参见作者原研究论文

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Mar 2017

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Imaging Gene Expression Dynamics in Pseudomonas fluorescens In5 during Interactions with the Fungus Fusarium graminearum PH-1
荧光假单胞菌In5与小麦赤霉菌PH-1互作过程中的基因表达动态成像   

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Abstract

Genomics, transcriptomics and metabolomics are powerful technologies for studying microbial interactions. The main drawback of these methods is the requirement for destructive sampling. We have established an alternative but complementary technique based on a microplate system combined with promoter fusions for visualizing gene expression in space and time. Here we provide a protocol for measuring spatial and temporal gene expression of a bacterial reporter strain interacting with a fungus on a solid surface.

Keywords: Microbial interactions (微生物相互作用), Pseudomonas (假单胞菌), Fungi (真菌), Bioassay (生物测定), Imaging (成像), Spatial and temporal gene expression (基因时空表达), Living cells (活细胞)

Background

Microbial interactions underpin many important biotechnological applications spanning medicine, food development and processing, bioremediation and biocontrol. In order to study microbial interactions, a broad range of technologies exist including genomics, transcriptomics and mass spectrometry imaging to study gene expression and metabolites exchanged during interactions. While these methods advance our understanding of microbial interactions, they also have limitations namely the requirement for destructive sampling. We have developed a microplate reader-based system for visualizing gene expression dynamics in living bacterial cells in response to a fungus in space and real-time. Pseudomonas fluorescens In5 is a Gram-negative soil bacterium and potent producer of secondary metabolites with antifungal activity (Michelsen et al., 2015; Hennessy et al., 2015). We previously identified the LuxR-type regulator NunF as a key regulator for synthesis of the antifungal compounds nunamycin and nunapeptin (Hennessy et al., 2017a). In this protocol, we detail how in vivo monitoring of target gene expression in living bacterial cells interacting with living fungal cells can be performed using a microplate-reader based technique (Hennessy et al., 2017b). P. fluorescens In5 expressing the red fluorescent protein mCherry fused to the promoter region of a regulator gene nunF indicating activation of an antifungal secondary metabolite gene cluster was used as a reporter system. Time-lapse image recordings of the reporter red signal and a green signal from fluorescent metabolites naturally produced by the bacterium combined with microbial growth measurements showed that nunF-regulated gene transcription is switched on when the bacterium enters the deceleration growth phase and upon physical encounter with fungal hyphae. The established non-destructive method has many advantages notably the ability to provide detailed space and time information on the transcription of target genes in living organisms. Importantly, the technique is a fast and simple alternative and complementary tool to the many technologies already used for studying microbial interactions.

Here we present a detailed protocol for imaging microbial interactions. In this example, we describe the construction of a reporter strain of the antimicrobial isolate Pseudomonas fluorescens In5 coupled with imaging analysis of gene expression of the reporter strain during an interaction with the fungus Fusarium graminearum PH-1 on an agar surface.

Materials and Reagents

  1. Eclipse pipette refilling system (Labcon, catalog numbers: 1045-260-000, 1093-260-000, 1036-260-000)
  2. NuncTM OmniTrayTM Single-Well Plate (Fischer Scientific, catalog number: 140156)
  3. Gene Pulser® Cuvettes 0.2 cm gap (Bio-Rad, catalog number: 1652086)
  4. 2 ml Microcentrifuge tubes (Fischer Scientific, catalog number: 21-402-905)
  5. NEB® 5-alpha Competent E. coli (Subcloning Efficiency) (NEB, catalog number: C2988J)
  6. Restriction enzymes: BamHI (NEB, catalog number: R3136S ), EcoR1 (NEB, catalog number: R0101S )
  7. Gentra Puregene Yeast/Bact. kit (QIAGEN, catalog number: 158567)
  8. Reporter plasmid pSEVA237R expressing the red fluorescent protein mCherry (Hennessy et al., 2018)
  9. Phusion high-fidelity polymerase (Fisher Scientific, catalog number: F530S)
  10. Wizard® SV Gel and PCR Clean-Up System (Promega, catalog number: A9281)
  11. Plasmid Mini Kit QIAprep Spin Miniprep Kit (QIAGEN, catalog number: 27104)
  12. Gibson Assembly® Cloning Kit (NEB, catalog number: E5510S)
  13. BactoTM agar (Difco, catalog number: 214050)
  14. Luria-Bertani broth (LB) (Difco, catalog number: 244610)
  15. LB agar (LBA) (Difco, catalog number: 244520)
  16. SOC outgrowth medium (NEB, catalog number: B9020S)
  17. Defined Fusarium Medium (DFM) (Hennessy et al., 2017b)
  18. Kanamycin sulfate monohydrate (Duchefa Biochemie, catalog number: K0126)
  19. Sucrose (Sigma-Aldrich, catalog number: S0389)
  20. Bacto-tryptone (VWR Chemicals, catalog number: 84610)
  21. Yeast extract (VWR Chemicals, catalog number: 84601)
  22. NaCl (Millipore, catalog number: 1.06404.1000)
  23. Glucose (Sigma-Aldrich, catalog number: 49159)
  24. Asparagine (Difco, catalog number: 214410)
  25. MgSO4 (VWR Chemicals, catalog number: 25164.265)
  26. KH2PO4 (Sigma-Aldrich, catalog number: NIST200B)
  27. KCl (Sigma-Aldrich, catalog number: P9333)
  28. Na2B4O7·10H2O (Sigma-Aldrich, catalog number: S9640)
  29. CuSO4·5H2O (Sigma-Aldrich, catalog number: C8027)
  30. FeSO4·7H2O (Sigma-Aldrich, catalog number: 1270355)
  31. MnSO4·H2O (Sigma-Aldrich, catalog number: M7634)
  32. NaMoO2·2H2O (Sigma-Aldrich, catalog number: 331058)
  33. ZnSO4·7H2O (Sigma-Aldrich, catalog number: Z0251)
  34. 300 mM sucrose (see Recipes)
  35. LB medium (see Recipes)
  36. DFM agar (see Recipes)
  37. 1,000x Trace solution (see Recipes)

Equipment

  1. Pipettes (VWR, catalog numbers: 613-2696E, 613-2702E, 613-2704E)
  2. Microplate reader (BMG LABTECH, catalog number: FLUO-star Omega)
  3. Eppendorf® Mastercycler® (Sigma-Aldrich, catalog number: EP6313000018-1EA)
  4. Gene Pulser® II (Bio-Rad, catalog number: 165-2105)
  5. Sterile inoculation loops (10 μl) (VWR, catalog number: 12000-812)
  6. Cork borer (6 mm Ø) for making fungal plugs
  7. Water bath or a heat block

