Delivering "Chromatic Bacteria" Fluorescent Protein Tags to Proteobacteria Using Conjugation
利用接合生殖将产色细菌荧光蛋白标签导入变形杆菌   

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FEMS Microbiology Letters
Aug 2016

 

Abstract

Recently, we published a large and versatile set of plasmids, the chromatic bacteria toolbox, to deliver eight different fluorescent protein genes and four combinations of antibiotic resistance genes to Gram-negative bacteria. Fluorescent tags are important tools for single-cell microbiology, synthetic community studies, biofilm, and host-microbe interaction studies. Using conjugation helper strain E. coli S17-1 as a donor, we show how plasmid conjugation can be used to deliver broad host range plasmids, Tn5 transposons delivery plasmids, and Tn7 transposon delivery plasmids into species belonging to the Proteobacteria. To that end, donor and recipient bacteria are grown under standard growth conditions before they are mixed and incubated under non-selective conditions. Then, transconjugants or exconjugant recipients are selected on selective media. Mutant colonies are screened using a combination of tools to ensure that the desired plasmids or transposons are present and that the colonies are not containing any surviving donors. Through conjugation, a wide range of Gram-negative bacteria can be modified without prior, often time-consuming, establishment of competent cell and electroporation procedures that need to be adjusted for every individual strain. The here presented protocol is not exclusive for the delivery of Chromatic bacteria plasmids and transposons, but can also be used to deliver other mobilizable plasmids to bacterial recipients.

Keywords: Fluorescent proteins (荧光蛋白), GFP (绿色荧光蛋白), Plasmid (质粒 ), Tn5 transposon ( Tn5转座子), Tn7 transposon (Tn7转座子), Conjugation (接合生殖), Mobilisable plasmids (可移动质粒)

Background

Fluorescent proteins have become an important tool for the study of bacteria, e.g., in biofilms, gene expression studies, or host-microbe interactions (Tomlin et al., 2004; Monier and Lindow, 2005; Ma and Bryers, 2010; Remus-Emsermann et al., 2011; Schada von Borzyskowski et al., 2015; Remus-Emsermann et al., 2016a; Remus-Emsermann and Schlechter, 2018; Schlechter et al., 2018).

In a recently published study, we have described the construction of 96 plasmids, each different in fluorescent protein gene, antibiotic resistance marker combination, or plasmid backbone. The complete collection offers eight different fluorescent protein genes, each with its own unique excitation and emission spectrum, four different antibiotic resistance marker combinations, a broad host range plasmid, or delivery plasmids based on two different transposon systems, Tn5 and Tn7 (Schlechter et al., 2018).

The broad-host plasmid backbone and the two transposon systems have different advantages and disadvantages: 1) The plasmid usually exists in several copies in the recipient cell, which results in multiple gene copies and thereby often higher fluorescent signals. However, plasmids may be lost more easily, leading to subpopulations of cells that might lose their ability to express fluorescent proteins (Bahl et al., 2004). 2) The Tn5 transposon inserts into Proteobacteria with high efficiency and, as it integrates into the bacterial genome, the chance of losing the fluorescent protein tag is minimal. However, the Tn5 transposon randomly inserts DNA into the genome, thereby potentially disrupting genes that are important for bacterial fitness (de Lorenzo et al., 1990). 3) The Tn7 transposon integrates into the genome of the recipient bacterium in a site-specific manner, where no genes are disrupted and thereby should have minor impact on the bacterial fitness. However, the Tn7 transposon only inserts in a narrow range of bacteria, e.g., most γ-Proteobacteria, some α-Proteobacteria, and β-Proteobacteria. Outside of the γ-Proteobacteria, its insertion frequency may be low (McKenzie and Craig, 2006; Schlechter et al., 2018).

In this protocol, we describe the procedure to equip a bacterial strain with fluorescent labels using Pantoea eucalypti 299R (Remus-Emsermann et al., 2013; Tecon and Leveau, 2016) as an example recipient strain. P. eucalypti 299R is a competent recipient of all three different kinds of delivery systems, the broad host range plasmid and the two transposon systems. We could show that the broad-host plasmid as well as Tn5 and Tn7 transposon-based delivery of fluorescent proteins is feasible in this recipient strain (Schlechter et al., 2018). Other Enterobacteriaceae, such as E. coli, Erwinia amylovora and Pantoea vagans were also successfully tested with all three systems as well as Pseudomonas strains such as Pseudomonas citronellolis P3B5 (Remus-Emsermann et al., 2016b; Schlechter et al., 2018). Additionally, bacteria from other taxa, such as the α-Proteobacterial genera Sphingomonas and Methylobacterium were modified at least with the Tn5 transposon delivery system and in some cases with the broad-host-range plasmids and Tn7 transposons.

The here presented protocol is based on bacterial mating using the E. coli S17-1 helper strain, which is able to transfer mobilizable plasmids to recipients. Conjugation is a promiscuous process that crosses strain, genus, and phylum level and even works across domains of life (Heinemann and Sprague, 1989; Waters, 2001). Transformation methods such as heat-shock and electroporation are often used when transferring genetic material into laboratory model hosts, however, newly isolated strains are usually recalcitrant to these methods and the establishment of transformation protocols is often tedious and time-consuming. By relying on conjugation, transformation can be avoided and can be as simple as scraping recipient bacterial colonies from an agar plate and mixing them with the donor bacterium.

Materials and Reagents

  1. Autoclaved, untreated, wooden toothpicks (local supermarket of choice)
  2. Pipette tips, 100-1,000 μl
  3. Pipette tips, 1-200 μl
  4. Pipette tips, 0.1-10 μl
  5. 1.5 ml Eppendorf tubes (Corning, Axygen®, catalog number: MCT-150-C)
  6. 0.2 ml PCR tubes (Merck, Corning®, catalog number: CLS6531-960EA)
  7. Parafilm (Bemis, catalog number: PM996)
  8. Petri dishes 90 mm diameter (Merck, catalog number: P5731-500EA)
  9. Disposable cuvettes (Merck, catalog number: Z330418-1PAK)
  10. Disposable loops (Citotest Labware Manufacturing Co., catalog number: 2121-0001)
  11. Disposable spreaders (VWR, catalog number: 612-1560)
  12. 15 ml centrifuge tubes (Electron Microscopy Sciences, catalog number: 64760-01)
  13. 0.22 µm membrane filter (Merck, catalog number: GSTF02500)
  14. Donor strain E. coli S17-1 equipped with plasmids listed in Table 1 (strains can be acquired from Addgene, https://www.addgene.org/browse/article/28196767/)
  15. Recipient strain (e.g., Pantoea eucalypti 299R)
  16. Plasmids (Table 1) encoding for fluorescent proteins (Table 2)

    Table 1. Plasmids

    Source of all plasmids: Schlechter et al., 2018, plasmids can be obtained at Addgene plasmid number 118484 to 118579. X is a placeholder for either 0, 1, 2, 3, 4, 5, 6, 7 representing mTagBFP2, mTurquoise2, sGFP2, sYFP2, mOrange2, mScarlet-I, mCardinal, or mClover3 encoding versions of the plasmids, respectively. KanR = Kanamycin resistance; CamR = Chloramphenicol resistance; GentR = Gentamicin resistance; TetR = Tetracycline resistance; AmpR = Ampicillin resistance.

    Table 2. Fluorescent protein properties*

    *A more detailed spectrum figure can be found in Schlechter et al., 2018

  17. Lysogeny broth (Merck, catalog number: 110285)
  18. Agar No. 1 (Oxoid, catalog number: LP0011)
  19. Tris free base (Sigma, catalog number: T6791-500G)
  20. Glacial acetic acid (Sigma, catalog number: 695092-500ML)
  21. Glycerol (Sigma, catalog number: G6279-500ML)
  22. Succinate (Sigma, catalog number: S9637-500G)
  23. DMSO (Sigma, catalog number: D8418-100ML)
  24. dNTPs (Thermo Fisher, catalog number: N8080260)
  25. Multi-purpose HyAgaroseTM LE Agarose (HydraGene, catalog number: R9012LE-500g)
  26. Phusion polymerase (Thermo Fisher, catalog number: F530S)
  27. Oligonucleotides, synthesis amount 0.025 µmol, purified by desalting (see Table 3, Macrogen)

