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Feb 2017

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Construction of Antisense RNA-mediated Gene Knock-down Strains in the Cyanobacterium Anabaena sp. PCC 7120
反义RNA介导的鱼腥藻PCC 7120基因敲除株的构建   

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Abstract

Anabaena sp. PCC 7120 (hereafter Anabaena) is a model cyanobacterium to study nitrogen fixation, cellular differentiation and several other key biological functions that are analogous in plants. As with any other organism, many genes in Anabaena encode an essential life function and hence cannot be deleted, causing a bottleneck in the elucidation of its genomic function. Antisense RNA (asRNA) mediated approach renders the study of essential genes possible by suppressing (but not completely eliminating) expression of the target gene, thus allowing them to function to some extent. Recently, we have successfully implemented this approach using the strong endogenous promoter of the psbA1 gene (D1 subunit of Photosystem II) introduced into a high-copy replicative plasmid (pAM1956) to suppress the transcript level of the target gene alr0277 (encoding a sigma factor, SigJ/Alr0277) in Anabaena. This protocol represents an efficient and easy procedure to further explore the functional genomics, expanding the scope of basic and applied research in these ecologically important cyanobacteria.

Keywords: AntisenseRNA (asRNA) (反义RNA), Knock-down (敲除), Anabaena PCC 7120 (鱼腥藻PCC 7120), Cyanobacteria (蓝藻), Green fluorescent protein (绿色荧光蛋白), psbA1 promoter (psbA1启动子)

Background

Cyanobacteria, a diverse phylum of aerobic bacteria capable of photosynthesis, require light (solar energy), carbon dioxide and trace elements for growth. They are genetically amenable due to the availability of facile molecular biology techniques and efficient conjugation systems for the transfer of foreign genes into them (Wolk et al., 1984; Elhai et al., 1997). Anabaena is a filamentous cyanobacterium, which is capable of cellular differentiation, wherein specialised cells (termed heterocysts) carry out nitrogen fixation. Classical strategies such as gene knockout to disrupt the function of a “gene of interest” have been widely employed in cyanobacteria in order to understand their function. Following transformation, segregation of the polyploid genome is required to obtain homozygous mutants. In the case of many essential genes (for example, GroEL, LexA), however, the mutants are not viable or cannot segregate completely as the target protein is essential for its survival (Rajaram and Apte, 2008; Kumar et al., 2018). In such cases, an option is to knockdown the genes of interest by targeting them using asRNA (Blanco et al., 2011; Lin et al., 2013) or dCas9-based CRISPR approaches (Tian et al., 2017), and then study the down-regulated/knockdown phenotype.

The dCas9-based approach has its own limitations, for example, in some organisms, expression of the dCas9 protein may be toxic to cells wherein it is expressed (Lee et al., 2016; Zhang and Voigt, 2018). The reason for the toxicity of Cas9 in cyanobacteria remains unclear, nonetheless recently the employment of a novel RNA directed dsDNA nuclease Cpf1 from Francisella novicida showed far less toxicity in cyanobacteria (Ungerer and Pakrasi, 2016; Niu et al., 2018). Although CRISPR-based gene editing approaches are being rapidly optimized, they need further development to be efficiently used in future cyanobacterial gene knockdown procedures. In such cases, it would be more fruitful to use another methodology such as asRNA to create knockdown strains. The asRNA, which is the complement of its respective mRNA, binds specifically to the mRNA (i.e., forms a dsRNA), thereby reducing its ability to be translated by the ribosomal machinery. Due to decreased translation, a reduction in expression of the desired protein (knockdown) is achieved. Recently, we have used asRNA-mediated approach to downregulate a sigma factor SigJ (Alr0277) by using native psbA1 promoter for expression of asRNA and achieved a 3-fold reduction in the sigJ transcript (Srivastava et al., 2017). Similarly, this approach was also employed to downregulate in vivo expression of the Alr3183 protein (an atypical 2 cysteine-containing thiol peroxidase) in Anabaena (Tailor and Ballal, 2017). Here we present the detailed protocol to knockdown genes in Anabaena for applications in basic as well as applied research.

Materials and Reagents

  1. Vessel and consumable materials
    1. Pipette tips
    2. Petri dishes, 100 mm (Thermo Fisher Scientific, catalog number: FB0875713)
    3. 1.5 ml centrifuge tubes (Thermo Fisher Scientific, catalog number: 02-682-550)
    4. 15 ml conical centrifuge tubes (Thermo Fisher Scientific, catalog number: 05-527-90)
    5. 50 ml conical centrifuge tubes (Thermo Fisher Scientific, catalog number: 12-565-270)
    6. Supor® PES membrane disc filters (Pall corporation, catalog number: 60110)

  2. Biological materials
    1. pAM1956, a replicative vector in Anabaena (Yoon and Golden, 1998) (Addgene, plasmid number: 40251)
      Note: The map of pAM1956 is available on Addgene; https://www.addgene.org/40251/.
    2. pAM1956-PpsbA1 (control plasmid, available upon request)
    3. pRL443 (Elhai et al., 1997) (Addgene, plasmid number: 70261)
    4. pRL623 (Elhai et al., 1997) (Addgene, plasmid number: 58494)
    5. Anabaena PCC 7120 (can be obtained from Pasteur Culture Collection (PCC), Institut Pasteur, Paris, France)
    6. E. coli DH5α: F- recA41 endA1 gyrA96 thi-1 hsdR17 (rk- mk-) supE44 relAλ ΔlacU169 (Gibco-BRL)
    7. E. coli HB101: F- mcrB mrr hsdS20 (rB- mB-) recA13 leuB6 ara-14 proA2 lacY1 galK2 xyl-5 mtl-1 rpsL20 (SmR) glnV44 λ- (Promega, catalog number: L2015)
    8. E. coli HB101-R2: Donor strain carrying pRL623 (encoding methylases) and pRL443 (conjugal plasmid) (Elhai et al., 1997; Banerjee et al., 2012)

  3. Chemicals for preparing media
    1. MgSO4·7H2O (Sigma-Aldrich, catalog number: 63138)
    2. CaCl2·2H2O (Sigma-Aldrich, catalog number: C1016)
    3. Citric Acid (Sigma-Aldrich, catalog number: 251275)
    4. Ferric ammonium citrate (Sigma-Aldrich, catalog number: RES20400-A7)
    5. Na2-EDTA (Sigma-Aldrich, catalog number: E5134)
    6. Na2CO3 (Sigma- Aldrich, catalog number: 1613757)
    7. H3BO3 (Sigma-Aldrich, catalog number: B6768)
    8. Mn2Cl2·4H2O (Sigma-Aldrich, catalog number: 1375127)
    9. ZnSO4·7H2O (Sigma-Aldrich, catalog number: 1.08881)
    10. Na2MoO4·2H2O (Sigma-Aldrich, catalog number: M1003)
    11. CuSO4·5H2O (Sigma-Aldrich, catalog number: C8027)
    12. CoCl2·6H2O (Sigma-Aldrich, catalog number: 1.02539)
    13. NaNO3 (Sigma-Aldrich, catalog number: S5506) 
    14. NaHCO3 (Sigma-Aldrich, catalog number: S5761)
    15. K2HPO4 (Sigma-Aldrich, catalog number: 1551128)
    16. NaCl (Sigma-Aldrich, catalog number: 63138)
    17. Tryptone (Casein Hydrolysate) (Sigma-Aldrich, catalog number: 22090)
    18. Yeast extract (Sigma-Aldrich, catalog number: Y1625)
    19. Agar (for E. coli) (Sigma-Aldrich, catalog number: A1296)
    20. Bacto Agar (for Anabaena) (BD Diagonistics, catalog number: 90000-767)
    21. Co(NO3)2·6H2O (Sigma-Aldrich, catalog number: 239267)
    22. Sodium thiosulfate pentahydrate (Sigma-Aldrich, catalog number: 217247)
    23. BG11 medium (see Recipes)
    24. BG11 agar plate (see Recipes)
    25. 1 M TES (see Recipes)