Software

  1. BMG Omega Mars Software (Omega V5.11; BMG LABTECH) 
  2. ImageJ analysis software (https://imagej.nih.gov/ij/)
  3. "Text image to stack” macro (ImageJ, http://rsbweb.nih.gov/ij/macros/ImportTextImageSequence.txt)
  4. Bulk Rename Utility, (https://www.bulkrenameutility.co.uk)
  5. Batch converter software converting BMG Omega Mars exported well scan text files to image text files for a rectangle of adjacent wells (MofM.exe)

Procedure

  1. Selection of target gene and construction of reporter strains
    The first step of this protocol is to select a gene of interest (GOI) for gene expression analysis. Once selected, the promoter region located upstream of the GOI is PCR-amplified and cloned in front of a fluorescent protein-encoding gene. The promoter region (400 bp) upstream of the start codon is PCR-amplified using a high-fidelity polymerase and primers introducing overhangs to enable Gibson Assembly® cloning (Gibson et al., 2009) into the target plasmid.

  2. PCR to amplify promoter region located upstream of target gene
    1. Thaw the Phusion kit reagents and thoroughly mix after thawing.
    2. Make a 2x Phusion High-Fidelity (HF) DNA Polymerase PCR master mix using the following recipe:
      20 μl Phusion HF Buffer
      2 μl 10 mM dNTPs
      1 μl DMSO
      2 μl forward primer (10 μM) (5′-CACAGGAGGCCGCCTAGGCCGCGGCCGCGCGAATTCGCCGACCGTTGGTCGGCTTTGTC-3′)
      2 μl reverse primer (10 μM) (5′-GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCAAGCCTGCATACCAAAATCGCTG-3′)
      1 μl Phusion HF Taq Polymerase
      70 μl Nuclease-free water
    3. Dispense 49 μl of 2x Phusion PCR master mix into sterile 0.5 μl PCR tubes and add 1 μl genomic DNA (50 ng μl-1) purified from P. fluorescens In5 as previously described (Dichmann et al., 2018).
    4. PCR program:
      a. 98 °C 30 s
      b. 98 °C 10 s
      c. 62 °C 15 s
      d. 72 °C 15 s
      e. Repeat steps b-d, 30x
      f. 72 °C 10 min
    Note: Recommended running a water blank to ensure amplified bands are not due to contaminants in the PCR reagents.
    PAUSE POINT: PCR products can be stored at 4 °C or -20 °C.

  3. Preparation of reporter plasmid for Gibson assembly® cloning
    1. Digest plasmid DNA of the reporter plasmid pSEVA237R (Hennessy et al., 2017a) harboring a gene encoding the red fluorescent marker mCherry using the restriction enzymes BamHI-EcoR1. Once digested, purify the digested plasmid DNA using the Promega Gel extraction kit according to the manufacturer’s instructions.
    2. Set up a Gibson assembly® reaction on ice according to the manufacturer’s instructions. Incubated the Gibson assembly® reaction for 1 h at 50 °C without shaking. 
    3. Thaw 50 μl of E. coli DH5α chemically competent cells on ice for 30 min and add 5 μl of Gibson assembly® mix and incubate cells for further 30 min on ice. 
    4. Heat-shock cells using either a water bath for 45 s at 42 °C or a heat block warmed to 42 °C for 90 s. 
    5. Add 250 μl of SOC outgrowth medium to the heat-shocked cells and incubate shaking 200 x g at 37 °C for 1 h.
    6. Plate 100 μl and 200 μl on LBA supplemented with kanamycin (50 μg/ml). 
    7. Isolate antibiotic resistant colonies into 10 ml LB supplemented with kanamycin (50 μg/ml) and incubate shaking 200 x g overnight at 37 °C.
    8. Purify plasmid DNA and digest using BamHI-EcoR1 to identify positive clones.
    9. Perform DNA sequencing to confirm the integrity of the DNA. Once the plasmid DNA is confirmed by sequencing, it can be transformed into P. fluorescens In5.

  4. Transformation of P. fluorescens In5 with the reporter plasmid pSEVA237R::PGOI
    1. Inoculate P. fluorescens In5 in 10 ml LB broth shaking 200 x g overnight at 28 °C.
    2. Centrifuge 6 ml of overnight culture at 4,000 x g for 5 min at 20 °C.
    3. Wash cells three times each with 4 ml 300 mM sterile filtered sucrose.
    4. Resuspend the washed bacterial pellet in 400 μl of 300 mM sterile filtered sucrose.
    5. Add 300-500 ng plasmid DNA from each construct: tester plasmid (pSEVA237R::PGOI) and empty vector control (pSEVA237R) to 100 μl competent cells.
    6. Transfer mixture to 2 mm gap width electroporation cuvette. 
    7. Apply a pulse (25 μF, 200 Ω, 2.5 kV) in the electroporation apparatus. 
    8. Add 1 ml LB at room temperature.
    9. Transfer mixture to a 2 ml Eppendorf tube.
    10. Shake mixture at 200 x g 2 h at 28 °C.
    11. Isolate antibiotic resistant colonies into 10 ml LB supplemented with kanamycin (50 μg/ml) and incubate shaking 200 x g overnight at 28 °C.
    12. Purify plasmid DNA and digest using BamHI-EcoR1 to identify positive clones.
    Note: Recommend adding no DNA to 100 μl competent cells as a negative control. If a lawn appears on transformed plates but not on the negative control plate, streak cells onto a fresh LBA kanamycin (50 μg/ml) plate and incubate overnight with no shaking at 28 °C to isolate single antibiotic-resistant colonies.

  5. Pre-culturing of F. graminearum PH-1 and P. fluorescens In5
    1. Using a sterile cork borer, transfer a 6 mm Ø sized plug of F. graminearum PH-1 onto DFM media prepared as previously described (Hennessy et al., 2017b) and incubate for 5 days at 28 °C.
    2. Inoculate P. fluorescens In5 harboring either pSEVA237R::PGOI or pSEVA237R in 10 ml LB broth supplemented with 50 μg ml-1 kanamycin (antibiotic selectable marker on pSEVA237R backbone) and incubate shaking 200 x g overnight at 28 °C.

  6. Establishment of bacterial-fungal interaction on solid surface
    1. Add 35 ml of DFM agar to a sterile NuncTM OmniTrayTM. Place 10 plugs of F. graminearum PH-1 (6 mm Ø) down the center of the plate in a straight line. 
    2. Using a 10 μl sterile loop streak the bacterial reporter strains 3 cm away from the fungal plugs. Allow bacterial streaks to dry on the surface.