    Table 3. Primers


  28. Nucleic acid stain RedSafe (Intron, catalog number: 21141)
  29. Ampicillin (Duchefa Biochemie, catalog number: A0104)
  30. Gentamicin (Duchefa Biochemie, catalog number: G0124)
  31. Kanamycin (Duchefa Biochemie, catalog number: K0126)
  32. Chloramphenicol (Duchefa Biochemie, catalog number: C0113)
  33. Tetracycline (Duchefa Biochemie, catalog number: T0150)
  34. Ethanol (Carl Roth, catalog number: T171.5)
  35. K2HPO4 (Sigma, catalog number: 795496-100G)
  36. NaH2PO4·2H2O (Sigma, catalog number: 71505-250G)
  37. NH4Cl (Sigma, catalog number: A9434-500G)
  38. MgSO4·7H2O (Sigma, catalog number: 63138-250G)
  39. Na2EDTA·2H2O (Sigma, catalog number: E6635-500G)
  40. ZnSO4·7H2O (Sigma, catalog number: Z0251-100G)
  41. CoCl2·6H2O (Sigma, catalog number: C8661-25G)
  42. MnCl2 (Sigma, catalog number: 244589-10G)
  43. H3BO3 (Sigma, catalog number: 202878-10G)
  44. CaCl2 (Sigma, catalog number: C5670-500G)
  45. Na2MoO4·2H2O (Sigma, catalog number: 480967-25G)
  46. FeSO4·7H2O (Sigma, catalog number: F8263-500G)
  47. CuSO4·5H2O (Sigma, catalog number: 469130-50G)
  48. NaCl (Sigma, catalog number: S7653-1KG)
  49. KCl (Sigma, catalog number: P9333-500G)
  50. Na2HPO4 (Sigma, catalog number: S7907-500G)
  51. KH2PO4 (Sigma, catalog number: P5655-500G)
  52. NaOH (Sigma, catalog number: S8045-500G)
  53. Demineralized water
  54. Lysogeny broth (LB) (see Recipes)
  55. Lysogeny broth agar (LB agar) (see Recipes)
  56. Minimal medium (see Recipes)
    1. 10x ammonium solution 
    2. 10x phosphate buffer solution 
    3. 10x sulfate solution 
    4. 1,000x trace elements solution 
    5. 20% succinate solution 
    6. 3% agar 
  57. 1x Phosphate buffered saline (1x PBS) (see Recipes)
  58. Antibiotic stock solutions and working concentrations (see Recipes)
    1. Ampicillin 100 mg ml-1 stock solution
    2. Gentamicin 15 mg ml-1 stock solution
    3. Kanamycin 50 mg ml-1 stock solution
    4. Chloramphenicol 15 mg ml-1 stock solution
    5. Tetracycline 5 mg ml-1 stock solution
  59. TAE buffer (see Recipes)
    1. 0.5 M EDTA stock solution 
    2. 50x TAE stock solution
    3. 1x TAE working solution 

Equipment

  1. Forceps
  2. Pipettes (Eppendorf, P1000, catalog number: 3120000062; P200, catalog number: 3120000054; P10, catalog number: 3120000020)
  3. Erlenmeyer flasks 125 ml (e.g., Merck, catalog number: CLS4980125-12EA)
  4. Automatic micropipettes (Eppendorf or Gilson)
  5. Metal tweezers (VWR, catalog number: 232-0092)
  6. Bunsen burner (Eisco, catalog number: CH0087A)
  7. Vortex (Scientific Industries, catalog number: SI-0236)
  8. Autoclave (Heidolph, catalog number: 023210745CA)
  9. -20 °C Freezer (Fisher and Paykel, catalog number: E388LXFD1)
  10. Sterile bench (Labconco, catalog number: 302389100)
  11. Microcentrifuge for 1.5 ml centrifuge tubes with max speed > 6,000 x g (Eppendorf, catalog number: 5418000017)
  12. Centrifuge for 15 ml centrifuge tubes with max speed > 6,000 x g (Eppendorf, catalog number: 5805000327)
  13. Shaking incubators 30 °C and 37 °C (Infors, catalog number: Multitron Pro)
  14. Incubators 30 °C and 37 °C (Binder, catalog number: BD 56)
  15. Spectrophotometer (Biochrom WPA CO8000 Cell Density meter, catalog number: 80-3000-45)
  16. PCR thermocycler (Eppendorf, catalog number: 6311000010)
  17. Horizontal agarose gel electrophoresis setup including running chamber and power supply (VWR International, catalog number: 76196-448)
  18. Blue light or UV transilluminator (optional: camera for documentation) (Fisher Scientific, catalog number: UV95042001)
  19. Fluorescent microscope with appropriate filter sets for the excitation and emission spectra of the fluorescent protein tags and equipped with a 5x objective with a long working distance (Zeiss Axio Imager.M1 equipped with Objective A-Plan 5x/0.12 M27 [Zeiss, catalog number: 421030-9900-000])

Procedure

An overview of the workflow is shown in Figure 1.


Figure 1. Flowsheet of protocol

Prepare or have ready media and stock solutions for the conjugation

  1. Prepare 2 lysogeny broth (LB) agar plates without antibiotics.
  2. Prepare 1 LB plate containing kanamycin when working with a pMRE1XX plasmid or 1 LB plate containing ampicillin and chloramphenicol when working with transposon bearing plasmids (Ampicillin will select for the plasmid and chloramphenicol for the transposon).
  3. Prepare 3 ml and 50 ml LB broth without antibiotics.
  4. Prepare 3 ml and 50 ml LB broth containing kanamycin when working with a pMRE1XX plasmid or 3 ml and 50 ml LB broth containing ampicillin and chloramphenicol when working with transposon bearing plasmids. 
  5. Prepare ~10 minimal media plates containing succinate as the sole carbon source and antibiotics selecting for the plasmids or transposons as explained in Table 1. In case you are selecting for transposon insertions, do not include ampicillin, since it selects for the plasmid, not the transposon. Store at 4 °C and in the dark if the light-sensitive tetracycline is used in the plates.
    Note: Select a carbon source for minimal media plates that can be metabolized/utilized by the recipient strain.
  6. Prepare 100 ml sterile 1x PBS.

Day 1
  1. Streak donor strain E. coli S17-1 containing pMRE1XX, pMRE-Tn5-1XX, or pMRE-Tn7-1XX on LB agar plates containing appropriate antibiotics from a glycerol stock using a disposable loop and incubate overnight at 37 °C or 30 °C when working with pMRE-Tn7-1XX plasmids.
    Note: pMRE-Tn7-1XX plasmids do not replicate above 32 °C. Thus, to maintain it in the bacterial culture, it must be grown at lower temperatures.
  2. Streak recipient strain P. eucalypti 299R on an LB agar plate from a glycerol stock using a disposable loop and incubate overnight at 30 °C.

Day 2
  1. Inoculate 3 ml LB broth containing the same antibiotics as before with an individual colony of donor E. coli S17-1 containing pMRE1XX, pMRE-Tn5-1XX, or pMRE-Tn7-1XX using a sterile toothpick and ethanol flamed forceps. Incubate overnight at 200 rounds per minute (rpm) and 37 °C or 30 °C when working with pMRE-Tn7-1XX plasmids.
  2. Inoculate 3 ml LB broth without antibiotics with an individual colony of P. eucalypti 299R using a sterile toothpick and ethanol flamed forceps. Incubate overnight at 30 °C and 200 rpm.