  4. Molecular biology reagents
    1. Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9393)
    2. Chloramphenicol (Sigma-Aldrich, catalog number: C0378)
    3. Kanamycin sulfate (Sigma-Aldrich, catalog number: 60615)
    4. Neomycin trisulfate salt (Sigma-Aldrich, catalog number: N1876)
    5. Wizard® SV Gel and PCR Clean-Up System (Promega, catalog number: A9281)
    6. QIAGEN Plasmid Mini Kit (100) (Qiagen, catalog number: A9281)
    7. Agarose (Sigma-Aldrich, catalog number: A9539)
    8. Cfr9I (XmaI) (10 U/μl) (Thermo Fisher Scientific, catalog number: ER0171)
    9. Reverse Transcriptase (Roche Diagnostics GmbH, catalog number: 38823100)
    10. AmbionTM DNase I (RNase-free) (Thermo Fisher Scientific, catalog number: AM2222)
    11. Gibson assembly master mix (NEB, catalog number: E2611L)
    12. Phenol (Sigma-Aldrich, catalog number: P1037)
    13. Glycerol (Sigma-Aldrich, catalog number: G5516)
    14. 8-hydroxyquinoline (Sigma-Aldrich, catalog number: 252565)
    15. Na2-EDTA (Sigma-Aldrich, catalog number: E5134)
    16. Sodium acetate (Sigma-Aldrich, catalog number: S2889)
    17. Guanidine thiocyanate (Sigma-Aldrich, catalog number: G9277)
    18. Guanidine hydrochloride (Sigma-Aldrich, catalog number: G3272)
    19. Triton X-100 (Sigma-Aldrich, catalog number: X100)
    20. PGTX solution (see Recipes; compositions listed points 12-19)

Equipment

  1. Pipetts
  2. Tweezers, 120 mm (Ideal-Tek, catalog number: 36A-SA)
  3. Erlenmeyer flask (Thermo Fisher Scientific, catalog number: FB-500-150)
  4. NanoDrop (Thermo Fisher Scientfic, catalog number: ND-1000)
  5. Gel documentation machine (Bio-Rad, Gel Doc 2000 Imaging System)
  6. Fluorescence microscope, excitation filter BP470/40 nm, emission filter BP 525/50, Leica DFC360FX black and white camera (Leica Camera Inc., catalog number: DM5500B)
  7. Light microscope, 100/1.3 oil objective, Leica DFC420C camera (Leica Camera Inc., catalog number: DM2500)
  8. PCR thermocycler (Thermo Fisher Scientific, Veriti® Thermal Cycler)
  9. UV-Vis spectrophotometer (Analytik Jena AG, Germany, SPECORD® 205)
  10. Water bath sonicator (Bandelin Souvrex NK51)
  11. Centrifuge (Eppendorf 5415R Refrigerated Centrifuge)
  12. Incubator (New BrunswickTM Innova® 43)
  13. Vacuum filter system (Thermo Fisher Scientific, NalgeneTM 300-4100)

Software

  1. ImageJ (NIH, USA, https://imagej.nih.gov/ij/)
  2. Geneious (Biomatters Ltd., https://www.geneious.com)

Procedure


Figure 1. Schematic representation of steps for construction of asRNA mediated gene knockdown mutant in Anabaena. Abbreviations: Km, Kanamycin; Neo12.5, Neomycin (12.5 µg/ml); Neo25, Neomycin (25 µg/ml); npt, neomycin phosphotransferase; bla, beta-lactamase; cat, chloramphenicol acetyltransferase.

Note: The handling of cyanobacterial cultures should be done exclusively under sterile conditions.


  1. DNA manipulation
    1. Design primers for (a) promoter e.g., PpsbA1 (alr4866; encoding Photosystem II protein D1) and (b) target gene in reverse orientation (hereafter asGENE) and amplify the DNA fragments by PCR. All the steps in this section are illustrated in Figure 1A. The primers can be designed either manually or using programs such as Geneious (https://www.geneious.com). Primers used for construction and confirmation of knock-down mutant of alr0277 gene in Anabaena (Srivastava et al., 2017) are listed in Table 1.
      Note: In this protocol, use of the Gibson assembly, as described by the manufacturer (Gibson et al., 2009), was employed. Primer designing in the Gibson assembly method does not require the addition of restriction sites in primers.

      Table 1. Primers used for construction and confirmation of knock-down mutant of alr0277 gene in Anabaena (Srivastava et al., 2017)


    2. Linearize the promoterless-gfpmut2 containing replicative shuttle vector pAM1956 (Yoon and Golden, 1998) using the XmaI restriction enzyme.
    3. Check all three fragments on 1.5% (w/v) agarose gel for the desired size and recover them by gel-purification. Measure the concentration and purity of the purified fragments on a NanoDrop instrument.
    4. Combine promoter, asGENE and linearized plasmid (pAM1956-XmaI), in the Gibson assembly reaction (contains the enzymes: T5 Exonuclease, Phusion DNA Polymerase and Taq DNA Ligase) and follow the manufacturer's instructions.
    5. Transform the ligation mix into competent E. coli HB101, spread on a LB agar plate (1.5% agar) supplemented with kanamycin (50 μg/ml) and wait for the colonies to grow overnight at 37 °C. The competent E. coli HB101 cells can be prepared by treatment with CaCl2 (Sambrook et al., 2006).
    6. Once the colonies have appeared, they can be confirmed by colony-PCR by using the forward primer of PpsbA1 and the reverse primer of asGENE.

  2. Construction of recombinant Anabaena strain
    1. Grow Anabaena in 25 ml BG11 medium (Rippka et al., 1979) (Recipe 1) under continuous light illumination (40 μmol photons/m2/s) at 30 °C and shaking at 150 rpm for 4-5 days until it is in the exponential phase (OD730 ~0.5). All the steps in this section are illustrated in Figure 1B.
    2. Collect the cells by centrifugation at 4,000 x g for 10 min at room temperature and resuspend the cell pellet in 25 ml BG11 media in a 150 ml Erlenmeyer flask.
    3. Break filaments by sonicating the cultures for 60-120 s, using a water bath sonicator to an average 3-5 cell length and confirm it by bright field microscopy.
    4. Grow the broken filaments for one day under continuous light illumination (40 μmol photons/m2/s) at 30 °C and shaking at 150 rpm.
    5. On the same day, inoculate the colonies of E. coli HB101 bearing the cargo plasmid (KmR), which has been constructed in the step 5 (section A), and E. coli HB101-R2 harboring pRL443 (ApR) and pRL623 (CmR) in 2 ml LB broth with 50 μg/ml kanamycin and 100 μg/ml ampicillin + 25 μg/ml chloramphenicol respectively for 12-16 h at 37 °C at 200 rpm.
    6. Next day, sub-inoculate 100 μl of the overnight-grown cultures of the above-mentioned E. coli strains into 5 ml of fresh LB liquid media supplemented with appropriate antibiotics and allow them to grow until they reach an exponential phase (OD600 ~0.5).
    7. Harvest the 5 ml culture by centrifugation at 4,000 x g in a table centrifuge for 10 min at 25 °C (room temperature).
    8. Wash the cell pellets three times with 1 ml LB to remove antibiotics completely, and then add 200 μl LB to re-suspend the pellets in both. Do not vortex the cells and if required, use a sterile pipette to gently re-suspend the E. coli cells.
    9. For the mating experiment, mix 100 μl HB101 bearing the cargo plasmid with 100 μl HB101-R2 (harboring pRL443 and pRL623) and incubate at room temperature for 30 min (experimental group). For negative control, mix 100 μl LB with 100 μl HB101-R2 and incubate at room temperature for 30 min (control group).
      Note: We have used an E. coli strain (HB101) that contains both plasmids pRL443 and pRL623, which differs from the original method (Elhai and Wolk, 1988) where one strain contains conjugal plasmid (pRL443), and other strain contains the helper plasmid (pRL623) and the cargo plasmid.
    10. The same day, harvest 15 ml Anabaena culture by centrifugation at 4,000 x g for 10 min at room temperature. Re-suspend the cell pellet in 1 ml BG11, transfer the cells into a 1.5 ml Eppendorf tube, and then centrifuge at 6,000 x g for 1 min at room temperature. Re-suspend the pellet in 400 μl of 2x BG11; then divide cells equally into two tubes.
    11. Mix 200 μl of Anabaena resuspension with 200 μl of the mated E. coli mixture (experimental group) or the control mixture (control group), respectively, and incubate at room temperature and low light (20 μmol photons/m2/s) overnight.
    12. Spread the conjugal mixtures onto the autoclaved Supor® PES Membrane Disc Filters (Supor-800, 0.8 μm, 47 mm) placed on BG11 agar plate (1.5% agar, Recipe 2) containing 12.5 μg/ml neomycin and incubate at 30°C, with continuous low light illumination (20 μmol photons/m2/s).
    13. Transfer the membrane to a fresh BG11 agar plate (25 μg/ml neomycin) every fourth day until neomycin-resistant colonies gradually appear after 15-20 days.
    14. Observe the Anabaena colonies under a fluorescent microscope and select the colonies emitting GFP fluorescence (100x/1.3 oil objective lens, an excitation filter BP 470/40 nm and emission filter BP 525/50 nm) for further experiments.
    15. Pick up the desired colony with a sterile inoculation loop or a toothpick and inoculate into a sterile glass tube (3-4 ml total volume) containing 250 μl of BG11 medium with 25 μg/ml neomycin. Keep this tube under continuous low light illumination (20 μmol photons m2/s). After 3-4 days, once the visual increase in growth is observed, add another 250-500 μl of BG11 medium (25 μg/ml neomycin). Once the OD730 of this culture reaches 0.3-0.4, transfer cells into 5 ml of BG11 medium (in a 25 ml sterile glass tube) containing neomycin as mentioned above. Now the tube can be placed under continuous light illumination of 40 μmol photons/m2/s. After sufficient growth is observed (OD730 ~0.6-1.0), cells can be transferred to a 150 ml sterile Erlenmeyer flask containing 25 ml BG11 medium (25 μg/ml neomycin). Now cells can be subcultured as required in the above-mentioned medium for routine maintenance or for performing experiments. All these steps can be performed at 24-26 °C.