  7. Experimental set-up
  1. The bacterial reporter strains and fungus are then co-cultured in the microplate reader, programmed to simultaneously measure red and green fluorescence respectively in addition to cell density (Figures 1 and 2). 
  2. For step-by-step instructions for defining plates and time-series recording of images using the plate reader software BMG Omega Mars Software see Hennessy et al. (2017b).


    Figure 1. Schematic of experimental setup. A. Plate reader programmed to measure fluorescence and cell density and serves as an incubator to co-cultivate microbes at selected temperatures. B. 96-well microplates exchanged for OmniTray enabling the addition of agar medium providing a solid surface for interactions. C. Bacterial reporter strains are constructed whereby the promoter region located upstream of a gene of interest is cloned in front of a gene encoding a fluorescent protein (e.g., mCherry) to measure gene transcription in living cells. D. Biological interaction is established where fungal plugs are placed down the center of the OmniTray and bacterial reporter strains streaked either side of the fungal plugs.


    Figure 2. Schematic of scanning function. A script can be written to instruct the plate reader to measure multiple parameters simultaneously (A) and perform a scanning function “imaging” a specific area of the plate measuring gene expression and growth. Bacteria streaked on the left-hand side of the fungal strains is the reporter strain (pSEVA237R::PGOI) and the bacterial streak on the right-hand side of the fungal plugs is the control strain (pSEVA237R). “Well map” is shown using a rainbow (blue-green-yellow-red) representation of intensities of signals for one of the protocols from low to high (B).

Data analysis

Once the runs are complete, a series of scans or images are obtained which must then be exported as text files prior to batch conversion of the files from Mars formatted well image text files into image text files for the whole plate. The text files can then be imported into the free image analysis software ImageJ. Once in ImageJ, the time-series image stack can be converted into an animation presentation and quantitative data can be extracted from specific areas of interest (Figures 3 and 4).


Figure 3. Schematic showing data processing. Upon completion of imaging, a time series of scans or images are (A) exported as MARS text files. (B) Batch converted into a time series of text image formatted files using the program MofM.exe. (C) The text image time series files are imported into the free image analysis software ImageJ (D) to make a TIF formatted time-series image stack retaining all the intensity levels of the original scans (32-bit TIF image stack used). Once in ImageJ, the time-series image stack can be converted into an animation presentation to visualize spatial and temporal gene expression or using the image analysis procedures to extract numerical data from the image stack (E).


Figure 4. System enables real-time imaging of transcription. Example of time-series stack converted into an animation video (available at https://www.nature.com/articles/s41598-017-00296-4#Sec9). Top image shows a bacterial reporter strain emitting red fluorescence. Middle image shows fluorescent compounds produced by the bacterial reporter strains and a weak signal of fungal produced fluorescent compounds seen accompanying the expanding fungal mycelium growth. Bottom image shows fungal growth.


  1. Export of imaging runs into text files
    1. Open the MARS Omega Software.
    2. Import files (or scans) from the plate reader by selecting the .RUC files (note the plate reader files should reflect the number of parameters run e.g., 141 files divided by 3 measurements [cell density, red fluorescence, green fluorescence] should give 47 files per run).
    3. Once the files are imported select all the runs, select the set of stored test runs using the BMG Omega MARS program “Open function” (use the program version V3.20 R2 that includes the new “Export multiple Ascii files” function).
    4. Next sort the files in order (i.e., mCherry runs, GFP runs etc.).
    5. Click “sort according to name” (sort the test runs so that it is easy to select all test runs with the same protocol).
    6. Mark the set of test runs and right-click. Select “Export multiple ASCII files”. Check settings by clicking the “Settings” button: Untick boxes in the “File content options”. In the “File Name and Locations options” do the following:
      1. Select folder to store the set of output text files (there will be one file for each reading time). 
      2. File name: use the < automatic filename creation > option. 
      3. Select “File Extension”= TXT “Separator”=TAB and “If file exist”=”rename old file adding date/time”. In “Auto mode and Manage Test Runs File Export options” only tick the “Export microplate view” option. Then press the “OK” button. 
    7. Next, replace the ‘o’ s with ‘0’ to ensure files are ordered correctly.
    Note: For step 3, export only files from one protocol (i.e., export mCherry files). In other words, for every protocol a new analysis has to be performed independently e.g., analysis 1 (mCherry), 2 (cell density) and so on.

  2. Bulk rename utility tool for renaming files
    1. Download software Bulk Rename Utility tool (http://www.bulkrenameutility.co.uk/Main_Intro.php).
    2. Open Bulk Rename Utility.
    3. Open txt. Files.
    4. Highlight only those to change (only 2 digit files e.g., o98 not 100 or above).
    5. Go into documents folder where .txt files saved and check that they have been changed from noo98 to no098.
    6. Prepare destination folder for these files by removing all spaces e.g., Imaging Test Run to ImagingTestRun (necessary for converter program to function) and also find the location for this folder through my Computer: H:\Documents\ImagingTestRun. Then insert additional forward slashes for converter program to read: H:\\Documents\\ImagingTestRun
    7. Double-click the converter program MofM.exe (in supplementary file S2) that can be placed in any folder and need no installation. In the dialogue enter full path name (note that use double backslash \\ in the path name) of the folder containing the text files saved in step 6 (and eventually renamed by giving a prefix for the converted files. The program is now run and there is created a subfolder containing the converted files that can be used for text image series import to stack using the ImageJ Macro in supplementary file S3).
    8. Copy location address by right-clicking in program > edit > paste then press enter and enter a prefix, e.g., pl (as the program is converting to a single well plate layout) > enter. You should find a new sub-folder under TestImagingRun called ‘plfolder’ with all the files.

  3. Installation of ImageJ analysis software and macro retrieval
    1. Download and install ImageJ.
    2. Go to the following link to retrieve the macro: http://rsbweb.nih.gov/ij/macros/ImportTextImageSequence.txt.
    3. Save the macro as a macro file (.ijm) in the macros folder of the ImageJ folder as “Text to images. ijm”.
    4. Open ImageJ software as follows:
      > plugins > macros > install > select and open Text images to stack.ijm
    5. The macro is now installed.
    Note: Recommend verifying the macro is correctly installed by the following: > plugins >macros > “text to images stack” under the macros tab.