Day 3
  1. Inoculate 50 ml LB broth in a 125 ml Erlenmeyer flask containing the same antibiotics as before with 500 µl overnight culture E. coli S17-1 containing pMRE1XX, pMRE-Tn5-1XX, or pMRE-Tn7-1XX using a sterile pipette tip and a P1000 pipette. Incubate at 200 rpm and 37 °C or 30 °C when working with pMRE-Tn7-1XX plasmids. Grow to an optical density at 600 nm (OD600nm) of ~0.5, which will take between 1.5 h and 3 h depending on the incubation temperature.
    Note: Measure OD600nm regularly using disposable cuvettes and a spectrophotometer and note down the final OD600nm.
  2. Inoculate 50 ml LB broth in a 125 ml Erlenmeyer flask containing no antibiotics with 500 µl overnight culture P. eucalypti 299R using a sterile pipette tip and a P1000 pipette. Incubate at 200 rpm and 30 °C. Grow until P. eucalypti 299R has reached late exponential/early stationary phase, which will take ~3-4 h (OD600nm of 0.7 or above). Measure OD600nm using disposable cuvettes and a spectrophotometer. Alternatively, recipient strain P. eucalypti 299R can be scraped off directly from the plate using a sterile disposable loop and resuspended in a 1.5 ml microcentrifuge tube containing 1 ml sterile 1x PBS by gentle vortexing.
    Note: P. eucalypti 299R is a fast growing strain with a similar growth rate to E. coli S17-1. For others, such as slow-growing strains, the inoculation volume or time of inoculation may vary accordingly. For other strains, growth rate, exponential growth phase and early/late stationary phase will have to be determined.
  3. Harvest 10 ml of both cultures by centrifugation at 2,000 x g for 5 min in a centrifuge. Discard the supernatants, resuspend each culture in 5 ml sterile 1x PBS by gentle vortexing and measure OD600nm.
    Note: Vortex gently for less than 5 s to avoid shearing of the donor which could impact conjugation efficiency. Mixing by inversion is also recommended.
  4. Mix the cultures to reach similar amounts of donor and recipient cells.
    Example: If the 10 ml E. coli S17-1 culture had an OD600nm of 0.5, it should be concentrated to OD600nm of 1. If the 10 ml P. eucalypti 299R culture had an OD600nm of 0.4, it should be concentrated to OD600nm of 0.8. The P. eucalypti 299R culture, therefore, contains only 80% of the cells that the E. coli S17-1 culture contains. Therefore, mix the 5 ml P. eucalypti 299R culture with 4 ml (80% of 5 ml) E. coli S17-1 culture.
    Note: For other strains, the number of CFU per OD600nm will have to be determined beforehand.
  5. Harvest the combined cultures by centrifugation at 2,000 x g for 5 min in a centrifuge, discard the supernatant and resuspend in 100 µl sterile 1x PBS using a vortex.
    Note: Vortex gently for less than 5 s to avoid shearing of donor pili which could impact conjugation efficiency. Mixing by inversion is also recommended.
  6. Dry an LB agar plate containing no antibiotics in a sterile bench for 15 min. In the sterile bench, dropspot the resuspended culture mix onto the agar using a sterile pipette tip and a 200 µl pipette. Keep the plate open and wait until all the liquid evaporates. Afterwards, close the plate and incubate overnight at 30 °C.

Day 4
  1. Harvest the culture drop using a wire loop and transfer to a 1.5 ml microcentrifuge tube containing 1 ml sterile 1x PBS. Resuspend the bacteria using a vortex. Plate 10 µl, 100 µl and the rest of the bacterial suspensions on the minimal media plates containing antibiotics selecting for the used plasmid or transposon using a disposable or flame-sterilized spreader. Minimal media will select against the auxotrophic E. coli donor.
  2. Incubate at 30 °C until visible colonies appear on the plates.

Days 5-8
Note: Slow growing strains may appear weeks after the conjugation.

  1. Check morphology and fluorescence emission in emerging colonies. Fluorescence can often be seen using a transilluminator (e.g., UV or Blue light) that is also used for agarose gel electrophoresis (e.g., for most green, yellow, orange, and red emitting proteins see Figure 2). Otherwise, and additionally, it is useful to investigate emerging colonies with a fluorescent microscope equipped with a very low magnification and long working distance objectives. Use filters that have appropriate excitation and emission filters. Often colonies are made up of both, the donor and the recipient strain, for an example see Figure 3A. Select colonies that appear homogeneously fluorescent under the fluorescent microscope and streak them on minimal medium (as above) to individual colonies (Figure 3B).
    Note: A fun and beautiful example of a forced consortium. The recipient strain provides nutrients for the otherwise auxotroph E. coli and E. coli is providing antibiotic resistance to the recipient strain.
  2. Restreak the colonies at least three times using a disposable loop in the subsequent days on minimal media plates.


    Figure 2. Bacterial colonies expressing fluorescent proteins. A and B. Bacteria were streaked as crude streaks on agar plates. Expressed fluorescent proteins were visualized on a blue light gel reader. Green, yellow, orange, red and far-red fluorescent colonies were easily detectable on gel readers, blue and cyan fluorescent colonies were difficult to distinguish from non-fluorescent wild type colonies. C. Expressed fluorescent proteins were visualized using a UV light tray with attached gel documentation system. Fluorescent protein expressing colonies appear brighter than the wild type.


    Figure 3. Overlay of fluorescent and brightfield microscope channels. A. Example of unsuccessful conjugation that yielded colonies. The colonies are mixes of a Sphingomonas species and red fluorescent protein expressing E. coli S17-1. B. Clonal colony of P. eucalypti 299R::MRE-Tn7-145. Scale bars = 500 µm.

Day 9, or after subsequent subcultivations
At this stage, you will have to follow different procedures, depending on the delivery systems you choose:
pMRE1XX plasmids

  1. As final check, perform PCRs to test for E. coli contamination. For that, perform a crude DNA extraction from a freshly grown individual colony by picking a part of the colony with an autoclaved toothpick and dislodging the cells in a 0.2 ml PCR tube filled with 100 µl 0.05 M NaOH. 
  2. Boil the sample for 15 min at 95 °C in a PCR machine. Use 1 µl of crude DNA extract as template for a PCR using Phusion polymerase and primers FWD_uidA and REV_uidA following the recommendations of the manufacturer (for an example refer to Figure 4).
    Note: Presence of E. coli will yield a ~500 bp PCR product. To check for the presence of uidA in a bacterial strain, the sequence of the E. coli gene (https://www.ncbi.nlm.nih.gov/gene/946149) can be blasted against the genome of the target strain.


    Figure 4. Colony PCR of uidA for detection of Escherichia coli. uidA is an E. coli and Shigella sp. specific gene, thereby amplification of this gene fragment indicates mixed colony or false positive colony after restreak and colony isolation. Primers used were FWD_uidA and REV_uidA (Table 3) and yield a 500 bp product. Lane M: Molecular marker (HyperLadderTM, Bioline); Sample 1: P. eucalypti 299R::MRE-Tn5-145; Sample 2: E. coli (pMRE-Tn5-145); Sample 3: P. eucalypti 299R::MRE-Tn7-145; Sample 4: E. coli (pMRE-Tn7-145); C-: water control. In this example, samples #1 and #3 showed no detection of E. coli, while sample #2 and #4 showed presence of uidA.

pMRE-Tn5-1XX–Tn5 transposon delivery plasmids
  1. Perform the E. coli contamination check as described above for pMRE1XX plasmids.
  2. Additionally, use primers FWD_Tn5_gt, REV_Tn5_gt, FWD_Tn5/7_gt, and REV_Tn5/7_gt in a multiplex PCR. The primer combination tests of the presence of the delivery plasmid and for the presence of the fluorescent protein coding sequence in the sample (for an example refer to Figure 5). Samples containing the plasmid will yield two bands (~700 bp and ~400 bp), which suggests E. coli contamination. Samples containing only the fluorescent protein coding sequence yield only the larger band (~700 bp).


    Figure 5. Colony PCR to confirm Tn5 insertions. Multiplex PCR result using primers FWD_Tn5_gt, REV_Tn5_gt, FWD_Tn5/7_gt, and REV_Tn5/7_gt. M: Molecular marker (HyperLadder, Bioline); Sample 1: P. eucalypti 299R::MRE-Tn5-145; Sample 2: E. coli (pMRE-Tn5-145); Sample 3: pMRE-Tn5-145; C-: water control.

pMRE-Tn7-1XX–Tn7 transposon delivery plasmids
  1. Curing of plasmid (depending on the recipient strain)
    pMRE-Tn7-1XX plasmid series are conditional suicide plasmids in many, but not all, Enterobacteriaceae. P. eucalypti belongs to the Enterobacteriaceae, i.e., the plasmid is able to replicate in P. eucalypti since the plasmids origin of replication pSC101 is functional in the strain. pSC101 is however heat instable and does not replicate at temperatures above 32 °C (McKenzie and Craig, 2006). Therefore, to cure the strain from the plasmid, it has to be grown at high temperatures.
    1. Inoculate a 5 ml LB broth culture using an individual fluorescent colony and grow it overnight at 37 °C. 
    2. Streak out the culture on an LB agar plate using an inoculation loop and incubate overnight at 37 °C. 
    3. Select 10 individual colonies and streak them each onto a new LB agar plate containing Amp and on an LB agar plate containing no antibiotic. Colonies that still contain the plasmid will be able to grow on ampicillin, while colonies where the transposition occurred will only grow on the LB agar plate without antibiotics.
  2. Perform the E. coli contamination check as described above for pMRE1XX plasmids.
  3. Additionally, perform a PCR to identify the presence of Tn7 insertion and/or Tn7-derived plasmid. Use FWD_Tn5/7_gt and REV_Tn5/7_g for the presence of the fluorescent protein coding sequence in the sample (~700 bp) and primers Tn7_gt1, Tn7_gt2, and Tn7_gt3 for the presence of the delivery plasmid (~1,000 bp). Tn7_gt1, Tn7_gt2, and Tn7_gt3 are needed because pMRE-Tn7-1XX plasmids were constructed by blunt-end cloning, so the insert orientation may vary between plasmids (Tn7_gt2, and Tn7_gt3 anneal to a flanking sequence of the insert in the plasmid backbone). For an example refer to Figure 6.