  3. Confirmation of expression of asRNA and downregulation of target gene
    1. To confirm the expression of asRNA by a semi-quantitative PCR approach, design the forward primer internal to asGENE and reverse primer internal to gfpmut2 gene. To confirm the downregulation of the target gene, design the forward primer from the upstream sequence of the target gene that is present in the genome but absent in the antisense construct so that it can be selectively amplified from cDNA derived from GENE mRNA and not from its asRNA. All the steps in this section are illustrated in Figure 1C.
      Note: A regular PCR was used for amplification after the reverse transcriptase reaction. However, qRT-PCR can also be employed to evaluate differences in the expression of mRNA between the wild-type and the knockdown strain, which is a more sensitive technique. Alternatively, if antibodies against the proteins that are knocked down are available, then Western blotting may also be employed to monitor the reduction in the protein content.
    2. Filter 20 ml of exponential phase (OD730 ~0.5) Anabaena culture on the membrane filter (Supor® PES membrane disc filters-supor-800, 0.8 μm, 47 mm) using a vacuum pump assembly for harvesting the cells. Alternatively, cells may be also harvested by centrifugation (4,000 x g, 10 min) and reagents such as TRI Reagent (Sigma)/Trizol (Invitrogen) may be used to isolate total RNA using the manufacturer’s protocol.
    3. Insert the filter containing cells in the vial containing PGTX solution (see Recipes) using tweezers and vortex until the filter is broken into small flakes and mixed with phenol properly. Keep samples on ice.
    4. Freeze the samples in liquid nitrogen immediately, store at -80 °C and perform the RNA extraction exactly as described by Pinto et al. (2009).
    5. Detect the RNA on a denaturing formaldehyde-agarose gel (1.5-2.0% w/v agarose). After staining with ethidium bromide, intact total RNA on migration will show sharp, clear 23S and 16S rRNA bands. Measure the RNA concentration and purity using the NanoDrop instrument as described by the manufacturer.
    6. Treat the RNA (usually 1:10 dilution of original stock) with the DNase I enzyme and subsequently inactivate the DNase I by DNase inactivation reagent according to the manufacturer’s instruction.
    7. DNA contamination of RNA preparations is not necessarily detected by gel electrophoresis or similar methods. To test if a detectable amount of genomic DNA is absent, use the diluted RNA sample as a template for PCR using primer pairs for a constitutive gene rnpB. Measure the RNA concentration and purity using the NanoDrop instrument.
    8. Prepare cDNA using Reverse transcriptase with 1 μg of RNA as template and 0.5 μM of random primers, following the manufacturer's instructions. 
    9. PCR amplify a small fragment of the housekeeping gene rnpB as a control and target gene using specific primers in different PCR reactions, increasing the number of cycles from 25 to 36.
    10. Detect the PCR products on an agarose gel and document the image.
    11. Measure the intensity of PCR bands using ImageJ software for comparison of the transcript level.

Data analysis

The gene expression data can be analyzed using ImageJ software (NIH, USA, https://imagej.nih.gov/ij/) as following:

  1. Record the picture of gel with a gel documentation machine and "invert" image color so that the cDNA bands appear black. Open your final gel image file (tiff) in ImageJ software for analysis.
  2. All the bands in the image should be selected individually. Choose the “rectangular” selection tool to select the widest band as the first band. The height of the selected area should be almost double of this band.
  3. Mark the selected box as first by pressing CTRL + 1. As a result, number 1 will be displayed inside the selected box.
  4. Move the pointer (cursor) inside the first selected box, click and drag the box area to the next band in the image.
  5. Mark the selected box as second by pressing CTRL + 2. Number 2 will be displayed inside the selected box.
  6. Repeat steps 4 and 5 to select rest of the bands in the image.
  7. Once all the bands are selected press CTRL + 3 to display the histogram of each band.
  8. To measure the area below each peak, first separate the histogram peaks by choosing the “draw line” tool to draw a line across the lowest part of each of the histogram. Then choose the “Magic wand” tool and click inside the histogram. The selected area will be outlined in yellow color and a “Result” window will appear.
  9. The Result window will show the quantitative intensity (value) of the each band.

The size of the peak will be registered as a percentage of the total size of the entire highlighted peaks. The peak percentage of test gene should be divided by the peak percentage of the housekeeping gene (control gene) to demonstrate the relative percentage of expression of each experimental gene. The relative expression should be measured in triplicates and statistical analyses should be performed considering the mean, standard deviation, minimum and maximum of the values of each sample. For details of the ImageJ procedure please refer to the user manual, section 30.13 (Ferreira and Rasb, 2012).

Notes

For appropriate comparison with the knockdown strain that has been constructed as described above, it is recommended to construct a control strain by conjugating into the wild-type Anabaena, a plasmid that contains only the PpsbA1 promoter fused with promoter-less gfpmut2 gene in pAM1956 (i.e., vector control).

Recipes

  1. BG11 media (modified from Rippka et al., 1979)
    Note: This medium is used for growing Anabaena.
    1. Prepare 200 ml of each stock solution, 100 ml of trace element stock solution and 50 ml of 1 M NaHCO3 (Table 2, Table 3 and Table 4 respectively) and sterilize them by autoclaving.
      Note: Store citric acid and ferric citrate at room temperature, protected from light; whereas other solutions should be stored at 4 °C. Filter sterilize the trace elements and NaHCO3 stock.
    2. To prepare 1 L BG11 media (1x), add 5 ml of each 200x st.ock solution and 1 ml of trace element stock solution (1,000x) in 964 ml Milli-Q water

      Table 2. Stock solutions (200x) for BG11 media


      Note: The modified BG11 media includes adjustment in the level of following reagents:
      Na2CO3: Modified: 0.04 g/L; Original: 0.02 g/L
      Co(NO3)2·6H2O: Modified: 0.078 g/L; Original: 0.0494 g/L

      Table 3. Trace elements stock solution (1,000x)