  4. ImageJ data analysis
    1. First open ImageJ then click plugins > macros > text image to stack > select plfolder.
    2. To reorder files, open image > stacks > tools > reverse.
    3. This results in a pixelated image so click image > adjust > brightness and contrast > auto.
    4. Once steps 1-3 are completed, the files are ready for analysis and the next step is extraction of data from Regions of Interest (ROI).
    5. Enlarge the image using the magnifying glass and click on the image (right click enlarges, left click reduces).
    6. Click on ROI and export data as a table at a specific slice (i.e., time-point slice or image 31/47).
    7. A picture shows the cumulative expression at a specific time over the whole area. To decide which time-point to investigate, scroll along e.g., 24/27 and draw on the image.
    8. To measure distance, the first step is to calibrate the image. For example, to measure the known distance between two streaks (e.g., 5 cm) first press analyze, set scale. Calculate the distance in pixels. Known distance 50 mm.
    9. To investigate expression over time on a plate reader, first mark the ROI, then select area > image > stacks > plot Z axis profile > save > save as excel (Figure 5).
    10. Click Live > File > save as > selection (save ROI file e.g., stack.roi).
    11. Go to “Files ready for analysis” and save as single TIFF file e.g., stack. tif.
    12. To open the file, click “File> Open stack.tif > select a time-point e.g., 34/47 > File > save as >JPEG to get an image.
    13. For saving marked files (i.e., marking a specific area): File > save as selection > stack.roi. This step can be repeated to add a second ROI.
    Note: For step 10, remember to also take a background “control” value. Recap: highlight the area of interest then take a Z-plot and save the image for methods.


    Figure 5. Extraction of quantitative data. Example of regions of interest (ROI) of two reporter strains: Tester (bacterium with mCherry behind nunF promoter) and the Control (bacterium with mCherry and no promoter). Using the free image analysis software ImageJ (https://imagej.nih.gov/ij/) ROIs are selected and a z-axis profile is plotted to obtain quantitative data.

Notes

  1. How to add color: work of the TIFF file created above then click image > look-up tables > red. Note that if the file is saved in TIFF format, the color will not be red so remember to save the file in PNG format.
  2. How to export a video: save image as an AVI file then compress as a JPEG and define the frame rate e.g., 7 fps (this can be manually decided based on the scrolling time through image).
  3. How to overlay images: The above method describes the data processing required for analyzing one set of files from the plate reader (i.e., red fluorescence readings). Before overlaying, a TIFF file must be created for each parameter measured on the plate reader:
    3x TIFF files for each “channel”
    1. Channel 1: red fluorescence signal stack to TIFF (color red)
    2. Channel 2: blue fluorescence signal stack to TIFF (color blue)
    3. Channel 3: cell density signal stack to TIFF (color grey or black/white)
  4. Click image > colour > merge channels > create composite > ok
  5. Save the super-imposed file.

Note: For “create composite” step, tick “create composite” and leave the other parameters blank. Note this type of image can only be used for distance or visualization. Red fluorescence e.g., mCherry signal cannot be measured, for this the single TIFF file must be used. It is also important to measure the distance between strains before and after the runs and to take a scan or photo of the plate.

Recipes

  1. 300 mM sucrose (Filter sterilized)
    Add 102.69 g sucrose to 800 ml sterilized MilliQ water and stir until the sucrose is dissolved, and make the final volume to 1 L
  2. LB
    1. Add 10 g Bacto-tryptone, 5 g yeast extract, 10 g NaCl to 800 ml MilliQ water and stir until the reagents are dissolved
    2. Autoclave at 121 °C for 15 min
    3. To make LBA, add 15 g BactoTM agar
  3. DFM agar
    1. Add 20 g BactoTM agar to 716 ml MilliQ water and autoclave at 121 °C for 15 min
    2. Following autoclaving add the following:
      20 ml 20% w/v glucose
      1 ml of 1,000x Trace solution
      50 mM asparagin
      10 ml 0.21 M MgSO4
      10 ml (1.12 M KH2PO4 + 0.7 M KCl), pH 6
  4. 1,000x Trace solution
    Add 40 mg Na2B4O7·10H2O, 400 mg CuSO4·5H2O, 1.2 mg FeSO4·7H2O, 700 mg MnSO4·1H2O, 800 mg NaMoO2·2H2O, 10 g ZnSO4·7H2O to 800 ml sterilized MilliQ water and autoclave at 121 °C for 15 min

Acknowledgments

This work was supported by the Villum Foundation grant VKR7310 (Microbial Communication-A Key to the Development of Novel Sustainable Agri- and Aquaculture Practices Using Biological Control Bacteria) awarded to P. Stougaard.

Competing interests

None declared.

References

  1. Hennessy, R. C., Christiansen, L., Olsson, S. and Stougaard, P. (2018). A broad-host range dual-fluorescence reporter system for gene expression analysis in Gram-negative bacteria. J Microbiol Methods 144: 173-176.
  2. Dichmann, S. I., Park, B., Pathiraja, D., Choi, I. G., Stougaard, P. and Hennessy, R. C. (2018). Draft genome sequence of a novel Serratia sp. strain with antifungal activity. Microbiol Resour Announc 7(22) pii: e01340-18.
  3. Gibson, D. G., Young, L., Chuang, R. Y., Venter, J. C., Hutchison, C. A., 3rd and Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5): 343-345.
  4. Hennessy, R. C., Glaring, M. A., Michelsen, C. F., Olsson, S. and Stougaard, P. (2015). Draft genome sequence of Pseudomonas sp. strain in5 isolated from a greenlandic disease suppressive soil with potent antimicrobial activity. Genome Announc 3(6) pii: e01251-15.
  5. Hennessy, R. C., Phippen, C. B. W., Nielsen, K. F., Olsson, S. and Stougaard, P. (2017a). Biosynthesis of the antimicrobial cyclic lipopeptides nunamycin and nunapeptin by Pseudomonas fluorescens strain In5 is regulated by the LuxR-type transcriptional regulator nunF. Microbiologyopen 6(6).
  6. Hennessy, R. C., Stougaard, P. and Olsson, S. (2017b). A microplate reader-based system for visualizing transcriptional activity during in vivo microbial interactions in space and time. Sci Rep 7(1): 281.
  7. Michelsen, C. F., Jensen, H., Venditto, V. J., Hennessy, R. C. and Stougaard, P. (2015). Bioactivities by a crude extract from the Greenlandic Pseudomonas sp. In5 involves the nonribosomal peptides, nunamycin and nunapeptin. PeerJ 3: e1476.