    Figure 6. Colony PCRs to confirm Tn7 insertions. PCR result using primers FWD_Tn5/7_gt and REV_Tn5/7_gt for the amplification of Tn7-delivered insert (MRE-Tn7 insert), and PCR result using primers Tn7_gt1, Tn7_gt2, and Tn7_gt3 for amplification of a fragment of plasmid pMRE-Tn7 backbone (MRE-Tn7 backbone). M: Molecular marker. Sample 1: P. eucalypti 299R::MRE-Tn7-145; Lane 2: E. coli (pMRE-Tn7-145); Lane 3: pMRE-Tn7-145; C-: water control.

Notes

  1. Depending on the recipient strain, addition of 100 µl drop spotted 1 M CaCl2 onto the agar plate used to drop spot the bacterial mixture onto the CaCl2 might increase conjugation efficiencies (Wang and Jin, 2014; McCully et al., 2018).
  2. Conjugation efficiency can be optimized by mixing donor and recipient strains in different ratios (e.g., perform three conjugations by using a 1:3, 1:1, and 3:1 ratio of donor and recipient strain, respectively).

Recipes

  1. Lysogeny broth (LB)
    Add 25 g of lysogeny broth medium to 1 L of demineralized water
    Autoclave
  2. Lysogeny broth agar (LB agar)
    Add 25 g of lysogeny broth medium and 15 g agar no. 1 to 1 L of demineralized water
    Autoclave
  3. Minimal medium (Attwood and Harder, 1972)
    1. Prepare the following stock solutions:
      1. 10x ammonium solution
        Dissolve 16.2 g of NH4Cl in 1 L of demineralized water
        Autoclave
      2. 10x phosphate buffer solution
        Dissolve 15.9 g of K2HPO4 and 18 g of NaH2PO4·2H2O in 1 L of demineralized water (pH should be 6.7 without adjustment)
        Autoclave
      3. 10x sulfate solution
        Dissolve 2 g of MgSO4 in 1 L of demineralized water
        Filter sterilize using a 0.22 µm filter to avoid precipitation
      4. 1,000x trace elements solution (Harder and Veldkamp, 1968)
        Dissolve 15 g Na2EDTA2·2H2O, 4.5 g ZnSO4·7H2O, 3 g CoCl2·6H2O, 0.6 g MnCl2, 1 g H3BO3, 3 g CaCl2, 0.4 g Na2MoO4·2H2O, 3 g FeSO4·7H2O, and 0.3 g CuSO4·5H2O in 1 L of demineralized water
        Filter sterilize using a 0.22 µm filter
      5. 20% succinate solution
        Dissolve 20 g succinate in 100 ml of demineralized water
        Filter sterilize using a 0.22 µm filter
        Note: P. eucalypti is able to grow on succinate as the sole carbon source, other strains may require other carbon source supplements to grow on minimal medium.
      6. 3% agar
        Add 15 g Agar No. 1 in 500 ml of demineralized water
        Autoclave
        Note: 3% agar should be at a temperature below 60 °C, since higher temperatures might lead to salts precipitation. This also prevents boiling retardation. Pour quickly after thoroughly mixing the agar medium.
    2. To prepare 1 L of minimal medium agar supplemented with 0.5% succinate as sole carbon source, combine 100 ml of 10x ammonium solution with 100 ml 10x phosphate buffer solution, 100 ml 10x sulphate solution, 1 ml of 1,000x trace element solution and 25 ml 20% succinate solution into 500 ml of molten 3% agar. Top up with sterile demineralized water to a final volume of 1 L
  4. 1x Phosphate buffered saline (1x PBS)
    Dissolve 8 g NaCl, 0.24 g KCl, 1.42 g Na2HPO4, 0.24 g KH2PO4 in 1 L of demineralized water and autoclave
    The buffer can alternatively be prepared as a 10x concentrated stock which is diluted to a 1x concentration with sterile water prior use
  5. Antibiotic stock solutions and working concentrations


    1. Ampicillin stock solution
      Dissolve ampicillin in water to a concentration of 100 mg ml-1 and filter sterilized using a 0.22 µm filter
    2. Gentamicin stock solution
      Dissolve gentamicin in water to a concentration of 15 mg ml-1 and filter sterilized using a 0.22 µm filter
    3. Kanamycin stock solution
      Dissolve kanamycin in water to a concentration of 50 mg ml-1 and filter sterilized using a 0.22 µm filter
    4. Chloramphenicol stock solution
      Dissolve chloramphenicol in ethanol to a concentration of 15 mg ml-1
    5. Tetracycline stock solution
      Dissolve tetracycline in ethanol to a concentration of 5 mg L-1
    Note: Store all antibiotic stock solutions in 1.5 ml aliquots at -20 °C.
  6. TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA)
    1. 0.5 M EDTA stock solution
      Dissolve 93.05 g Na2EDTA·2H2O in 400 ml of demineralized water and adjust the pH to 8.0 with NaOH
      Top up the solution to a final volume of 500 ml
      Note: EDTA will start dissolving when pH gets close to 8, use 10 M NaOH to adjust the pH.
    2. 50x TAE stock solution
      Dissolve 242 g Tris base in 750 ml of demineralized water
      Add 57.1 ml of 100% glacial acetic acid and 100 ml of 0.5 M EDTA solution
      Adjust the volume to 1 L with demineralized water
      This solution can be stored at room temperature
    3. 1x TAE working solution
      Dilute 20 ml of 50x TAE stock solution in 980 ml of demineralized water

Acknowledgments

The protocol presented here was adapted from two previously published studies (Remus-Emsermann et al., 2016a; Schlechter et al., 2018) and majorly influenced by a study by McKenzie and Craig (2006). The authors thank Michał Bernach, Hyunwoo Jun and the other authors of Schlechter et al., 2018 for their help during the original study.
  This work was funded by a seed grant of the Biomolecular Interaction Centre of the University of Canterbury to MR-E and the Royal Society of New Zealand Marsden Fast Start grant (UOC1704) to MR-E. RS was supported by an NZIDRS doctoral scholarship.

Competing interests

The authors declare no competing interests.

