板来加强载体的基因组整合。1c)通过抗生素抗性菌落的绿色荧光确认基因组整合。2a)确认的共整合物在选择培养基中繁殖,并用来自pQURE系列的辅助质粒进行电穿孔。2b)通过来自pQURE的I- SceI的表达来解决共整合。2c)通过稀释划线获得来自悬浮液的单个菌落,并且2d)通过不存在GFP荧光来证实共整合的分辨率。2e)通过菌落PCR确认所选菌落的基因型;菌落可以是回复体,也可以携带所需的基因组改变。3a)修饰的克隆在非选择性培养基中繁殖。3b)通过稀释划线获得单个菌落,并通过不存在红色荧光来鉴定丢失pQURE的克隆。


电镀
用卡那霉素将pSNW2涂在LB琼脂上; 庆大霉素用于pSNW6或链霉素用于pSNW4,并在30 °C下孵育平板约16小时。根据要编辑的菌株,可能必须调整生长条件。生长的菌落应足够大,以识别蓝光透射照明器上的荧光,并易于通过接种环进行拾取。

透照仪的荧光检查
整合了自杀质粒的菌落应显示绿色荧光(图3A )。在后续步骤中标记几个荧光菌落。

提示:电镀后,由于荧光团的成熟,荧光会随时间显着增加。因此,可将板在室温或在较低温度(4°C)下延长孵育时间,以增强信号强度。通常,所有抗生素抗性菌落都应显示绿色荧光。(不太可能)没有荧光可能表明存在污染物。


解决整合问题
证实的共整合体的繁殖和pQURE质粒的转化
拾取六个共整合菌落,通过荧光验证,然后将其生物量一起转移到装有适当抗生素的50 ml离心管中的10 ml LB培养基中。于30 °C振荡孵育约16 h。准备用于电穿孔的培养物,并使用约10 ng的pQURE质粒(表4 )。将2 mM的3- m Bz直接添加到回收介质中。

!!! 选择一个pQURE变体,其抗生素抗性与所使用的自杀质粒和生物相容(即,没有天然抗性,并且与用于pSNW的抗生素抗性标记不同)!

!!! 关键:从现在开始,省略用于选择pSNW共整合的抗生素,否则将选择pSNW内的突变,排除其成功解决的可能性!


表4.用于共整合的质粒


引起双链断裂和同源重组事件
继续培养细胞2小时,然后添加适当的抗生素以选择pQURE 。1-3小时后,无论是(我)板70的体积μ升到LB琼脂补充有2mM 3-米BZ和合适的抗生素以选择用于pQURE并在30过夜孵育所述板℃下,和/或(ii)将悬浮液转移至10 ml具有2 mM 3- m Bz的选择性LB培养基中,并在30 °C振荡下继续温育约16小时。

提示:在选择性液体培养基中生长的时间越长,分离效率越高,获得包含具有所需遗传改变的回复子和细胞的遗传异质菌落的机会就越少。分辨效率在不同位点之间可能有很大差异。

分离单个突变体
如果在上一步中选择了(ii),请在具有2 mM 3- m Bz的选择性(对于pQURE )固体培养基上进行培养物稀释。

提示:您需要数十个孤立的殖民地。您可以在单个平板上一次培养一次进行多次稀释,以增加分离菌落的数量。

检查蓝光透射灯上的荧光
分离出的菌落不显示绿色荧光,在蓝光照射下应显示为红色(来自pQURE编码的RFP ,图3B和3C )。通常,大多数殖民地都会得到解决。标记几个没有绿色荧光的菌落,以供进一步分析。

!!! 关键:平板中的3-mBz被某些微生物(例如恶臭假单胞菌)缓慢降解,导致菌落和周围培养基呈棕色。长时间存放(数天至数周)后,产生的棕色颜料会掩盖菌落的荧光,因此,应立即标记菌落!!!



图3.基因组工程过程不同步骤的示例板。A.自杀质粒共整合后,在孵育约24小时后,恶臭假单胞菌的菌落显示绿色荧光(标有绿色圆圈)。B.孵育约18小时后分辨培养。大多数菌落显示淡红色(带有绿色圆圈)。只有少数显示绿色荧光(带有红色圆圈标记)。后者仍包含共整合质粒。C.孵育约24小时后,培养带有固化菌落的培养板。保留几个没有红色荧光的分辨菌落(以绿色圆圈突出显示)以进行进一步分析。


检查已解析菌落中的基因组编辑
使用A3中设计的基因分型引物,通过菌落PCR(请参阅A8和表3 )检查至少八个菌落的目的基因座。对于基因组缺失或插入,两种基因型的扩增子大小不同,因此可以对其进行鉴定。对于小的插入或序列替换,对修饰的序列特异的引物与结合在相反链上的外部引物将产生仅对于修饰的基因型的产物。的比例回复体,即,未修饰的基因型,和修饰的基因型可以变化很大,这取决于无论是对两种基因型的一个健身优点或修饰的基因座的序列背景。

提示:扩增区域的等分试样可直接用于测序。为此,添加0.5微升的PCR产物,以14.5微升的DNA酶-free水和2微升用于其扩增相应的引物。对于对细菌的生长造成严重损害的基因组操作,使用CRISPR / Cas9反向选择可以促进获得所需的突变基因型[请参见Wirth等。(2020)和Volke等。(2020年)]。


辅助质粒的固化
繁殖具有所需基因型的菌落
用来自单个菌落的生物量接种10 ml不含任何添加剂(既无抗生素,又不含3- m Bz)的LB培养基。将培养物在30 °C下孵育,直至达到固定相(通常过夜)。