简介

基因组学,转录组学和代谢组学是研究微生物相互作用的强大技术。 这些方法的主要缺点是需要进行破坏性采样。 我们已经建立了一种替代但互补的技术,该技术基于微孔板系统与启动子融合相结合,用于在空间和时间中可视化基因表达。 在这里,我们提供了一种用于测量与固体表面上的真菌相互作用的细菌报道菌株的空间和时间基因表达的方案。
【背景】微生物相互作用是许多重要的生物技术应用的基础,涵盖医学,食品开发和加工,生物修复和生物防治。为了研究微生物相互作用,存在广泛的技术,包括基因组学,转录组学和质谱成像,以研究在相互作用过程中交换的基因表达和代谢物。虽然这些方法促进了我们对微生物相互作用的理解,但它们也有限制,即对破坏性取样的要求。我们开发了一种基于酶标仪的系统,用于可视化活细菌细胞中的基因表达动态,以响应空间和实时真菌。 荧光假单胞菌 In5是一种革兰氏阴性土壤细菌,是具有抗真菌活性的次级代谢产物的有效生产者(Michelsen et al。,2015; Hennessy et al。,2015)。我们之前已将LuxR型调节剂NunF鉴定为合成抗真菌化合物nunamycin和nunapeptin的关键调节因子(Hennessy et al。,2017a)。在这个协议中,我们详细说明了体内如何监测与活真菌细胞相互作用的活细菌细胞中的靶基因表达可以使用基于酶标仪的技术进行(Hennessy 等。,2017b)。 P上。表达与调节基因 nunF 的启动子区融合的红色荧光蛋白mCherry的荧光蛋白In5表示抗真菌次级代谢物基因簇的活化用作报告系统。记者红色信号的延时图像记录和细菌自然产生的荧光代谢物的绿色信号结合微生物生长测量结果表明,当细菌进入减速时, nunF 调节的基因转录被打开。生长阶段和与真菌菌丝的物理相遇。已建立的非破坏性方法具有许多优点,特别是能够提供关于生物体中靶基因转录的详细空间和时间信息。重要的是,该技术是已经用于研究微生物相互作用的许多技术的快速且简单的替代和补充工具。

在这里,我们提出了一个详细的微生物相互作在这个例子中,我们描述了抗菌分离株荧光假单胞菌 In5的报告菌株的构建,以及与真菌 Fusarium graminearum相互作用过程中报告菌株基因表达的成像分析 PH-1在琼脂表面上。

关键字:微生物相互作用, 假单胞菌, 真菌, 生物测定, 成像, 基因时空表达, 活细胞

材料和试剂

  1. Eclipse移液器补充系统(Labcon,目录号:1045-260-000,1093-260-000,1036-260-000)
  2. Nunc TM OmniTray TM 单孔板(Fischer Scientific,目录号:140156)
  3. Gene Pulser ®比色皿0.2 cm间隙(Bio-Rad,目录号:1652086)
  4. 2 ml Microcentrifuge试管(Fischer Scientific,目录号:21-402-905)
  5. NEB ® 5-alpha Competent E.大肠杆菌(亚克隆效率)(NEB,目录号:C2988J)
  6. 限制酶: Bam HI(NEB,目录号:R3136S), Eco R1(NEB,目录号:R0101S)
  7. Gentra Puregene Yeast / Bact。套件(QIAGEN,目录号:158567)
  8. 报告质粒pSEVA237R表达红色荧光蛋白mCherry(Hennessy et al。,2018)
  9. Phusion高保真聚合酶(Fisher Scientific,目录号:F530S)
  10. Wizard ® SV凝胶和PCR清洁系统(Promega,目录号:A9281)
  11. Plasmid Mini Kit QIAprep Spin Miniprep Kit(QIAGEN,目录号:27104)
  12. Gibson Assembly ®克隆试剂盒(NEB,目录号:E5510S)
  13. Bacto TM 琼脂(Difco,目录号:214050)
  14. Luria-Bertani肉汤(LB)(Difco,目录号:244610)
  15. LB琼脂(LBA)(Difco,目录号:244520)
  16. SOC向外生长介质(NEB,目录号:B9020S)
  17. 定义的镰刀菌培养基(DFM)(Hennessy et al。,2017b)
  18. 硫酸卡那霉素一水合物(Duchefa Biochemie,目录号:K0126)
  19. 蔗糖(西格玛奥德里奇,目录号:S0389)
  20. Bacto-tryptone(VWR Chemicals,目录号:84610)
  21. 酵母提取物(VWR Chemicals,目录号:84601)
  22. NaCl(Millipore,目录号:1.06404.1000)
  23. 葡萄糖(西格玛奥德里奇,目录号:49159)
  24. 天冬酰胺(Difco,目录号:214410)
  25. MgSO 4 (VWR Chemicals,目录号:25164.265)
  26. KH 2 PO 4 (Sigma-Aldrich,目录号:NIST200B)
  27. KCl(西格玛奥德里奇,目录号:P9333)
  28. Na 2 B 4 O 7 ·10H 2 O(Sigma-Aldrich,目录号:S9640)
  29. CuSO 4 ·5H 2 O(Sigma-Aldrich,目录号:C8027)
  30. FeSO 4 ·7H 2 O(Sigma-Aldrich,目录号:1270355)
  31. MnSO 4 ·H 2 O(Sigma-Aldrich,目录号:M7634)
  32. NaMoO 2 ·2H 2 O(Sigma-Aldrich,目录号:331058)
  33. ZnSO 4 ·7H 2 O(Sigma-Aldrich,目录号:Z0251)
  34. 300毫克蔗糖(见食谱)
  35. LB培养基(见食谱)
  36. DFM琼脂(见食谱)
  37. 1,000x Trace解决方案(参见食谱)

设备

  1. 移液器(VWR,目录号:613-2696E,613-2702E,613-2704E)
  2. 微孔板读板机(BMG LABTECH,目录号:FLUO-star Omega)
  3. Eppendorf ® Mastercycler ®(Sigma-Aldrich,目录号:EP6313000018-1EA)
  4. Gene Pulser ® II(Bio-Rad,目录号:165-2105)
  5. 无菌接种环(10μl)(VWR,目录号:12000-812)
  6. 软木钻孔机(6毫米直径),用于制作真菌塞
  7. 水浴或加热块

软件

  1. BMG欧米茄Mars软件(Omega V5.11; BMG LABTECH)&nbsp;
  2. ImageJ分析软件( https://imagej.nih.gov/ij/ )
  3. “文本图像堆叠”宏(ImageJ, http://rsbweb.nih.gov/ij /macros/ImportTextImageSequence.txt )
  4. 批量重命名工具,( https://www.bulkrenameutility.co.uk )
  5. 批量转换软件转换BMG Omega Mars导出的井扫描文本文件到图像文本文件的矩形相邻井(MofM.exe)

程序

  1. 靶基因的选择和报告菌株的构建
    该方案的第一步是选择感兴趣的基因(GOI)进行基因表达分析。一旦选择,位于GOI上游的启动子区域被PCR扩增并克隆在荧光蛋白编码基因的前面。启动密码子上游的启动子区(400 bp)使用高保真聚合酶进行PCR扩增,引入突出端的引物可以进行Gibson Assembly ®克隆(Gibson et al。,2009)进入目标质粒。