References

  1. Bahl, M. I., Sorensen, S. J. and Hestbjerg Hansen, L. (2004). Quantification of plasmid loss in Escherichia coli cells by use of flow cytometry. FEMS Microbiol Lett 232(1): 45-49.
  2. de Lorenzo, V., Herrero, M., Jakubzik, U. and Timmis, K. N. (1990). Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol 172(11): 6568-6572.
  3. Attwood, M. M. and Harder, W., (1972). A rapid and specific enrichment procedure for Hyphomicrobium spp. Antonie van Leeuwenhoek 38: 369–377.
  4. Harder, W. and Veldkamp, H. (1968). Physiology of an obligately psychrophilic marine Pseudomonas species. J Appl Microbiol 31: 12-23.
  5. Heinemann, J. A. and Sprague, G. F., Jr. (1989). Bacterial conjugative plasmids mobilize DNA transfer between bacteria and yeast. Nature 340(6230): 205-209.
  6. Ma, H. and Bryers, J. D. (2010). Non-invasive method to quantify local bacterial concentrations in a mixed culture biofilm. J Ind Microbiol Biotechnol 37(10): 1081-1089.
  7. McCully, L. M., Bitzer, A. S., Seaton, S. C., Smith, L. M. and Silby, M. W. (2018). Social Motility: Interaction between two sessile soil bacteria leads to emergence of surface motility. bioRxiv: 296814.
  8. McKenzie, G. J. and Craig, N. L. (2006). Fast, easy and efficient: site-specific insertion of transgenes into enterobacterial chromosomes using Tn7 without need for selection of the insertion event. BMC Microbiol 6: 39.
  9. Monier, J. M. and Lindow, S. E. (2005). Spatial organization of dual-species bacterial aggregates on leaf surfaces. Appl Environ Microbiol 71(9): 5484-5493.
  10. Remus-Emsermann, M. N. P., de Oliveira, S., Schreiber, L. and Leveau, J. H. (2011). Quantification of lateral heterogeneity in carbohydrate permeability of isolated plant leaf cuticles. Front Microbiol 2: 197.
  11. Remus-Emsermann, M. N. P., Gisler, P. and Drissner, D. (2016a). MiniTn7-transposon delivery vectors for inducible or constitutive fluorescent protein expression in Enterobacteriaceae. FEMS Microbiol Lett 363(16).
  12. Remus-Emsermann, M. N. P., Kim, E. B., Marco, M. L., Tecon, R. and Leveau, J. H. (2013). Draft genome sequence of the phyllosphere model bacterium Pantoea agglomerans 299R. Genome Announc 1(1).
  13. Remus-Emsermann, M. N. P., Schmid, M., Gekenidis, M. T., Pelludat, C., Frey, J. E., Ahrens, C. H. and Drissner, D. (2016b). Complete genome sequence of Pseudomonas citronellolis P3B5, a candidate for microbial phyllo-remediation of hydrocarbon-contaminated sites. Stand Genomic Sci 11: 75.
  14. Remus-Emsermann, M. N. P. and Schlechter, R. O. (2018). Phyllosphere microbiology: at the interface between microbial individuals and the plant host. New Phytol 218(4): 1327-1333.
  15. Schada von Borzyskowski, L., Remus-Emsermann, M., Weishaupt, R., Vorholt, J. A. and Erb, T. J. (2015). A set of versatile brick vectors and promoters for the assembly, expression, and integration of synthetic operons in Methylobacterium extorquens AM1 and other alphaproteobacteria. ACS Synth Biol 4(4): 430-443.
  16. Schlechter, R. O., Jun, H., Bernach, M., Oso, S., Boyd, E. F., Muñoz-Lintz, D. A., Dobson, R. C., Remus, D. M. and Remus-Emsermann, M. (2018). Chromatic Bacteria - A broad host-range plasmid and chromosomal insertion toolbox for fluorescent protein expression in bacteria. bioRxiv: 402172.
  17. Tecon, R. and Leveau, J. H. (2016). Symplasmata are a clonal, conditional, and reversible type of bacterial multicellularity. Sci Rep 6: 31914.
  18. Tomlin, K. L., Clark, S. R. and Ceri, H. (2004). Green and red fluorescent protein vectors for use in biofilm studies of the intrinsically resistant Burkholderia cepacia complex. J Microbiol Methods 57(1): 95-106.
  19. Wang, X. K. and Jin, J. L. (2014). Crucial factor for increasing the conjugation frequency in Streptomyces netropsis SD-07 and other strains. FEMS Microbiol Lett 357(1): 99-103.
  20. Waters, V. L. (2001). Conjugation between bacterial and mammalian cells. Nat Genet 29(4):375-6.

简介

最近,我们发布了一套大型多功能质粒,即色素细菌工具箱,可向革兰氏阴性菌提供8种不同的荧光蛋白基因和4种抗生素抗性基因组合。荧光标签是单细胞微生物学,合成社区研究,生物膜和宿主 - 微生物相互作用研究的重要工具。使用缀合辅助菌株 E.大肠杆菌S17-1作为供体,我们展示了质粒缀合如何用于提供广泛的宿主范围质粒,Tn 5 转座子递送质粒和Tn 7 转座子将质粒转移到属于变形菌门的物种中。为此,供体和受体细菌在标准生长条件下生长,然后将它们混合并在非选择性条件下培养。然后,在选择性培养基上选择转接合子或结合前接受者。使用工具的组合筛选突变菌落以确保存在所需的质粒或转座子,并且菌落不含任何存活的供体。通过缀合,可以修改多种革兰氏阴性细菌而无需事先(通常是耗时的)建立需要针对每个个体菌株调整的感受态细胞和电穿孔程序。此处提出的方案不仅用于递送色素细菌质粒和转座子,还可用于向细菌接受者递送其他可移动的质粒。
【背景】荧光蛋白已成为研究细菌的重要工具,例如,生物膜,基因表达研究或宿主 - 微生物相互作用(Tomlin et al。,2004; Monier和Lindow,2005; Ma和Bryers,2010; Remus-Emsermann et al。,2011; Schada von Borzyskowski et al。,2015; Remus-Emsermann et al。,2016a; Remus-Emsermann和Schlechter,2018; Schlechter et al。,2018)。

在最近发表的一项研究中,我们描述了96种质粒的构建,每种质粒在荧光蛋白基因,抗生素抗性标记组合或质粒骨架中各不相同。完整的系列提供八种不同的荧光蛋白基因,每种基因都有自己独特的激发和发射光谱,四种不同的抗生素抗性标记组合,广泛的宿主范围质粒,或基于两种不同转座子系统的递送质粒,Tn 5 和Tn 7 (Schlechter et al。,2018)。

广宿主质粒骨架和两个转座子系统具有不同的优点和缺点:1)质粒通常在受体细胞中以几个拷贝存在,这导致多个基因拷贝,从而通常具有更高的荧光信号。然而,质粒可能更容易丢失,导致细胞亚群可能失去表达荧光蛋白的能力(Bahl et al。,2004)。 2)Tn 5 转座子高效插入Proteobacteria,并且当它整合到细菌基因组中时,失去荧光蛋白标签的机会很小。然而,Tn 5 转座子将DNA随机插入基因组中,从而可能破坏对细菌适应性很重要的基因(de Lorenzo et al。,1990)。 3)Tn 7 转座子以位点特异性方式整合到受体细菌的基因组中,其中没有基因被破坏,因此应该对细菌适应性具有轻微影响。然而,Tn 7 转座子仅插入窄范围的细菌,例如,,大多数γ-变形菌,一些α-变形菌和β-变形菌。在γ-变形菌门之外,其插入频率可能较低(McKenzie和Craig,2006; Schlechter等,等,2018)。

在本协议中,我们描述了使用 Pantoea eucalypti 299R为细菌菌株配备荧光标记的程序(Remus-Emsermann et al。,2013; Tecon和Leveau,2016)作为受体菌株的一个例子。 P上。 eucalypti 299R是所有三种不同类型的递送系统,广泛宿主范围质粒和两种转座子系统的有效接受者。我们可以证明广泛宿主质粒以及Tn 5 和Tn 7 基于转座子的荧光蛋白的递送在该受体菌株中是可行的(Schlechter et al。,2018)。其他肠杆菌科,如 E.大肠杆菌, Erwinia amylovora 和 Pantoea vagans 也成功地用所有三种系统以及假单胞菌菌株如 Pseudomonas citronellolis P3B5进行了测试(Remus-Emsermann et al。,2016b; Schlechter et al。,2018)。此外,来自其他分类群的细菌,例如α-变形杆菌属 Sphingomonas 和 Methylobacterium 至少用Tn 5 转座子传递系统进行了修饰,在某些情况下,使用广宿主范围的质粒和Tn 7 转座子。

这里提出的协议基于使用 E的细菌交配。大肠杆菌 S17-1辅助菌株,能够将可移动的质粒转移到受体。共轭是一个跨越菌株,属和门水平的混杂过程,甚至可以跨越生命领域(Heinemann和Sprague,1989;沃特斯,2001)。当将遗传物质转移到实验室模型宿主中时经常使用诸如热激和电穿孔的转化方法,然而,新分离的菌株通常难以接受这些方法,并且转化方案的建立通常是繁琐且耗时的。通过依赖缀合,可以避免转化,并且可以像从琼脂平板上刮下受体细菌菌落并将它们与供体细菌混合一样简单。

关键字:荧光蛋白, 绿色荧光蛋白, 质粒 , Tn5转座子, Tn7转座子, 接合生殖, 可移动质粒

材料和试剂

  1. 高压灭菌,未经处理的木制牙签(当地超市选择)
  2. 移液器吸头,100-1,000μl
  3. 移液器吸头,1-200μl
  4. 移液器吸头,0.1-10μl
  5. 1.5毫升Eppendorf管(Corning,Axygen ®,目录号:MCT-150-C)
  6. 0.2 ml PCR管(Merck,Corning ®,目录号:CLS6531-960EA)
  7. Parafilm(Bemis,目录号:PM996)
  8. 培养皿直径90毫米(默克,目录号:P5731-500EA)
  9. 一次性比色皿(默克,目录号:Z330418-1PAK)
  10. 一次性环(Citotest Labware Manufacturing Co.,目录号:2121-0001)
  11. 一次性吊具(VWR,目录号:612-1560)
  12. 15ml离心管(Electron Microscopy Sciences,目录号:64760-01)
  13. 0.22μm膜滤器(默克,目录号:GSTF02500)
  14. 供体菌株 E.大肠杆菌 S17-1配有表1中列出的质粒(菌株可从Addgene获得, https://www.addgene.org/browse/article/28196767/
  15. 受体菌株(例如, Pantoea eucalypti 299R)
  16. 编码荧光蛋白的质粒(表1)(表2)