      Table 4. 1 M NaHCO3 stock solution


  2. BG11 1.5% (w/v) Agar plates
    1. To prepare 500 ml of 2x concentrated BactoTM agar in 1,000 ml bottle, add 30 g BactoTM Agar in 500 ml of Milli-Q water
    2. Then prepare 2x BG11 medium in 500 ml bottle by adding 464 ml Milli-Q water and 5 ml of each 200x stock, 1 ml of trace element stock solution (1,000x)
    3. In addition, add 3 g/L Sodium thiosulfate pentahydrate and add 10 ml 1 M (1:100) TES, pH 7.6-8.2
    4. Autoclave both 2x BG11 media and 2x agar
    5. Combine both solutions when cooled to approx. 60 °C under the sterile hood
    6. Add NaHCO3 to a final concentration of 5 mM before pouring the plates
    7. Add antibiotics if desired
    Note: Plates should be thick (use 30-40 ml of BG11 agar for 100 mm diameter plates) since cyanobacteria have long generation time and water evaporates throughout incubation. Additionally, the plates may also be sealed with parafilm.
  3. TES buffer
    1. To prepare 1 M TES buffer, add 229.25 g of TES [N-{Tris(hydroxymethyl)methyl}-2-aminoethanesulfonic acid] in 750 ml of Milli-Q water
    2. Adjust to pH 8.2 using 10 N NaOH
    3. Fill to final volume of 1 L with dH2O
    4. Filter sterilize (recommended) or autoclave
    5. Store at 4 °C
  4. PGTX solution (100 ml)
    Phenol
    39.6 g
    Glycerol
    6.9 ml
    8-hydroxyquinoline
    0.1 g
    Na2-EDTA
    0.58 g
    Sodium acetate
    0.8 g
    Guanidine thiocyanate
    9.5 g
    Guanidine hydrochloride
    4.6 g
    Triton X-100
    2 ml
    1.  Weigh all components of the PGTX solution and add dH2O to 100 ml
    2. The solution should be stirred at 50-60 °C to create a homogenous solution
    3. This mixture forms a monophasic solution at room temperature
    4. The final pH of the solution should reach ~4.2 without any manual adjustment

Acknowledgments

The protocol is based on the publications “Conjugal transfer of DNA to cyanobacteria” (Elhai and Wolk, 1988) and “Down-regulation of the alternative sigma factor SigJ confers a photoprotective phenotype to Anabaena PCC 7120” (Srivastava et al., 2017). This work is supported by the Department of Science and Technology (DST), New Delhi (grant sanctioned to A.K.T., A.B. and A.S.). A.S. is also supported by German Academic Exchange Service (DAAD) and Ministry of Education, Youth and Sports of the Czech Republic (project LO1416).

Competing interests

The authors have no conflicts of interest to declare.

References

  1. Blanco, N. E., Ceccoli, R. D., Segretin, M. E., Poli, H. O., Voss, I., Melzer, M., Bravo-Almonacid, F. F., Scheibe, R., Hajirezaei, M. R. and Carrillo, N. (2011). Cyanobacterial flavodoxin complements ferredoxin deficiency in knocked-down transgenic tobacco plants. Plant J 65(6): 922-935.
  2. Elhai, J. and Wolk, C. P. (1988). Conjugal transfer of DNA to cyanobacteria. Methods Enzymol 167: 747-754.
  3. Elhai, J., Vepritskiy, A., Muro-Pastor, A. M., Flores, E. and Wolk, C. P. (1997). Reduction of conjugal transfer efficiency by three restriction activities of Anabaena sp. strain PCC 7120. J Bacteriol 179(6): 1998-2005.
  4. Ferreira, T. and Rasb, W. (2012). ImageJ user guide: IJ 1.46 r. (Most recent accessed date: 9/15/2019)
  5. 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.
  6. Kumar, A., Kirti, A. and Rajaram, H. (2018). Regulation of multiple abiotic stress tolerance by LexA in the cyanobacterium Anabaena sp. strain PCC7120. Biochim Biophys Acta Gene Regul Mech 1861(9): 864-877.
  7. Lee, Y. J., Hoynes-O'Connor, A., Leong, M. C. and Moon, T. S. (2016). Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system. Nucleic Acids Res 44(5): 2462-2473.
  8. Lin, C. H., Tsai, Z. T. and Wang, D. (2013). Role of antisense RNAs in evolution of yeast regulatory complexity. Genomics 102(5-6): 484-490.
  9. Niu, T. C., Lin, G. M., Xie, L. R., Wang, Z. Q., Xing, W. Y., Zhang, J. Y. and Zhang, C. C. (2018). Expanding the potential of CRISPR-Cpf1-based genome editing technology in the cyanobacterium Anabaena PCC 7120. ACS Synth Biol 8(1): 170-180.
  10. Pinto, F. L., Thapper, A., Sontheim, W. and Lindblad, P. (2009). Analysis of current and alternative phenol based RNA extraction methodologies for cyanobacteria. BMC Mol Biol 10: 79.
  11. Rajaram, H. and Apte, S. K. (2008). Nitrogen status and heat-stress-dependent differential expression of the cpn60 chaperonin gene influences thermotolerance in the cyanobacterium Anabaena. Microbiology 154(Pt 1): 317-325.
  12. Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M. and Stainer, R. Y. (1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111(1): 1-61.
  13. Sambrook, J. and Russell, D. W. (2006). Preparation and transformation of competent E. coli using calcium chloride. Cold Spring Harb Protoc (1): pdb-prot3932.
  14. Srivastava, A., Brilisauer, K., Rai, A. K., Ballal, A., Forchhammer, K. and Tripathi, A. K. (2017). Down-regulation of the alternative sigma factor SigJ confers a photoprotective phenotype to Anabaena PCC 7120. Plant Cell Physiol 58(2): 287-297.
  15. Tailor, V. and Ballal, A. (2017). Novel molecular insights into the function and the antioxidative stress response of a Peroxiredoxin Q protein from cyanobacteria. Free Radic Biol Med 106: 278-287.
  16. Tian, P., Wang, J., Shen, X., Rey, J. F., Yuan, Q. and Yan, Y. (2017). Fundamental CRISPR-Cas9 tools and current applications in microbial systems. Synth Syst Biotechnol 2(3): 219-225.
  17. Ungerer, J. and Pakrasi, H. B. (2016). Cpf1 is a versatile tool for CRISPR genome editing across diverse species of cyanobacteria. Sci Rep 6: 39681.
  18. Wolk, C. P., Vonshak, A., Kehoe, P. and Elhai, J. (1984). Construction of shuttle vectors capable of conjugative transfer from Escherichia coli to nitrogen-fixing filamentous cyanobacteria. Proc Natl Acad Sci U S A 81(5): 1561-1565.
  19. Yoon, H. S. and Golden, J. W. (1998). Heterocyst pattern formation controlled by a diffusible peptide. Science 282(5390): 935-938.
  20. Zhang, S. and Voigt, C. A. (2018). Engineered dCas9 with reduced toxicity in bacteria: implications for genetic circuit design. Nucleic Acids Res 46(20): 11115-11125.

简介

[摘要] 鱼腥藻。PCC 7120(以下称Anabaena )是一种蓝藻细菌模型,用于研究固氮,细胞分化以及与植物相似的其他几种关键生物学功能。与其他任何生物一样,鱼腥藻中的许多基因编码必不可少的生命功能,因此无法删除,从而在阐明其基因组功能方面造成了瓶颈。反义RNA(asRNA )介导的方法使得可以通过抑制(但不能完全消除)靶基因的表达来研究必需基因,从而使它们发挥一定的功能。最近,我们已经成功地使用引入高拷贝复制质粒(pAM1956)的psbA1 基因(Photosystem II的D1亚基)的强内源性启动子来实现该方法,以抑制目标基因alr0277 (编码sigma因子)的转录水平,SigJ / Alr0277)在鱼腥。该协议代表了一种有效且简便的程序,可以进一步探索功能基因组学,扩大这些具有生态学意义的蓝细菌的基础研究和应用研究的范围。