  2. PCR扩增位于靶基因上游的启动子区域
    1. 解冻Phusion试剂盒试剂并在解冻后彻底混合。
    2. 使用以下配方制作2x Phusion高保真(HF)DNA聚合酶PCR主混合物:
      20μlPhusionHF Buffer
      2μl10mM dNTPs
      1μlDMSO
      2μl正向引物(10μM)(5'-CACAGGAGGCCGCCTAGGCCGCGGCCGCGCGAATTCGCCGACCGTTGGTCGGCTTTGTC-3')
      2μl反向引物(10μM)(5'-GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCAAGCCTGCATACCAAAATCGCTG-3')
      1μlPhusionHF Taq聚合酶
      70μl无核酸酶水
    3. 将49μl2xPhusion PCR预混合物分配到无菌0.5μlPCR管中,并加入从 P纯化的1μl基因组DNA(50ngμl -1 )。如前所述(Dichmann et al。,2018),荧光素 In5。
    4. PCR程序:
      一个。 98°C 30秒
      湾98°C 10秒
      C。 62°C 15 s
      d。 72°C 15秒
      即重复步骤b-d,30x
      F。 72°C 10分钟
    注意:建议运行水空白以确保扩增条带不是由于PCR试剂中的污染物。
    PAUSE POINT:PCR产物可以在4°C或-20°C下储存。

  3. 用于Gibson组装®克隆的报告质粒的制备
    1. 使用限制酶 Bam HI- Eco消化含有编码红色荧光标记mCherry的基因的报告质粒pSEVA237R(Hennessy et al。,2017a)的质粒DNA。 R1。消化后,根据制造商的说明使用Promega Gel提取试剂盒纯化消化的质粒DNA。
    2. 根据制造商的说明在冰上建立Gibson装配®反应。将Gibson组装®反应在50°C下孵育1小时而不摇动。&nbsp;
    3. 解冻50μl E.将大肠杆菌DH5α化学感受态细胞在冰上孵育30分钟,加入5μl吉布森装配混合物,在冰上再孵育细胞30分钟。&nbsp;
    4. 在42°C下使用水浴45秒或使用加热块在42°C下加热90秒的热激电池。&nbsp;
    5. 向热休克细胞中加入250μlSOC向外生长培养基,并在37℃下振荡孵育200μlg/ g,持续1小时。
    6. 在补充有卡那霉素(50μg/ ml)的LBA上加入100μl和200μl的平板。&nbsp;
    7. 将抗生素抗性菌落分离到补充有卡那霉素(50μg/ ml)的10ml LB中,并在37℃下孵育200μlxg/ ml过夜。
    8. 纯化质粒DNA并使用 Bam HI- Eco R1进行消化以鉴定阳性克隆。
    9. 进行DNA测序以确认DNA的完整性。一旦通过测序确认质粒DNA,就可以将其转化为 P.荧光灯 In5。

  4. P的转换。荧光素 In5与报告质粒pSEVA237R :: P GOI
    1. 接种 P.荧光素 In5在10ml LB肉汤中,在28℃下振荡200 x g 过夜。
    2. 在4℃下,在20℃下离心6ml过夜培养物5分钟。
    3. 用4ml 300mM无菌过滤的蔗糖洗涤细胞三次。
    4. 将洗过的细菌沉淀重悬于400μl300mM无菌过滤的蔗糖中。
    5. 从每个构建体添加300-500ng质粒DNA:测试质粒(pSEVA237R :: P GOI )和空载体对照(pSEVA237R)至100μl感受态细胞。
    6. 将混合物转移至2 mm间隙宽度的电穿孔比色杯。&nbsp;
    7. 在电穿孔装置中施加脉冲(25μF,200Ω,2.5kV)。&nbsp;
    8. 在室温下加入1ml LB.
    9. 将混合物转移至2ml Eppendorf管中。
    10. 在28℃下以200 x g 2小时摇动混合物。
    11. 将抗生素抗性菌落分离到补充有卡那霉素(50μg/ ml)的10ml LB中,并在28℃下孵育200μlxg/ ml过夜。
    12. 纯化质粒DNA并使用 Bam HI- Eco R1进行消化以鉴定阳性克隆。
    注意:建议不要在100μl感受态细胞中添加DNA作为阴性对照。如果草坪出现在转化平板上但不出现在阴性对照平板上,则将细胞划线到新鲜的LBA卡那霉素(50μg/ ml)平板上,在28°C下无振荡孵育过夜,以分离单个抗生素抗性菌落。

  5. F的预培养。禾本科植物 PH-1和 P.荧光素 In5
    1. 使用无菌软木钻孔器,转移6 mm直径的 F插头。将禾谷镰刀菌PH-1置于如前所述制备的DFM培养基上(Hennessy et al。,2017b),并在28℃下孵育5天。
    2. 接种 P.荧光素 In5含有pSEVA237R :: P GOI 或pSEVA237R在10 ml LB肉汤中添加50μg/ ml -1 卡那霉素( pSEVA237R骨架上的抗生素选择标记)并在28°C下孵育200 xg 过夜。

  6. 在固体表面上建立细菌 - 真菌相互作用
    1. 将35ml DFM琼脂加入无菌Nunc TM OmniTray TM 中。放置10个 F的插头。禾本科 PH-1(6mmØ)沿着板的中心向下成直线。&nbsp;
    2. 使用10μl无菌环条纹,细菌报告菌株距离真菌栓塞3厘米。让细菌条纹在表面干燥。

  7. 实验设置
  1. 然后将细菌报告菌株和真菌在酶标仪中共培养,编程以分别同时测量红色和绿色荧光以及细胞密度(图1和2)。&nbsp;
  2. 有关使用平板读取器软件BMG Omega Mars Software定义印版和图像时间序列记录的分步说明,请参阅Hennessy et al。(2017b)。


    图1.实验装置的示意图。 A.用于测量荧光和细胞密度的板读取器,用作在选定温度下共培养微生物的培养箱。 B.交换OmniTray的96孔微孔板能够添加琼脂培养基,为相互作用提供固体表面。 C.构建细菌报告菌株,其中位于目的基因上游的启动子区克隆在编码荧光蛋白(例如,,mCherry)的基因前面,以测量活细胞中的基因转录。 D.建立生物相互作用,将真菌栓塞放在OmniTray的中心,细菌报告菌株在真菌栓的两侧划线。