    表1.质粒

    所有质粒的来源:Schlechter et al。,2018,质粒可以从Addgene质粒:118484到118579获得.X是0,1,2,3,4,5,6的占位符,7分别代表质粒的mTagBFP2,mTurquoise2,sGFP2,sYFP2,mOrange2,mScarlet-I,mCardinal或mClover3编码形式。 Kan R =卡那霉素抗性; Cam R =氯霉素抗性; Gent R =庆大霉素抗性; Tet R =四环素抗性; Amp R =氨苄青霉素抗性。

    表2.荧光蛋白质特性*

    * 更详细的频谱图可以在Schlechter et al。,2018中找到

  17. Lysogeny肉汤(默克,目录号:110285)
  18. 琼脂1号(Oxoid,目录号:LP0011)
  19. Tris free base(Sigma,目录号:T6791-500G)
  20. 冰醋酸(Sigma,目录号:695092-500ML)
  21. 甘油(Sigma,目录号:G6279-500ML)
  22. 琥珀酸盐(Sigma,目录号:S9637-500G)
  23. DMSO(Sigma,目录号:D8418-100ML)
  24. dNTPs(Thermo Fisher,目录号:N8080260)
  25. 多用途HyAgarose TM LE琼脂糖(HydraGene,目录号:R9012LE-500g)
  26. Phusion聚合酶(Thermo Fisher,目录号:F530S)
  27. 寡核苷酸,合成量0.025μmol,通过脱盐纯化(参见表3,Macrogen)

    表3.引物


  28. 核酸染色RedSafe(内含子,目录号:21141)
  29. 氨苄青霉素(Duchefa Biochemie,目录号:A0104)
  30. 庆大霉素(Duchefa Biochemie,目录号:G0124)
  31. 卡那霉素(Duchefa Biochemie,目录号:K0126)
  32. 氯霉素(Duchefa Biochemie,目录号:C0113)
  33. 四环素(Duchefa Biochemie,目录号:T0150)
  34. 乙醇(Carl Roth,目录号:T171.5)
  35. K 2 HPO 4 (Sigma,目录号:795496-100G)
  36. NaH 2 PO 4 ·2H 2 O(Sigma,目录号:71505-250G)
  37. NH 4 Cl(Sigma,目录号:A9434-500G)
  38. MgSO 4 ·7H 2 O(Sigma,目录号:63138-250G)
  39. Na 2 EDTA·2H 2 O(Sigma,目录号:E6635-500G)
  40. ZnSO 4 ·7H 2 O(Sigma,目录号:Z0251-100G)
  41. CoCl 2 ·6H 2 O(Sigma,目录号:C8661-25G)
  42. MnCl 2 (Sigma,目录号:244589-10G)
  43. H 3 BO 3 (Sigma,目录号:202878-10G)
  44. CaCl 2 (Sigma,目录号:C5670-500G)
  45. Na 2 MoO 4 ·2H 2 O(Sigma,目录号:480967-25G)
  46. FeSO 4 ·7H 2 O(Sigma,目录号:F8263-500G)
  47. CuSO 4 ·5H 2 O(Sigma,目录号:469130-50G)
  48. NaCl(Sigma,目录号:S7653-1KG)
  49. KCl(Sigma,目录号:P9333-500G)
  50. Na 2 HPO 4 (Sigma,目录号:S7907-500G)
  51. KH 2 PO 4 (Sigma,目录号:P5655-500G)
  52. NaOH(Sigma,目录号:S8045-500G)
  53. 脱矿水
  54. Lysogeny肉汤(LB)(见食谱)
  55. Lysogeny肉汤琼脂(LB琼脂)(见食谱)
  56. 最小介质(见食谱)
    1. 10x铵溶液 
    2. 10x磷酸盐缓冲溶液 
    3. 10倍硫酸盐溶液 
    4. 1,000x微量元素解决方案 
    5. 20%琥珀酸盐溶液 
    6. 3%琼脂 
  57. 1x磷酸盐缓冲盐水(1x PBS)(见食谱)
  58. 抗生素储备溶液和工作浓度(见食谱)
    1. 氨苄青霉素100mg / ml -1 储备溶液
    2. 庆大霉素15mg ml -1 储备溶液
    3. 卡那霉素50mg ml -1 储备溶液
    4. 氯霉素15mg ml -1 储备溶液
    5. 四环素5mg ml -1 储备溶液
  59. TAE缓冲区(见食谱)
    1. 0.5 M EDTA原液 
    2. 50倍TAE库存解决方案
    3. 1x TAE工作解决方案 

设备

  1. 钳子
  2. 移液器(Eppendorf,P1000,目录号:3120000062; P200,目录号:3120000054; P10,目录号:3120000020)
  3. Erlenmeyer烧瓶125毫升(例如,默克,产品目录号:CLS4980125-12EA)
  4. 自动微量移液器(Eppendorf或Gilson)
  5. 金属镊子(VWR,目录号:232-0092)
  6. 本生灯(Eisco,目录号:CH0087A)
  7. Vortex(科学工业,目录号:SI-0236)
  8. 高压灭菌器(Heidolph,目录号:023210745CA)
  9. -20°C冰箱(Fisher and Paykel,目录号:E388LXFD1)
  10. 无菌工作台(Labconco,目录号:302389100)
  11. 用于1.5ml离心管的微量离心机,最大速度> 100℃。 6,000 x g (Eppendorf,目录号:5418000017)
  12. 离心15ml离心管,最大速度> 100℃。 6,000 x g (Eppendorf,目录号:5805000327)
  13. 摇动培养箱30°C和37°C(Infors,目录号:Multitron Pro)
  14. 孵化器30°C和37°C(粘合剂,目录号:BD 56)
  15. 分光光度计(Biochrom WPA CO8000细胞密度计,目录号:80-3000-45)
  16. PCR热循环仪(Eppendorf,目录号:6311000010)
  17. 水平琼脂糖凝胶电泳装置,包括运行室和电源(VWR International,目录号:76196-448)
  18. 蓝光或紫外透射仪(可选:文件相机)(Fisher Scientific,目录号:UV95042001)
  19. 荧光显微镜,具有适当的滤光片组,用于荧光蛋白标签的激发和发射光谱,并配备5倍物镜,工作距离长(Zeiss Axio Imager.M1配有Objective A-Plan 5x / 0.12 M27 [Zeiss,目录号: 421030-9900-000])

程序

工作流程概述如图1所示。


图1.协议流程表

准备或准备好用于结合的培养基和储备溶液

  1. 准备2个不含抗生素的溶原肉汤(LB)琼脂平板。
  2. 当使用携带转座子的质粒时,使用pMRE1XX质粒或含有氨苄青霉素和氯霉素的1个LB平板制备含有卡那霉素的1个LB平板(氨苄青霉素选择质粒,氯霉素选择转座子)。
  3. 准备3毫升和50毫升没有抗生素的LB肉汤。
  4. 当使用携带转座子的质粒时,使用pMRE1XX质粒或含有氨苄青霉素和氯霉素的3 ml和50 ml LB肉汤时,准备含有卡那霉素的3 ml和50 ml LB肉汤。 
  5. 准备~10个含有琥珀酸作为唯一碳源的基本培养基平板和选择质粒或转座子的抗生素,如表1所示。如果您选择转座子插入,不包括氨苄青霉素,因为它选择质粒,而不是转座子。如果在板中使用光敏四环素,则在4°C和黑暗中储存。
    注意:选择一种碳源,用于接受菌株代谢/利用的基本培养基板。
  6. 准备100毫升无菌1x PBS。

第1天
  1. 条纹供体菌株 E.含有pMRE1XX,pMRE-Tn 5 -1XX或pMRE-Tn 7 -1XX的大肠杆菌S17-1在LB琼脂平板上含有来自甘油的适当抗生素使用一次性环并在使用pMRE-Tn 7 -1XX质粒时在37°C或30°C温育过夜。
    注意:pMRE-Tn 7 -1XX质粒不会在32°C以上复制。因此,为了将其保持在细菌培养物中,它必须在较低温度下生长。
  2. 条纹受体菌株 P.使用一次性环从甘油原液在LB琼脂平板上的桉树酸299R,并在30℃下孵育过夜。

第2天
  1. 接种含有与以前相同的抗生素的3ml LB肉汤和单独的供体 E菌落。使用无菌牙签和乙醇燃烧的镊子,含有pMRE1XX,pMRE-Tn 5 -1XX或pMRE-Tn 7 -1XX的大肠杆菌S17-1。当使用pMRE-Tn 7 -1XX质粒时,以200转/分钟(rpm)和37℃或30℃孵育过夜。
  2. 接种3毫升不含抗生素的LB肉汤,单个菌落 P. eucalypti 299R使用无菌牙签和乙醇火焰镊子。在30°C和200 rpm下孵育过夜。