[背景] 蓝细菌是能够光合作用的多种需氧细菌门,它需要光(太阳能),二氧化碳和微量元素才能生长。由于易用的分子生物学技术和将外源基因转移到它们中的有效缀合系统,它们在遗传上是适宜的(Wolk 等,1984;Elhai 等,1997)。鱼腥是丝状蓝藻,其能够细胞分化,其特征在于,专门的细胞(称为heterocys TS )进行固氮。为了了解其功能,蓝藻中广泛采用了经典策略,例如基因敲除以破坏“目的基因” 的功能。转化后,需要分离多倍体基因组以获得纯合突变体。在许多必需基因的情况下(考试PLE ,GroEL的,LexA的),然而,该突变体是不可行的或作为靶蛋白是埃塞不能完全分离周期边值问题PBVP其生存(拉贾拉姆和阿普特,2008;库马尔等人, 2018)。在这种情况下,一种选择是通过使用asRNA (Blanco 等,2011; Lin 等,2013)或基于dCas9的CRISPR方法(Tian 等,2017)靶向目标基因来敲除目标基因,然后研究下调/击倒表型。

基于dCas9的方法有其自身的局限性,例如,在某些生物中,dCas9 蛋白的表达可能对其表达的细胞具有毒性(Lee 等人,2016; Zhang和Voigt,2018)。其原因Cas9的蓝藻的毒性仍不清楚,最近仍然针对双链DNA的新型RNA的就业核酸酶Cpf1从弗朗西斯新凶手弗朗西斯菌显示在蓝藻中少得多的毒性(昂格雷尔和Pakrasi ,2016; Ñ IU 。等人,201 8 )。尽管基于CRISPR的基因编辑方法正在快速优化,但它们需要进一步开发才能有效地用于未来的蓝细菌基因敲除程序。在这种情况下,使用另一种方法(如asRNA)创建敲低菌株会更加富有成效。的asRNA ,这是它的各自的mRNA的互补物,特别是与mRNA结合(即,形成的dsRNA),从而降低其是TRA能力nslated由核糖体机械。由于翻译减少,因此所需蛋白质的表达减少(敲除)。最近,我们已使用asRNA 介导的方法通过使用天然psbA1 启动子表达asRNA 来下调sigma因子SigJ (Alr0277),并实现sigJ 转录本减少3倍(Srivastava et al。,2017)。同样,该方法也被用于下调鱼腥藻中Alr3183蛋白(一种非典型的含2个半胱氨酸的硫醇过氧化物酶)的体内表达(Tailor and Ballal ,2017)。在这里,我们介绍了详细的协议,以减少鱼腥藻基因在基础以及应用研究中的应用。

关键字:反义RNA, 敲除, 鱼腥藻PCC 7120, 蓝藻, 绿色荧光蛋白, psbA1启动子

材料和试剂


 


娜维斯E1和消耗材料
P ipette提示
培养皿,100毫米(Thermo Fisher Scientific,目录号:FB0875713)
1.5 ml离心管(Thermo Fisher Scientific,目录号:02-682-550)
15 ml锥形离心管(Thermo Fisher Scientific,目录号:05-527-90)
50 ml锥形离心管(Thermo Fisher Scientific,目录号:12-565-270)
泊尔® PES 米embrane d ISC ˚F ilters(Pall公司,目录号:60110)
 


生物材料
pAM1956,是鱼腥藻中的复制载体(Yoon和Golden,1998年)(Addgene ,质粒号:40251)
注意:pAM1956的图谱可在Addgene 上找到;https://www.addgene.org/40251/。


pAM1956-P psbA1 (对照质粒,可应要求提供)
pRL443(Elhai 等,1997)(Addgene ,质粒号:70261)
pRL623 (Elhai et al。,1997)(Addgene ,质粒编号:58494)
Anabaena P CC 7120(可从法国巴黎巴斯德研究所的巴斯德文化收藏(PCC)获得)
大肠杆菌DH5α:F - 的recA 41 恩达1 的gyrA 96 THI -1 HSD R17(RK - MK - )SUPE 44 RELA λ Δ LACU 169(GIBCO-BRL)
大肠杆菌HB101:˚F - MCRB MRR hsdS20 (rB中- 的mB - )recA13 leuB6 ARA-14 proA2 lacY1的galK2 XYL-5 MTL-1 rpsL20 (钐- [R )glnV44 λ- (Promega公司,目录号:L2015)
大肠杆菌HB101-R2:携带pRL623(编码甲基化酶)和pRL443(接合质粒)的供体菌株(Elhai 等,1997; Banerjee 等,2012)
 


用于制备介质的化学品
MgSO 4 · 7H 2 O (Sigma - Aldrich,目录号:63138)
CaCl 2 · 2H 2 O(Sigma - Aldrich,目录号:C1016)
柠檬酸(Sig ma - Aldrich,目录号:251275)
柠檬酸铁铵(Sigma - Aldrich,目录号:RES20400-A7)
Na 2 -EDTA(Sigma - Aldrich,产品目录号:E5134)
Na 2 CO 3 (Sigma - Aldrich,目录号:1613757)
H 3 BO 3 (Sigma - Aldrich,目录号:B6768)
Mn 2 Cl 2 · 4H 2 O(Sigma - Aldrich,目录号:1375127)
ZnSO 4 · 7H 2 O(Sigma - Aldrich,目录号:1.08881)
Na 2 MoO 4 · 2H 2 O(Sigma - Aldrich,目录号:M1003)
CuSO 4 · 5H 2 O (Sigma - Aldrich,目录号:C8027)
CoCl 2 · 6H 2 O(Sigma - Aldrich,目录号:1.02539)
NaNO 3 (Sigma - Aldrich,目录号:S5506)
NaHCO 3 (Sigma - Aldrich,目录号:S5761)
K 2 HPO 4 (Sigma - Aldrich,目录号:1551128)
NaCl (Sigma - Aldrich,目录号:63138)
胰蛋白((酪蛋白水解产物)(Sigma - Aldrich,目录号:22090)
酵母提取物(Sigma - Aldrich,目录号:Y1625)
琼脂(f 或E. coli )(Sigma - Aldrich,目录号:A1296)
细菌用琼脂(˚F 或鱼腥)(BD Diagonistics ,目录号:90000-767)
Co(NO 3 )2 · 6H 2 O(Sigma - Aldrich,目录号:239267 )
五水硫代硫酸钠(Sigma - Aldrich,目录号:217247)
BG11中号(请参阅食谱)
BG11琼脂板(请参阅食谱)
1 M TES(请参阅食谱)
 


分子生物学试剂
氨苄西林钠盐(Sigma - Aldrich,目录号:A9393)
氯霉素(Sigma - Aldrich,目录号:C0378)
硫酸卡那霉素(Sigma - Aldrich,目录号:60615)
新霉素三硫酸盐(Sigma - Aldrich,目录号:N1876)
向导® SV凝胶和PCR净化系统(Promega公司,目录号:A9281)
QIAGEN质粒迷你试剂盒(100)(Qiagen,目录号:A9281)
琼脂糖(Sigma - Aldrich,目录号:A9539)
Cfr9I位(XmaI位)(10U / μ 升)(赛默飞世尔科技,产品目录号:ER0171)
逆转录酶(Roche Diagnostics GmbH,目录号:38823100)
Ambion TM DNase I(无RNA酶)(Thermo Fisher Scientific,目录号:AM2222)
吉布森装配预混料(NEB,目录号:E2611L)
苯酚(Sigma - Aldrich,目录号:P1037)
甘油(Sigma - Aldrich,目录号:G5516)
8-羟基喹啉(Sigma - Aldrich,目录号:252565)
Na 2 -EDTA (Sigma-Aldrich,目录号:E5134)
醋酸钠(Sigma - Aldrich,目录号:S2889)
硫氰酸胍(Sigma - Aldrich,目录号:G9277)
盐酸胍(Sigma - Aldrich,目录号:G3272)
海卫一X-100(Sigma - Aldrich,目录号:X100)
PGTX解决方案(请参阅食谱;组成列于第12-19点)
 


设备


 


P ipetts
120毫米镊子(Ideal-Tek,目录号:36A-SA)
锥形瓶(Thermo Fisher Scientific,目录号:FB-500-150)
NanoDrop (Thermo Fisher Scientfic ,目录号:ND-1000)
凝胶记录仪(Bio - Rad,Gel Doc 2000成像系统)
荧光显微镜,激发滤光片BP470 / 40 nm,发射滤光片BP 525/50,Leica DFC360FX黑白相机(Leica C amera I nc。,目录号:DM5500B )
光镜,100 / 1.3油物镜,徕卡DFC420 Ç相机(徕卡ç 相机我NC,目录号:DM2500)
PCR热循环仪(赛默飞世尔科技,Veriti ® 热循环仪)
紫外可见分光光度计(Analytik的耶拿AG,德国,SPECORD ® 205)
水浴超声波仪(Bandelin Souvrex NK51)
离心机(Eppendorf 5415R冷冻离心机)
培养箱(新泽西不伦瑞克TM 伊诺® 43)
真空过滤系统(Thermo Fisher Scientific,Nalgene TM 300-4100)
软件