    图2.扫描功能示意图。可编写脚本以指示读板器同时测量多个参数(A)并执行扫描功能“成像”板测量基因表达的特定区域和增长。在真菌菌株左侧划线的细菌是报告菌株(pSEVA237R :: P GOI )和细菌条纹真菌栓的右侧是对照菌株(pSEVA237R)。使用彩虹(蓝 - 绿 - 黄 - 红)表示从低到高(B)的协议之一的信号强度的“井图”。

数据分析

运行完成后,将获得一系列扫描或图像,然后必须将这些扫描或图像导出为文本文件,然后将文件从Mars格式化的井图像文本文件批量转换为整个板的图像文本文件。然后可以将文本文件导入到免费图像分析软件ImageJ中。一旦进入ImageJ,时间序列图像堆栈可以转换为动画呈现,并且可以从感兴趣的特定区域提取定量数据(图3和图4)。


图3.显示数据处理的示意图。 完成成像后,扫描或图像的时间序列(A)导出为MARS文本文件。 (B)使用程序MofM.exe批量转换为文本图像格式化文件的时间序列。 (C)将文本图像时间序列文件导入到自由图像分析软件ImageJ(D)中,以使TIF格式化的时间序列图像堆栈保留原始扫描的所有强度等级(使用32位TIF图像堆栈)。一旦进入ImageJ,时间序列图像堆栈可以转换为动画呈现,以显示空间和时间基因表达,或使用图像分析程序从图像堆栈中提取数值数据(E)。


图4.系统可实现转录的实时成像。 转换为动画视频的时间序列堆栈示例(可在 https://www.nature.com/articles/s41598-017-00296-4#Sec9 )。顶部图像显示发出红色荧光的细菌报告菌株。中间图像显示由细菌报告菌株产生的荧光化合物和伴随着真菌菌丝体生长扩展所见的真菌产生的荧光化合物的弱信号。底部图像显示真菌生长。


  1. 导出成像会运行到文本文件中
    1. 打开MARS Omega软件。
    2. 通过选择.RUC文件从平板阅读器导入文件(或扫描)(注意平板阅读器文件应反映运行的参数数量例如,141个文件除以3个测量值[细胞密度,红色荧光,绿色荧光]每次运行应该给出47个文件)。
    3. 导入文件后,选择所有运行,使用BMG Omega MARS程序“打开功能”选择存储的测试运行集(使用包含新的“导出多个Ascii文件”功能的程序版本V3.20 R2)。
    4. 接下来按顺序对文件进行排序(即。,mCherry运行,GFP运行等。)。
    5. 单击“根据名称排序”(对测试运行进行排序,以便使用相同的协议轻松选择所有测试运行)。
    6. 标记测试运行集并右键单击。选择“导出多个ASCII文件”。单击“设置”按钮检查设置:“文件内容选项”中的取消框。在“文件名和位置选项”中,执行以下操作:
      1. 选择文件夹以存储输出文本文件集(每个阅读时间将有一个文件)。&nbsp;
      2. 文件名:使用&lt;自动文件名创建&gt;选项&NBSP;
      3. 选择“文件扩展名”= TXT“分隔符”= TAB和“如果文件存在”=“重命名旧文件添加日期/时间”。在“自动模式和管理测试运行文件导出选项”中,只勾选“导出微孔板视图”选项。然后按“确定”按钮。&nbsp;
    7. 接下来,将'o'替换为'0'以确保正确排序文件。
    注意:对于步骤3,仅导出一个协议中的文件(即导出mCherry文件)。换句话说,对于每个协议,必须独立地执行新的分析,例如分析1(mCherry),2(细胞密度)等。

  2. 批量重命名实用工具,用于重命名文件
    1. 下载软件批量重命名实用程序工具( http://www.bulkrenameutility.co .UK / Main_Intro.php )。
    2. 打开批量重命名实用程序。
    3. 打开txt。文件。
    4. 仅突出显示要更改的文件(仅限2位数文件例如,o98不是100或更高)。
    5. 进入保存.txt文件的文档文件夹,检查它们是否已从 o 98更改为无 0 98。
    6. 通过删除所有空格例如,将成像测试运行到 ImagingTestRun (转换器程序运行所需)来为这些文件准备目标文件夹,并通过我的计算机找到该文件夹的位置: H:\ Documents \ ImagingTestRun 。然后为转换器程序插入其他正斜杠以读取: H:\\ Documents \\ ImagingTestRun 。&nbsp;
    7. 双击可放置在任何文件夹中的转换器程序MofM.exe(在补充文件S2中),无需安装。在对话框中输入包含在步骤6中保存的文本文件的文件夹的完整路径名(注意在路径名中使用双反斜杠\\)(并最终通过为转换后的文件提供前缀来重命名。该程序现在运行并且创建了一个子文件夹,其中包含转换后的文件,可以使用补充文件S3中的ImageJ宏将文本图像序列导入到堆栈中。
    8. 右键单击 program&gt;复制位置地址编辑&gt;粘贴然后按回车并输入前缀,例如,pl(因为程序转换为单个井 p 晚 l ayout)&gt;输入。您应该在TestImagingRun下找到一个名为“ plfolder ”的新子文件夹,其中包含所有文件。

  3. 安装ImageJ分析软件和宏检索
    1. 下载并安装ImageJ。
    2. 转到以下链接以检索宏: http://rsbweb.nih.gov/ij /macros/ImportTextImageSequence.txt 。
    3. 将宏作为宏文件(.ijm)保存在ImageJ文件夹的宏文件夹中作为“文本到图像”。怡保工程”。
    4. 打开ImageJ软件如下:
      &GT;插件&gt;宏&gt;安装&gt;选择并打开文本图像到stack.ijm
    5. 宏现在已安装。
    注意:建议通过以下方式验证宏是否已正确安装:&gt;插件&gt;宏&gt; “宏标签”下的“文本到图像堆栈”。