第3天
  1. 用含有与之前相同的抗生素的125ml锥形瓶中的500μl过夜培养物 E接种50ml LB肉汤。使用无菌移液管尖端和P1000移液管,含有pMRE1XX,pMRE-Tn 5 -1XX或pMRE-Tn 7 -1XX的大肠杆菌S17-1。当与pMRE-Tn 7 -1XX质粒一起使用时,在200rpm和37℃或30℃下孵育。生长至600nm的光密度(OD 600nm )〜0.5,这将需要1.5小时至3小时,这取决于孵育温度。
    注意:使用一次性比色皿和分光光度计定期测量OD 600nm 并记下最终的OD 600nm的
  2. 在不含抗生素的125ml Erlenmeyer烧瓶中接种50ml LB肉汤,用500μl过夜培养物 P. eucalypti 299R使用无菌移液管吸头和P1000移液器。在200 rpm和30°C孵育。一直长到 P. eucalypti 299R已经达到指数/早期稳定期,需要约3-4小时(OD 600nm 为0.7或更高)。使用一次性比色皿和分光光度计测量OD 600nm 。或者,受体菌株 P.可以使用无菌一次性环直接从板上刮下桉树藻299R,并通过温和涡旋将其重悬于含有1ml无菌1x PBS的1.5ml微量离心管中。
    注意: P. eucalypti 299R是一种快速生长的菌株,其生长速率与 E相似。大肠杆菌 S17-1。对于其他物种,例如生长缓慢的菌株,接种量或接种时间可相应地变化。对于其他菌株,必须确定生长速率,指数生长期和早期/晚期稳定期。
  3. 通过在离心机中以2,000μL离心5分钟离心收获10ml两种培养物。弃去上清液,通过温和涡旋将每种培养物重悬于5ml无菌1x PBS中,并测量OD 600nm 。
    注意:轻轻涡旋不到5秒,以避免剪切供体,这可能影响结合效率。还建议通过反演进行混合。
  4. 混合培养物以达到相似数量的供体和受体细胞。
    示例:如果是10毫升 E。大肠杆菌 S17-1培养物的OD 600nm 为0.5,应将其浓缩至OD 600nm 1.如果10毫升 P. eucalypti 299R培养物的OD 600nm 为0.4,应浓缩至OD 600nm 0.8。 P.因此,eucalypti 299R培养物仅含有 E的80%的细胞。大肠杆菌 S17-1培养物含有。因此,混合5毫升 P. eucalypti 299R培养物用4 ml(80%的5 ml) E。大肠杆菌 S17-1培养。
    注意:对于其他菌株,每个OD的CFU数 600nm 必须事先确定。
  5. 通过在离心机中以2,000μL离心5分钟收集组合的培养物,丢弃上清液并使用涡旋重悬于100μl无菌1x PBS中。
    注意:轻轻涡旋不到5秒,以避免剪切供体菌毛,这可能会影响结合效率。还建议通过反演进行混合。
  6. 在无菌工作台中干燥不含抗生素的LB琼脂平板15分钟。在无菌工作台中,使用无菌移液管尖端和200μl移液管将重悬浮的培养物混合物滴在琼脂上。保持板打开并等待所有液体蒸发。然后,关闭平板并在30℃下孵育过夜。

第4天
  1. 使用线环收获培养液滴并转移至含有1ml无菌1x PBS的1.5ml微量离心管中。使用涡旋重悬细菌。将10μl,100μl和其余细菌悬浮液平板置于含有抗生素的基本培养基平板上,使用一次性或火焰灭菌的涂布器选择使用的质粒或转座子。最小的媒体将针对营养缺陷型 E进行选择。大肠杆菌供体。
  2. 在30°C孵育直至可见菌落出现在平板上。

第5-8天
注意:在结合后几周可能出现生长缓慢的菌株。
  1. 检查新出现的菌落中的形态和荧光发射。通常可以使用透射仪(例如,UV或蓝光)观察荧光,该透射仪也用于琼脂糖凝胶电泳(例如,对于大多数绿色,黄色,橙色和红色发光蛋白见图2)。另外,另外,用配备有非常低放大率和长工作距离物镜的荧光显微镜研究新出现的菌落是有用的。使用具有适当激发和发射滤光片的滤光片。通常菌落由供体和受体菌株两者组成,例如参见图3A。选择在荧光显微镜下看起来均匀荧光的菌落,并将它们在基本培养基(如上所述)上划线到单个菌落(图3B)。
    注意:强迫联盟的一个有趣而美丽的例子。受体菌株为其他营养缺陷型 E提供营养。大肠杆菌 和 E.大肠杆菌 为受体菌株提供抗生素抗性。
  2. 在随后的几天中,在最小的培养基平板上使用一次性环将菌落重新划线至少三次。


    图2.表达荧光蛋白的细菌菌落。 A和B.细菌在琼脂平板上作为粗条纹划线。表达的荧光蛋白在蓝光凝胶读数器上可视化。绿色,黄色,橙色,红色和远红色荧光菌落在凝胶读数器上很容易检测到,蓝色和青色荧光菌落难以与非荧光野生型菌落区分开。 C.使用附有凝胶记录系统的UV光盘显现表达的荧光蛋白。表达菌落的荧光蛋白看起来比野生型更亮。


    图3.荧光和明场显微镜通道的叠加。 A.产生菌落的不成功缀合的实例。菌落是 Sphingomonas 物种和表达 E的红色荧光蛋白的混合物。大肠杆菌 S17-1。 B. P的克隆菌落。 eucalypti 299R :: MRE-Tn 7 -145。比例尺=500μm。

第9天,或随后的传播
在此阶段,您必须遵循不同的程序,具体取决于您选择的交付系统:
pMRE1XX质粒
  1. 作为最终检查,执行PCR以测试 E.大肠杆菌污染。为此,通过用高压灭菌的牙签挑取一部分菌落并从填充有100μl0.05MNaOH的0.2ml PCR管中移出细胞,从新生长的单个菌落中进行粗DNA提取。 
  2. 在PCR机中将样品在95℃下煮沸15分钟。根据制造商的建议,使用1μl粗DNA提取物作为模板进行PCR,使用Phusion聚合酶和引物FWD_ uidA 和REV_ uidA (参见图4) )。
    注意: E的存在。大肠杆菌 将产生约500bp的PCR产物。要检查细菌菌株中是否存在 uidA , E的序列。大肠杆菌 基因( https:// www.ncbi.nlm.nih.gov/gene/946149 )可以针对目标菌株的基因组进行爆破。


    图4. uidA 的菌落PCR检测 大肠杆菌 。 uidA 是 E。大肠杆菌和志贺氏菌属。特异性基因,因此该基因片段的扩增表明在重新分裂和菌落分离后混合菌落或假阳性菌落。使用的引物是FWD_ uidA 和REV_ uidA (表3)并产生500bp的产物。泳道M:分子标记(HyperLadder TM ,Bioline);样本1: P. eucalypti 299R :: MRE-Tn 5 -145;样本2: E.大肠杆菌(pMRE-Tn 5 -145);样本3: P. eucalypti 299R :: MRE-Tn 7 -145;样本4: E.大肠杆菌(pMRE-Tn 7 -145); C-:水控制。在该示例中,样本#1和#3未显示 E的检测。大肠杆菌,而样品#2和#4显示 uidA 的存在。

的 pMRE-Tn的 <强> 5 <强> -1XX-Tn的 5 转座子传递质粒
  1. 执行 E.如上所述对pMRE1XX质粒进行大肠杆菌污染检查。
  2. 此外,使用引物FWD_Tn 5 _gt,REV_Tn 5 _gt,FWD_Tn 5 / 7 _gt和REV_Tn 多重PCR中> 5 / 7 _gt。引物组合测试了递送质粒的存在和样品中荧光蛋白编码序列的存在(例如参见图5)。含有质粒的样品将产生两条带(~700bp和~400bp),这表明 E.大肠杆菌污染。仅含有荧光蛋白编码序列的样品仅产生较大的条带(~700bp)。


    图5.确定Tn 5 插入的菌落PCR。使用引物FWD_Tn 5 <的多重PCR结果/ em> _gt,REV_Tn 5 _gt,FWD_Tn 5 / 7 _gt,以及REV_Tn 5 / 7 _gt。 M:分子标记(HyperLadder,Bioline);样本1: P. eucalypti 299R :: MRE-Tn 5 -145;样本2: E.大肠杆菌(pMRE-Tn 5 -145);样品3:pMRE-Tn 5 -145; C-:水控制。