提示:使用尽可能少的生物质以在液体LB培养基中最大程度地复制细胞是有利的。

固化菌株的选择
在没有抗生素或3- m Bz的LB平板上稀释培养液的条纹,以获得分离的菌落。在30 °C下孵育平板约16小时。通过不存在红色荧光,可以在蓝光透射照明器上鉴定质粒固化的菌株。

提示小号:

蓝光透射灯创建的波长对于RFP可视化不是最佳的。因此,需要大量的成熟RFP。由于RFP的成熟时间较长,因此将板在4-8°C下孵育几个小时有助于增加荧光强度。该信号在几天内会逐渐成熟,甚至可以在4°C下孵育2天后用肉眼看到。
使用的光源的波长更靠近最大激发RFP的(即,558纳米)显著增加荧光信号,使菌落的选择具有相对低的RFP的水平。
储存固化的工程菌株
用治愈的菌落接种10 ml LB培养基。在30 °C下孵育约16小时后,您可以存储菌株或继续对其进行处理。


菜谱


3-甲基苯甲酸(3- m Bz)溶液(500 mM)
每250毫升成分           
3-甲基苯甲酸17克           
将3- m Bz加入200 ml蒸馏水中
用磁力搅拌器剧烈搅拌并插入pH探针
逐滴添加5 M NaOH溶液,直到所有3- m Bz溶解并且pH达到7.0
充满蒸馏水至250毫升
无菌过滤器具有0.2的溶液μ微米的过滤器
注:小号olution可在室温下保存数月。在使用前,将3-mBz以1:500的比例添加到培养基中。让熔化的琼脂冷却至〜50 °C,然后添加3-mBz并立即浇铸板。

蔗糖溶液(300 mM)
每500毫升成分           
蔗糖52.3克           
将蔗糖加到400毫升蒸馏水中
用磁力搅拌器剧烈搅拌直至溶解
加满蒸馏水至500毫升
无菌过滤器具有0.2 μ微米的过滤器
注意:溶液可以在室温下保存几个月。建议分装(50 ml)以防止污染。

聚合酶链反应(PCR)
每50μl [ μl ]中的成分           
Phusion U 0.5           
NTP 1           
缓冲液HF 10           
DMSO 1.5           
寡核苷酸各2.5           
模板10 ng质粒(或生物质)           
H 2 O填充至50μl           
电感受态假单胞菌细胞
在50 ml管中用单个菌落接种10 ml LB培养基。
在30 °C的振荡培养箱(200-250 rpm)中将培养物培养约16小时。
注意:除非另有说明,以下所有步骤均在室温下执行。

通过离心(4,000 × g ; 10分钟,室温)收集细胞,重悬于1 ml 300 mM蔗糖中,并转移至微反应管(1.5 ml或2 ml)中。
通过离心(10,000 × g ,1分钟,室温)沉淀细胞,并重悬于1 ml蔗糖溶液中。
重复洗涤步骤(重悬和离心)两次。最后,重悬在400颗粒μ升300 mM的蔗糖。细胞悬浮液可以在使用前在冰上保存数小时,也可以直接转化。
混合100 μ升的细胞悬浮液用10纳克或> 200分别纳克DNA为复制或自杀质粒,。电穿孔(2.5千伏,25 μ ˚F电容,200Ω电阻),立即加入1毫升的LB培养基,将悬浮液转移到一个试管(推荐:14毫升圆底试管)。将细胞在30 °C下以200-250 rpm摇动恢复2小时。
注意:在以下步骤中,如果不需要分离的克隆,则可以将培养物铺板以获得单个转化体,或将悬浮液转移到选择性液体培养基中。

抗生素原液
卡那霉素:将0.5 g卡那霉素溶解在10 ml M illiQ水中。

庆大霉素:将0.1克庆大霉素溶于10毫升M illiQ水中。

链霉素:将0.5 g链霉素溶解在10 ml M illiQ水中。

注意:无菌过滤溶液并等分1 ml。解决方案可以存放在- 20 ℃,数月。立即以1:1,000的比例将抗生素储备溶液添加到液体和固体介质中。在加入抗生素并立即浇铸板之前,让融化的琼脂冷却至〜50 °C 。

致谢


作者感谢实验室的所有成员对协议进行测试并提供了有益的改进建议,特别是对参与开发该协议所基于的原型的Laura Friis表示感谢。这项工作得到了诺和诺德基金会(NNF10CC1016517和NNF18CC0033664),丹麦独立研究理事会(SWEET ,DFF研究项目8021-00039B)和欧盟的Horizon2020研究与创新计划(资助协议号为814418(SinFonia )。该协议已改编自Volke等人。(2020)。


利益争夺


作者宣称没有潜在的利益冲突。


参考


Belda ,E.,van Heck,RGA,López-Sánchez,MJ,Cruveiller ,S.,Barbe,V.,Fraser,C.,Klenk ,HP,Petersen,J.,Morgat ,A.,Nikel ,PI,Vallenet ,D.,Rouy ,Z.,Sekowska ,A.,VAP,de Lorenzo,V.,Danchin ,A.和Médigue ,C.(2016) 假单胞菌(Pseudomonas putida)KT2440的再造基因组启发了其价值健壮的代谢底盘。环境微生物学18(10):3403-3424。
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引用:Volke, D. C., Wirth, N. T. and Nikel, P. I. (2021). Rapid Genome Engineering of Pseudomonas Assisted by Fluorescent Markers and Tractable Curing of Plasmids. Bio-protocol 11(4): e3917. DOI: 10.21769/BioProtoc.3917.
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