  4. ImageJ数据分析
    1. 首先打开ImageJ,然后单击插件&gt;宏&gt;文本图像到堆栈&gt;选择plfolder
    2. 要重新排序文件,请打开 image&gt;堆栈&gt;工具&gt;反向
    3. 这会产生像素化图像,因此请单击 image&gt;调整&gt; <亮度和对比度>自动
    4. 完成步骤1-3后,文件就可以进行分析,下一步是从感兴趣区域(ROI)中提取数据。
    5. 使用放大镜放大图像并单击图像(右键单击放大,左键单击缩小)。
    6. 单击ROI并将数据导出为特定切片(即,时间点切片或图像31/47)的表格。
    7. 图片显示了整个区域特定时间的累积表达式。要确定要调查的时间点,请沿例如,24/27滚动并在图像上绘图。
    8. 要测量距离,第一步是校准图像。例如,要测量两条条纹之间的已知距离(例如,5 cm),首先按分析,设置比例。以像素为单位计算距离。已知距离50毫米。
    9. 要在平板读数器上研究随时间的表达,首先标记ROI,然后选择区域&gt;图像&gt;堆栈&gt; &gt;保存&gt;另存为excel (图5)。
    10. 单击 Live&gt;文件&gt;另存为&gt;选择(保存ROI文件例如,stack.roi)。
    11. 转到“准备分析的文件”并保存为单个TIFF文件,例如,堆栈。 TIF。
    12. 要打开文件,请单击“文件&gt;打开stack.tif&gt;选择一个时间点 例如,34/47 &gt;文件&gt;保存为&gt; JPEG 以获取图像。
    13. 保存标记文件(即,标记特定区域):文件&gt;保存为选择&gt; stack.roi 。可以重复该步骤以添加第二ROI。
    注意:对于步骤10,请记住还要采用背景“控制”值。回顾:突出显示感兴趣的区域,然后拍摄Z图并保存图像以供方法使用。


    图5.定量数据的提取。两种报告菌株的感兴趣区域(ROI)的实例:测试仪(具有mCherry的细菌在 nunF 启动子后面的细菌)和对照(具有细菌的细菌) mCherry,没有推动者)。使用免费图像分析软件ImageJ( https://imagej.nih.gov/ij/ )投资回报率选择并绘制z轴轮廓以获得定量数据。

笔记

  1. 如何添加颜色:上面创建的TIFF文件的工作然后点击图片&gt;查找表&gt;红色。请注意,如果文件以TIFF格式保存,则颜色不会为红色,因此请记住以PNG格式保存文件。
  2. 如何导出视频:将图像保存为AVI文件然后压缩为JPEG并定义帧速率例如,7 fps(这可以根据滚动手动决定时间通过图像)。
  3. 如何叠加图像:上述方法描述了从读板器分析一组文件所需的数据处理(即,红色荧光读数)。在覆盖之前,必须为读板器上测量的每个参数创建一个TIFF文件:
    每个“频道”的3x TIFF文件
    1. 通道1:红色荧光信号堆栈到TIFF(红色)
    2. 通道2:蓝色荧光信号堆栈到TIFF(蓝色)
    3. 通道3:细胞密度信号堆栈为TIFF(颜色灰色或黑色/白色)
  4. 点击图片&gt;颜色&gt;合并渠道&gt;创建复合&gt;确定
  5. 保存超强文件。

注意:对于“创建复合”步骤,勾选“创建复合”并将其他参数留空。请注意,此类图像只能用于距离或可视化。不能测量红色荧光,例如mCherry信号,为此必须使用单个TIFF文件。测量运行前后的应变之间的距离以及对板进行扫描或照片也很重要。

食谱

  1. 300毫克蔗糖(过滤灭菌)
    将102.69g蔗糖添加到800ml灭菌的MilliQ水中并搅拌直至蔗糖溶解,并使最终体积达到1L。
    1. 将10 g Bacto-tryptone,5 g酵母提取物,10 g NaCl加入800 ml MilliQ水中并搅拌直至试剂溶解
    2. 在121℃下高压灭菌15分钟
    3. 为了制备LBA,加入15g Bacto TM 琼脂
  2. DFM琼脂
    1. 将20 g Bacto TM 琼脂加入716 ml MilliQ水中,并在121°C高压灭菌15分钟
    2. 高压灭菌后添加以下内容:
      20毫升20%w / v葡萄糖
      1毫升1,000x微量溶液
      50 mM天冬酰胺
      10ml 0.21M MgSO 4
      10毫升(1.12 M KH 2 PO 4 + 0.7 M KCl),pH 6
  3. 1,000x跟踪解决方案
    加入40毫克Na 2 B 4 O 7 ·10H 2 O,400 mg CuSO 4 < / sub>·5H 2 O,1.2 mg FeSO 4 ·7H 2 O,700 mg MnSO 4 1H 2 O,800mg NaMoO 2 ·2H 2 O,10g ZnSO 4 ·7H 2 O至800 ml灭菌MilliQ水并在121°C高压灭菌15分钟

致谢

这项工作得到了Villum基金会授予的VKR7310(微生物通信 - 使用生物控制细菌开发新型可持续农业和水产养殖实践的关键)的支持,授予P. Stougaard。

利益争夺

没有声明。

参考

  1. Hennessy,R。C.,Christiansen,L.,Olsson,S。和Stougaard,P。(2018)。 用于革兰氏阴性菌基因表达分析的广泛宿主双荧光报告系统。< / a> J Microbiol Methods 144:173-176。
  2. Dichmann,S。I.,Park,B.,Pathiraja,D.,Choi,I.G.,Stougaard,P。和Hennessy,R。C.(2018)。 新的 Serratia sp。的基因组序列草案。具有抗真菌活性的菌株。 Microbiol Resour Announc 7(22)pii:e01340-18。
  3. Gibson,D.G.,Young,L.,Chuang,R.Y.,Venter,J.C.,Hutchison,C.A。,3rd and Smith,H。O.(2009)。 DNA分子的酶组装,最高可达数百千碱基。 Nat Methods 6(5):343-345。
  4. Hennessy,R。C.,Glaring,M。A.,Michelsen,C.F.,Olsson,S。和Stougaard,P。(2015)。 Pseudomonas sp。的基因组序列草案。从具有强烈抗菌活性的绿地疾病抑制性土壤中分离出的菌株。 Genome Announc 3(6)pii:e01251-15。
  5. Hennessy,R。C.,Phippen,C。B. W.,Nielsen,K.F。,Olsson,S。和Stougaard,P。(2017a)。 荧光假单胞菌菌株In5对抗菌环状脂肽nunamycin和nunapeptin的生物合成由LuxR型转录调节因子 nunF 调节。 Microbiologyopen 6(6)。
  6. Hennessy,R。C.,Stougaard,P。和Olsson,S。(2017b)。 基于酶标仪的系统,用于可视化 in vivo 微生物过程中的转录活性空间和时间的相互作用。 Sci Rep 7(1):281。
  7. Michelsen,C.F.,Jensen,H.,Venditto,V。J.,Hennessy,R。C. and Stougaard,P。(2015)。 来自格陵兰 Pseudomonas sp。的粗提物的生物活性。 In5涉及非核糖体肽,nunamycin和nunapeptin。 PeerJ 3:e1476。
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引用:Hennessy, R. C., Stougaard, P. and Olsson, S. (2019). Imaging Gene Expression Dynamics in Pseudomonas fluorescens In5 during Interactions with the Fungus Fusarium graminearum PH-1. Bio-protocol 9(12): e3264. DOI: 10.21769/BioProtoc.3264.
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