 


ImageJ(美国国立卫生研究院,https://imagej.nih.gov/ij/ )
慷慨(Biomatters Ltd.,https ://www.geneious.com )
 


程序


 


D:\ Reformatting \ 2020-1-6 \ 1902930--1293 Amit Srivastava 828906 \ Figs jpg \图1.jpg


图1. 在鱼腥藻中构建asRNA 介导的基因敲低突变体的步骤示意图。缩写:Km,卡那霉素;Neo12.5,新霉素(12.5 µg / ml);Neo25,新霉素(25 µg / ml);npt ,新霉素磷酸转移酶;bla ,β-内酰胺酶;猫,氯霉素乙酰转移酶。


 


注意:仅在无菌条件下进行蓝细菌培养物的处理。


 


A.DNA 操作      


设计(a)启动子,例如P psbA1 (alr4866 ;编码Photosystem II蛋白D1)和(b)反向定向靶基因的引物(以下称为GENE ),并通过PCR扩增DNA片段。图1A说明了本节中的所有步骤。可以手动设计引物,也可以使用Geneious (https://www.geneious.com)等程序设计引物。表1列出了用于构建和确认鱼腥藻alr0277 基因敲除突变体的引物(Srivastava et al。,2017)。
注意:在该协议中,使用了制造商描述的Gibson组件(Gibson等,2009)。Gibson组装方法中的引物设计不需要在引物中添加限制位点。


 


表1.用于构建和确认鱼腥藻中alr0277 基因敲低突变体的引物(Srivastava et al。,2017)


底漆


取向


顺序(5 ' 至3 ' )


用于构建对照质粒(pAM1956-P psbA1 )的引物


PpsbA1-P1


向前


CCTGCAGGTCGACTGCTAGAGGCATCAATTCGAGCTCGGTACGGATTCCCAAAGATAGGG


PpsbA2-P2


相反


CATATGTATATCTCCTTCTTAAATCTAGAGGATCCCCGGTTTTTTATGATTGCTTTGGATTTG


用于构建表达asRNA 的重组质粒的引物(pAM1956-as_alr0277)


asAlr0277-P1


向前


CCTGCAGGTCGACTGCTAGAGGCATCAATTCGAGCTCGGTACGGATTCCCAAAGATAGGG


asAlr0277-P2


相反


GTAATGCCTACTGGTTCATAGTTTTTTATGATTGCTTTGG


asAlr0277-P3


向前


CCAAAGCAATCATAAAAAACTATGAACCAGTAGGCATTAC


asAlr0277-P4


相反


CCTTCTTAAATCTAGAGGATCCCCGGATGGCAGCAAGTGAGTCC


用于确认反义(as_alr0277)表达的引物


RT-asAlr0277-P1


向前


GCCGGTGTGTAATTGAACCA


RT-GFPmut2-P2


相反


CACCCTCTCCACTGACAGAG


用于确认基因(alr0277 )转录抑制的引物


RT-Alr0277-P1


向前


GAGTTACCACAAGGGAATTTTTATATG


RT-asAlr0277-P2


相反


GCCGGTGTGTAATTGAACCA


在半定量PCR分析中用作对照基因(rnpB )的引物


RT-rnpB-P1


向前


ACTCTTGGTAAAAGGGTGCAAAGGTG


RT-rnpB-P2


相反


AACCATAGTTCCTTCGGCCTTGCT


 


线性化promoterless- gfpmut2 含有复制穿梭载体pAM1956(尹和金,1998)使用XmaI位的限制酶。
在1.5%(w / v)琼脂糖凝胶上检查所有三个片段的所需大小,然后通过凝胶纯化回收。在NanoDrop 仪器上测量纯化片段的浓度和纯度。
在Gibson组装反应(包含酶:T5外切核酸酶,Phusion DNA Polym 擦除酶和Taq DNA连接酶)中结合启动子asGENE 和线性化质粒(pAM1956-X maI),并按照制造商的说明进行操作。
变换连接混合物到感受大肠杆菌HB101,传播上的LB琼脂平板(1.5 %琼脂),补充有卡那霉素(50 μ 克/ 毫升),并等待菌落在37生长过夜 ℃。感受态大肠杆菌HB101细胞可以通过用CaCl 2 处理来制备(Sambrook 等,2006)。
一旦菌落出现,它们可以通过菌落PCR通过使用P的正向引物证实psbA1 和反向引物asGENE 。
 


B. 重组鱼腥藻菌株的构建      


生长鱼腥在25ml BG11培养基(Rippka 等人,1979)(配方1)在连续光照(40 μ 摩尔相片纳秒/ 米2 / S)在30 ℃下和以150rpm振荡4-5天,直到它处于指数阶段(OD 730〜0.5 )。本节中的所有步骤在图1B中进行了说明。
通过在室温下以4,00 0 xg离心10分钟收集细胞,并将细胞沉淀重悬于150 ml锥形瓶中的25 ml BG11培养基中。
通过使用水浴超声仪对培养物进行60-120 s的超声处理来破坏细丝,使其平均3-5个细胞长度,并通过明场显微镜对其进行确认。
生长第一天Ë断裂长丝在连续光照(40 μ 摩尔光子/ 米2 / 在3秒)0 ℃,并在150rpm下振荡。
在同一天,接种在步骤5(A部分)中构建的带有货物质粒(Km R )的大肠杆菌HB101 的菌落,以及带有pRL443(Ap R )的大肠杆菌HB101-R2的菌落。pRL623(厘米- [R )在2ml LB肉汤与50 μ 克/ ml卡那霉素和100 μ 克/ ml氨苄青霉素+ 25 μ 克/ ml氯霉素分别在37 12-16小时℃下以200rpm。
第二天,子接种100 μ 升的上述的过夜生长的培养物的大肠杆菌菌株于5ml补充有合适的抗生素的新鲜LB液体培养基,并允许它们生长,直到它们达到指数期(OD 600 〜0.5)。
通过在25 °C(室温)下在台式离心机中以4,000 xg 离心10分钟收获5 ml培养物。
用1ml LB洗细胞沉淀三次,以彻底去除抗生素,然后添加200 μ 升LB以重悬浮在两种颗粒。不要涡旋细胞,如果需要,请使用无菌移液器轻轻重悬大肠杆菌细胞。
关于配合实验中,混合100 μ 升HB101承载货物质粒用100 μ 升在室温下HB101-R2(窝藏pRL443和pRL623)和温育30分钟(试验组)。对于阴性对照,混合100 μ 升与100 LB μ 升HB101-R2,并在室温下搅拌30分钟(对照组)进行培养。
注意:我们使用的大肠杆菌菌株(HB101)包含质粒pRL443和pRL623,这与原始方法(Elhai and Wolk ,1988)不同,原始方法是一个菌株包含接合质粒(pRL443),其他菌株包含辅助质粒质粒(pRL623)和货物质粒。