的 pMRE-Tn的 <强> 7 <强> -1XX-Tn的 7 转座子传递质粒
  1. 质粒的固化(取决于受体菌株)
    pMRE-Tn 7 -1XX质粒系列是许多但不是全部肠杆菌科的条件性自杀质粒。 P上。 eucalypti 属于肠杆菌科,即,质粒能够在 P中复制。 eucalypti ,因为质粒复制起点pSC101在菌株中是有功能的。然而,pSC101是热不稳定的,并且在高于32℃的温度下不会复制(McKenzie和Craig,2006)。因此,为了从质粒中固化菌株,它必须在高温下生长。
    1. 使用单个荧光菌落接种5ml LB肉汤培养物并在37℃下培养过夜。&nbsp;
    2. 使用接种环在LB琼脂平板上划线培养物并在37℃下孵育过夜。&nbsp;
    3. 选择10个单独的菌落,并将它们各自条纹放在含有Amp的新LB琼脂平板上和不含抗生素的LB琼脂平板上。仍然含有质粒的菌落将能够在氨苄青霉素上生长,而发生转座的菌落将仅在没有抗生素的LB琼脂平板上生长。
  2. 执行 E.如上所述对pMRE1XX质粒进行大肠杆菌污染检查。
  3. 另外,进行PCR以鉴定Tn 7 插入和/或源自Tn 7 的质粒的存在。使用FWD_Tn 5 / 7 _gt和REV_Tn 5 / 7 _g来检测荧光蛋白编码序列的存在样品(~700 bp)和引物Tn 7 _gt1,Tn 7 _gt2和Tn 7 _gt3表示是否存在传递质粒( ~1,000 bp)。由于pMRE-Tn 7 -1XX质粒,因此需要Tn 7 _gt1,Tn 7 _gt2和Tn 7 _gt3通过平末端克隆构建,因此质粒之间的插入方向可能不同(Tn 7 _gt2和Tn 7 _gt3与质粒中插入片段的侧翼序列退火骨干)。有关示例,请参见图6.


    图6.确定Tn 7 插入的菌落PCR。使用引物FWD_Tn 5的PCR结果 / 7 _gt和REV_Tn 5 / 7 _gt用于扩增Tn 7 -delivered insert(MRE) -Tn 7 插入),使用引物Tn 7 _gt1,Tn 7 _gt2和Tn 7 的PCR结果_gt3用于扩增质粒pMRE-Tn 7 骨架(MRE-Tn 7 骨架)的片段。 M:分子标记。样本1: P. eucalypti 299R :: MRE-Tn 7 -145;第2道: E.大肠杆菌(pMRE-Tn 7 -145);泳道3:pMRE-Tn 7 -145; C-:水控制。

笔记

  1. 根据受体菌株的不同,在琼脂平板上加入100μl滴点1M CaCl 2 用于将细菌混合物滴到CaCl 2 上可能会提高结合效率( Wang和Jin,2014; McCully et al。,2018)。
  2. 通过以不同比例混合供体和受体菌株可以优化缀合效率(例如,通过分别使用1:3,1:1和3:1比例的供体和受体菌株进行三次缀合)。

食谱

  1. Lysogeny肉汤(LB)
    将25g溶原肉汤培养基加入1升软化水中 高压灭菌器
  2. Lysogeny肉汤琼脂(LB琼脂)
    加入25克溶原肉汤培养基和15克琼脂。 1至1升软化水
    高压灭菌器
  3. 最小的媒介(Attwood和Harder,1972)
    1. 准备以下库存解决方案:
      1. 10x铵溶液
        将16.2克NH 4 Cl溶于1升软化水中 高压灭菌器
      2. 10x磷酸盐缓冲溶液
        溶解15.9g K 2 HPO 4 和18g NaH 2 PO 4 ·2H 2 O在1升软化水中(pH值应为6.7,无需调整)
        高压灭菌器
      3. 10x硫酸盐溶液
        将2g MgSO 4 溶于1L软化水中 使用0.22μm过滤器过滤灭菌以避免沉淀
      4. 1,000x微量元素解决方案(Harder和Veldkamp,1968)
        溶解15g Na 2 EDTA 2 ·2H 2 O,4.5 g ZnSO 4 ·7H 2 O,3 g CoCl 2 ·6H 2 O,0.6 g MnCl 2 ,1 g H 3 BO 3 ,3 g CaCl 2 ,0.4 g Na 2 MoO 4 ·2H 2 O,3 g FeSO 4 ·7H 2 O,和0.3 g CuSO 4 ·5H 2 1升的去离子水中的水分
        使用0.22μm过滤器过滤灭菌
      5. 20%琥珀酸盐溶液
        将20克琥珀酸盐溶于100毫升软化水中 使用0.22μm过滤器过滤灭菌
        注意: P. eucalypti 能够以琥珀酸作为唯一碳源生长,其他菌株可能需要其他碳源补充剂才能在基本培养基上生长。
      6. 3%琼脂
        在500毫升软化水中加入15克琼脂1号
        高压灭菌器
        注意:3%琼脂的温度应低于60°C,因为较高的温度可能导致盐沉淀。这也可以防止沸腾延迟。充分搅拌琼脂培养基后快速倒入。
    2. 为了制备1L补充有0.5%琥珀酸盐作为唯一碳源的基本培养基琼脂,将100ml 10x铵溶液与100ml 10x磷酸盐缓冲溶液,100ml 10x硫酸盐溶液,1ml 1,000x微量元素溶液和25ml混合。将20%琥珀酸盐溶液加入500ml熔融的3%琼脂中。加入无菌软化水,最终体积为1升
  4. 1x磷酸盐缓冲盐水(1x PBS)
    溶解8g NaCl,0.24g KCl,1.42g Na 2 HPO 4 ,0.24g KH 2 PO 4 在1升软化水和高压灭菌器中 或者,可以将缓冲液制备成10倍浓缩的原液,在使用前用无菌水稀释至1倍浓度
  5. 抗生素储备液和工作浓度


    1. 氨苄青霉素原液
      将氨苄青霉素溶于水中至浓度为100 mg ml -1 并使用0.22μm过滤器过滤灭菌
    2. 庆大霉素原液解决方案
      将庆大霉素溶于水中至浓度为15 mg ml -1 并使用0.22μm过滤器过滤灭菌
    3. 卡那霉素原液
      将卡那霉素溶于水中至浓度为50 mg ml -1 并使用0.22μm过滤器过滤灭菌
    4. 氯霉素原液溶液
      将氯霉素溶解在乙醇中至浓度为15mg / ml -1
    5. 四环素库存解决方案
      将四环素溶解在乙醇中至浓度为5mg L-1
    注意:将所有抗生素储备溶液以1.5 ml等分试样储存在-20°C。
  6. TAE缓冲液(40mM Tris,20mM乙酸,1mM EDTA)
    1. 0.5 M EDTA原液
      将93.05g Na 2 EDTA·2H 2 O溶于400ml软化水中,用NaOH调节pH至8.0。 补充最终体积为500毫升的解决方案
      注意:当pH接近8时,EDTA将开始溶解,使用10 M NaOH调节pH值。
    2. 50x TAE库存解决方案
      将242克Tris碱溶于750毫升软化水中
      加入57.1毫升100%冰醋酸和100毫升0.5M EDTA溶液
      用去离子水将体积调节到1L
      该溶液可以在室温下储存
    3. 1x TAE工作解决方案
      在980ml软化水中稀释20ml 50x TAE储备溶液

致谢

这里介绍的协议改编自两项先前发表的研究(Remus-Emsermann et al。,2016a; Schlechter et al。,2018),并受McKenzie的一项研究的影响。和克雷格(2006年)。作者感谢MichałBernach,Hyunwoo Jun和Schlechter 等人的其他作者,2018在原始研究期间的帮助。
&NBSP;这项工作的资金来自坎特伯雷大学生物分子相互作用中心和MR-E的MR-E和新西兰皇家学会马斯登快速启动资助(UOC1704)。 RS获得NZIDRS博士奖学金的支持。

利益争夺

作者宣称没有竞争利益。

参考

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引用:Schlechter, R. O. and Remus-Emsermann, M. N. (2019). Delivering "Chromatic Bacteria" Fluorescent Protein Tags to Proteobacteria Using Conjugation. Bio-protocol 9(7): e3199. DOI: 10.21769/BioProtoc.3199.
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