同一天,通过在室温下以4,000 xg离心10分钟收获15 ml 鱼腥藻培养物。将细胞沉淀重悬于1 ml BG11中,将细胞转移至1.5 ml Eppendorf管中,然后在室温下以6,000 xg离心1分钟。重悬在400粒料μ 升2×BG11的; 然后将细胞平均分为两个试管。
混合200 μ 升的鱼腥resuspe nsion用200 μ 升的配合大肠杆菌分别混合物(实验组)或对照混合物(对照组),和在室温下孵育和低光(20 μ 摩尔光子/ 米2 / s)过夜。
传播夫妻混合物到高压灭菌的小号upor ® PES膜盘过滤器(苏泊尔-800,0.8 μ 米放置在BG11琼脂含有12.5板(1.5%琼脂,配方2),47 MM)μ 克/ ml新霉素,在30温育℃,用连续的低光照(20 μ 摩尔光子/ 米2 / S)。
所述膜转移到新鲜BG11琼脂平板上(25 μ 克/ ml新霉素)每第四天,直到新霉素抗性菌落之后15-20天逐渐显现。
在荧光显微镜下观察鱼腥藻菌落,并选择发出GFP荧光的菌落(100x / 1.3油物镜,激发滤光片BP 470/40 nm和发射滤光片BP 525/50 nm )进行进一步实验。
拾取用无菌接种环或牙签和接种所需的菌落到含有250无菌玻璃试管(3-4毫升总体积)微升BG11培养基的25 μ 克/ ml新霉素。保持此管在连续低光照(20 μ 摩尔光子米2 / S)。3-4天后,在一次生长视觉增加观察到的,添加另一个250-500 微升的BG11培养基(25 μ 克/ ml新霉素)。一旦该培养物的OD 730 达到0.3-0.4,就将细胞转移到5 ml含有新霉素的BG11培养基中(在25 ml无菌玻璃管中),如上所述。现在,管可以被放置在40连续光照明μ 摩尔光子/ 米2 / 秒。足够的生长,观察到(OD后730 〜0.6-1.0),可以将细胞转移到含有25ml BG11培养基150ml的无菌锥形瓶中(25 μ 克/ ml新霉素)。现在,可以根据需要在上述培养基中对细胞进行传代培养,以进行常规维护或进行实验。所有这些步骤都可以在24-26执行 ° Ç 。
 


C. 确认asRNA 的表达和靶基因的下调      


要通过半定量PCR方法确认asRNA 的表达,请设计asGENE 内部的正向引物和gfpmut2 基因内部的反向引物。要确认目标基因的下调,请从基因组中存在但反义构建体中不存在的目标基因的上游序列设计正向引物,以便可以从源自GENE mRNA的cDNA中选择性扩增而不是从其基因中扩增核糖核酸。图1C说明了本节中的所有步骤。
注意:逆转录酶反应后,使用常规PCR进行扩增。然而,qRT -PCR也可以用于评估野生型和敲低菌株之间mRNA表达的差异,这是一种更为敏感的技术。备选地,如果可获得抗被击倒的蛋白质的抗体,则也可以采用蛋白质印迹法来监测蛋白质含量的减少。


滤波器20毫升指数期(OD的730 〜0.5)鱼腥在膜过滤器培养(泊尔® PES 米embrane d ISC ˚F ilters- 小号upor-800,0.8 μ 米,47个1mm),使用真空泵组件收获细胞。可替代地,细胞也可以通过离心分离(4收获,000 ×g离心,10分钟)和试剂如TRI试剂(Sigma)/ Trizol试剂(Invitrogen公司)可以使用制造商的方案被用于分离总RNA。
使用镊子和涡旋将装有滤池的细胞插入装有PGTX溶液的小瓶中(请参阅“食谱”),直到滤池破碎成小薄片并与苯酚正确混合。将样品放在冰上。
立即将样品冷冻在液氮中,储存在-80 °C,并按照Pinto 等人的描述准确地进行RNA提取。(2009)。
在变性甲醛甲醛琼脂糖凝胶(1.5-2.0 %w / v琼脂糖)上检测RNA 。用溴化乙锭染色后,完整的总RNA在迁移时将显示清晰的23S和16S rRNA清晰条带。按照制造商所述,使用NanoDrop 仪器测量RNA浓度和纯度。
用DNase I酶处理RNA(通常是原始库存的1:10稀释液),然后根据制造商的说明用DNase灭活剂灭活DNaseI 。
RNA制品的DNA污染不一定通过凝胶电泳或类似方法检测。要测试是否不存在可检测量的基因组DNA,请使用稀释的RNA样品作为PCR模板,并使用组成型基因rnpB的引物对进行PCR 。使用NanoDrop 仪器测量RNA浓度和纯度。
使用cDNA逆转录酶与1准备μ 克RNA为模板以及0.5 μ 中号的随机引物,按照生产商的说明进行操作。
PCR 在不同的PCR反应中使用特异性引物,扩增管家基因rnpB 的小片段作为对照和目标基因,将循环数从25增加到36。
在琼脂糖凝胶上检测PCR产物并记录图像。
使用ImageJ软件测量PCR带的强度 比较成绩单水平。
 


数据分析


 


可以使用ImageJ软件(NIH,美国,https://imagej.nih.gov/ij/)对基因表达数据进行如下分析:


用凝胶记录仪记录凝胶图片并“反转”图像颜色,以使cDNA条带显示为黑色。在ImageJ软件中打开最终的凝胶图像文件(tiff)进行分析。
图像中的所有波段都应单独选择。选择“矩形”选择工具以选择最宽的波段作为第一波段。所选区域的高度应几乎是此范围的两倍。
通过按CTRL + 1 将选中的框标记为第一个。结果,数字1将显示在选中的框内。
将指针(光标)移动到第一个选定的框内,单击并将框区域拖到图像中的下一个区域。
通过按CTRL + 2将选中的框标记为第二个。数字2将显示在选择框内。
重复步骤小号4和5 来选择图像中的频带的其余部分。
选择所有频段后,按CTRL + 3以显示每个频段的直方图。
要测量每个峰下方的面积,请首先通过选择“绘制线”工具在每个直方图的最下部绘制一条线来分离直方图的峰。然后选择“魔术棒”工具并在直方图中单击。所选区域将以黄色框出轮廓,并出现“结果”窗口。
结果窗口将显示每个条带的定量强度(值)。
 


峰的大小将记录为整个突出显示的峰的总大小的百分比。测试基因的峰值百分比应除以管家基因(对照基因)的峰值百分比,以证明每个实验基因表达的相对百分比。相对表达应一式三份进行,并应考虑每个样品的平均值,标准差,最小值和最大值进行统计分析。有关ImageJ过程的详细信息,请参阅用户手册第30.13节(Ferreira和Rasb ,2012年)。


 


笔记


 


为了与如上所述构建的敲除菌株进行适当比较,建议通过与野生型鱼腥藻缀合来构建对照菌株,该质粒仅包含p psbA1 启动子,并与pAM1956中的无启动子gfpmut2 基因融合(即,矢量控制)。


 


菜谱


 


BG11介质(从Rippka 等人,1979年修改)
注意:此培养基用于种植鱼腥藻。


准备每种储备溶液200 ml ,100 ml微量元素储备溶液和50 ml 1 M NaHCO 3 (分别为表2 ,表3 和表4 ),并通过高压灭菌进行灭菌。
注意:柠檬酸和柠檬酸铁应在室温下避光保存;其他溶液应在4 °C下储存。网络滤波器消毒微量元素和NaHCO 3 股。


要准备1 L BG11培养基(1x),则每200x st 加入5 ml 。玉珠溶液和964毫升Milli-Q水将1ml微量元素储备溶液(1000倍)
 


表2 。适用于BG11媒体的储备液(200x)


物质


克/摩尔


克/升在1× 米edium


1x介质中的糖度


200x储备溶液,g / L


200x储备液,g / 200 ml


200x储液中的摩尔比


硝酸钠3


84.99


1.5


17.65毫米


300


60


3.53百万


K 2 HPO 4 · 3H 2 O


228.21


0.04


175.3 μ 中号


8


1.6


35毫米


MgSO 4 · 7H 2 O


246.48


0.075


304.3 μ 中号


15


3


60毫米


氯化钙2 · 2H 2 O


147.02


0.036


244.8 μ 中号


7.2


1.44


49毫米


柠檬酸


柠檬酸铁(III)


192.13


0.006


31.2 μ 中号


1.2


0.24


6.25毫米


244.94


0.006


24.5 μ 中号


1.2


0.24


4.9毫米


Na 2 -EDTA


292.24


(372.24)


0.001


3.42 μ 中号


0.2


0.04


(0.051克)


0.68毫米


 


Na 2 CO 3


105.99


0.04


377.4 μ 中号


8


1.6


75.5毫米


 


注意:改良的BG11介质可调节以下试剂的水平:


Na 2 CO 3 :修改:0.04 g / L;原稿:0.02克/升


Co(NO 3 )2 · 6H 2 O:修改:0.078 g / L;原始:0.0494克/升


 


表3 。微量元素原液(1,000x)


物质


克/摩尔


1 M 培养基中的μM


1,000x储备溶液,g / L


1,000x储备溶液,每100毫克毫克


^ h 3 BO 3


61.83


46.3


2.86


286


MnCl 2 · 4H 2 O


197.91


9.2


1.81


181


ZnSO 4 · 7H 2 O


287.53


0.77


0.222


22.2


Na 2 MoO 4 · 2H 2 O


241.95


1.6


0.390


39


CuSO 4 · 5H 2 O


249.7


0.32


0.079


7.9


Co(NO 3 )2 · 6H 2 O


291.04


0.267


0.078


7.8


 


表4 。1 M NaHCO 3 储备溶液


物质


克/摩尔


在1x培养基中的g / L


1M培养基中的毫米


1M储备溶液,g / L


1M储备溶液,g / 50 ml


稀释系数


碳酸氢钠3


84.01


0.42


5


84


4.2


1:200


 


BG11 1.5 %(w / v)琼脂板
要在1,000 ml瓶中制备500 ml 2x浓缩Bacto TM 琼脂,请在500 ml Milli-Q水中添加30 g Bacto TM 琼脂
然后通过添加464 ml Milli-Q水和5 ml每种200x储备液,1 ml微量元素储备液(1,000x)在500 ml瓶中制备2x BG11培养基
此外,添加3 g / L 五水硫代硫酸钠并添加10 ml pH 7.6-8.2的1 M(1:100)TES
高压灭菌2x BG11培养基和2x琼脂
将两种溶液冷却至大约50℃时合并。无菌罩下60°C
在倒板之前,添加NaHCO 3 至终浓度为5 mM
如果需要,添加抗生素
注意:板应厚(对于直径为100 mm的板,请使用30-40 ml BG11琼脂),因为蓝细菌的生成时间长,并且在整个孵育过程中水会蒸发。另外,板也可以用石蜡膜密封。


TES缓冲液
要准备1 M TES缓冲液,请在750 ml Milli-Q水中添加229.25 g TES [[(N- {Tris(羟甲基)甲基} -2-氨基乙磺酸]]
广告只是至pH 8.2使用10N NaOH将
用dH 2 O 填充至1 L的最终体积
过滤消毒(推荐)或高压灭菌器
储存在4 °C
PGTX解决方案(100毫升)
苯酚39.6克             


甘油6.9毫升             


8-羟基喹啉0.1克             


Na 2 - EDTA 0.58克             


醋酸钠0.8克             


硫氰酸胍9.5 g             


盐酸胍4.6克             


海卫一X-100 2毫升             


称量PGTX解决方案的所有成分,并将dH 2 O加入100 ml
溶液应在50-60搅拌℃下吨o创建均匀的溶液
该混合物在室温下形成单相溶液
溶液的最终pH应达到〜4。2无需任何手动调整
 


致谢


 


该协议是基于出版物“DNA的接合转移到蓝藻”(Elhai 和德沃尔克,1988)和“替代σ因子的下调SigJ 赋予光保护表型鱼腥藻PCC 7120”(塔瓦等人。,2017) 。这项工作得到了新德里科学技术部(DST)的支持(由AKT,AB和AS批准)。AS还得到了德国学术交流服务(DAAD)和捷克共和国教育,青年与体育部的支持(项目LO1416)。


 


利益争夺


 


作者没有利益冲突要声明。


[R eferences


 


Blanco,NE,Ceccoli ,RD,Segretin ,ME,Poli ,HO,Voss,I.,Melzer,M.,Bravo- Almonacid ,FF,Scheibe ,R.,Hajirezaei ,MR和Carrillo,N.(2011)。蓝细菌黄素毒素可以弥补转基因烟草植物中铁氧还蛋白的缺乏。植物J 65(6):922-935。
Elhai ,J。和Wolk ,CP(1988)。将DNA结合转移至蓝细菌。方法酶学方法167:747-754。
Elhai ,J.,Vepritskiy ,A.,Muro- Pastor,AM,Flores,E。和Wolk ,CP(1997)。鱼腥藻的三种限制活性降低了夫妻转移效率。应变PCC 7120 Ĵ 细菌学179(6):1998- 2005年。
Ferreira,T.和Rasb ,W.(2012年)。ImageJ用户指南:IJ 1.46 r。(最近访问日期:9/ 15 /2019年)
Gibson,DG,Young,L.,Chuang,RY,Venter,JC,Hutchison,CA,3rd and Smith,HO(2009)。DNA分子的酶促组装高达数百千个碱基。Nat Methods 6(5):343-345。
Kumar,A.,Kirti,A.和Rajaram,H.(2018)。蓝藻鱼腥藻中LexA对多种非生物胁迫耐受性的调控。菌株PCC7120。Biochim Biophys Acta Gene Regul Mech 1861 (9):864-877。
Lee,YJ,Hoynes -O'Connor,A.,Leong,MC和Moon,TS(2016)。CRISPR和反义RNA组合系统可对细菌基因表达进行可编程控制。Nucleic Acids Res 44(5):2462-2473。
Lin CH,Tsai,ZT和Wang D.(2013)。反义RNA在酵母调控复杂性进化中的作用。基因组学102(5-6):484-490。
牛TC,Li n,GM,谢,LR,王,ZQ,邢,WY,张,JY和张CC(2018)。扩展基于CRISPR-Cpf1的基因组编辑技术在蓝藻鱼腥藻PCC 7120中的潜力。ACS Synth Biol 8(1):170-180。
佛罗里达州Pinto,Thapper ,A.,Sontheim ,W。和Lindblad,P。(2009年)。分析当前和替代的基于酚的蓝细菌RNA提取方法。BMC分子生物学10:79。
Rajaram,H.和Apte ,SK(2008)。氮状态和的热应力有关的差异表达cpn60 伴侣蛋白基因影响耐热性在蓝藻鱼腥藻。微生物学154(Pt 1):317-325。
Rippka ,R.,Deruelles ,J.,Waterbury,JB,Herdman ,M。和Stainer,RY(1979)。蓝细菌的纯属培养的一般任务,菌株历史和特性。微生物学杂志111(1):1-61。
Sambrook,J。和Russell,DW(2006)。用氯化钙制备和转化感受态大肠杆菌。Cold Spring Harb Protoc (1):pdb-prot3932。
Srivastava,A.,Brilisauer ,K.,Rai,AK,Ballal ,A.,Forchhammer ,K.和Tripathi,AK(2017)。下调替代σ因子的SigJ赋予光保护表型鱼腥藻PCC 7120。植物细胞生理学58(2):287-297。
Tailor,V.和Ballal ,A.(2017年)。来自蓝细菌的Peroxiredoxin Q蛋白的功能和抗氧化应激反应的新型分子洞察力。Free Radic Biol Med 106:278-287。
田平,王建,沉X.,雷伊,JF,袁Q.和严Y.(2017)。CRISPR-Cas9基础工具和微生物系统中的当前应用。Synth Syst Biotechnol 2(3):219-225。
Ungerer ,J.和Pakrasi ,HB(2016)。Cpf1是用于跨多种蓝细菌物种进行CRISPR基因组编辑的多功能工具。科学代表6 :39681。
Wolk ,CP,Vonshak ,A.,Kehoe,P。和Elhai ,J。(1984)。构建能够从大肠杆菌到固氮丝状蓝细菌的共轭转移的穿梭载体。PROC国家科科学院科学USA 81(5):1561-1565。
Yoon,HS和Golden,JW(1998)。由可扩散肽控制的异型囊形成。科学282(5390):935-938。
Zhang,S.和Voigt,CA(2018)。经改造的dCas9,具有降低的细菌毒性:对遗传电路设计的影响。Nucleic Acids Res 46(20):11115-11125。
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引用:Srivastava, A., Ballal, A., Forchhammer, K. and Tripathi, A. K. (2020). Construction of Antisense RNA-mediated Gene Knock-down Strains in the Cyanobacterium Anabaena sp. PCC 7120. Bio-protocol 10(4): e3528. DOI: 10.21769/BioProtoc.3528.
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