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Section II:

Introduction of a broad host range plasmid into various Pseudomonas syringae  pv. syringae strains. 

     
     
   

Electroporation conditions can differ not only for bacterial species but also for strains within a species. Therefore, it is important to define the optimal parameters of electroporation for each bacterial strain. In this exercise, the parameters of electroporation are varied for the introduction of broad host range plasmids pRK415 or pUCP26 into three strains of Pseudomonas syringae pv. syringae: B301D, HS191, and B728a. The electroporation efficiencies for each set of conditions can be calculated, and optimal electroporation conditions for each strain can be determined.

MATERIALS:

    Media

  • NBY agar plates
  • NBY broth
  • NBY agar plates plus tetracycline (Tc) (25 µg/ml)
  • SOC medium

    CLICK HERE FOR MEDIA RECIPES:

   Bacterial strains

P. syringae pv. syringae strains

  • B301D
  • HS191
  • B728a

   Broad host range plasmid DNA

  • pRK415, an RK2-derived broad host range vector 13 kb in size, Tcr (6).
  • pUCP26, a pUC-derived vector, 4.9 kb in size, carrying the 1.9-kb PstI fragment enabling the plasmid to be maintained in Pseudomonas, Tcr (13).

   Equipment

  • Micropipetors (1000 µl, 250 µl, and 25 µl capacity)
  • Sterile pipet tips
  • Gene Pulser II electroporation system with Pulse Controller II Unit (Bio-Rad Laboratories, Hercules, CA)
  • cuvettes (0.1 cm) (store these at -20ºC and keep on ice until you are ready to use them)

    CLICK HERE FOR Instructor's Note 1

  • Sterile glass test tubes.

  • Shaking incubator (25°C)

  • Glass hockey stick for spreading bacteria

  • Microfuge tubes (1.5 and 0.5 ml capacity)

  • Sterile distilled water

     CLICK HERE FOR Instructor's Note 2

PROCEDURE:

1. Inoculate an NBY plate with freshly growing cells of P. syringae pv. syringae (strains B301D, HS191, B728a) (<24 h old) using a sterile loop, so that a lawn of bacteria will grow. Incubate the petri plate at 25°C overnight (~16 h). The bacteria should be in exponential to early stationary phase. Using bacterial cells in late stationary phase can reduce the efficiency of electroporation; therefore, cultures greater than 24-h old should not be used.

Figure 1. Bacterial lawn of P. syringae  pv. syringae strain B301D. The NBY plate was inoculated with the bacterial strain B301D and incubated at 25ºC for ~ 16 h.

2. Resuspend the bacterial lawn using 4 ml of cold sterile distilled water (SDW). Be careful not to resuspend small pieces of agar, because the presence of agar in the bacterial suspension will decrease the electroporation efficiency. It is important to keep the bacterial cells on ice throughout the procedure to increase the electroporation efficiency.

3. Transfer the suspension to 1.5 ml microfuge tubes and pellet the cells (14,000 rpm for 1 min).

4. Resuspend each microfuge tube of cells in 1 ml cold SDW and repeat step 3 twice. After the second wash, resuspend the cells in ~400 µl of cold SDW for each petri plate of cells and place the microfuge tube of cells on ice. Remove a 5 µl aliquot and determine the cell concentration by dilution plating.

    CLICK HERE FOR Instructor's Note 3

5. Electroporator settings.

Experimentally evaluate the best electroporator settings for P. syringae pv. syringae strains B301D, HS191 and B728a by varying the resistance and the voltage using the same bacterial strain and broad host range plasmid.

Suggested settings:

Resistance

Voltage

Resistance

Voltage

200 Ω

1.8 kV/cm

200 Ω

1.2 kV/cm

400 Ω

1.8 kV/cm

400 Ω

1.2 kV/cm

600 Ω

1.8 kV/cm

600 Ω

1.2 kV/cm

800 Ω

1.8 kV/cm

800 Ω

1.2 kV/cm

   CLICK HERE FOR Instructor's Note 4

6. Pipet 60 µl of bacterial cells into a 0.5 ml microfuge tube with 100-500 ng of plasmid pRK415 or pUCP26 DNA and mix gently.

7. Pipet bacterial cells and the DNA suspension into an electroporation cuvette and electroporate.

    CLICK HERE FOR Instructor's Note 5

8. Dilute the electroporation mix with 1 ml of SOC and place it in a test tube with 4 ml of SOC. Incubate with shaking at 25°C for 4 h.

9. Remove 1 ml aliquots and pellet the entire 5 ml culture in 1.5 ml microfuge tubes (14,000 rpm for 1 min).

10. Resuspend the bacterial cells in 200 µl of NBY broth per microfuge tube. Transfer 100 µl aliquots onto NBY agar plus appropriate selection (i.e., Tc for pRK415 and pUCP26) and spread evenly over the agar surface using a sterile glass hockey stick. Incubate the plates at 25°C for 48 h.

11. Pick colonies that grow after 48 h and streak for single colonies onto NBY agar plus appropriate selection to confirm the transformation. Incubate the petri plates at 25°C for 48 h.

12. Colonies that grow and are Tcr should carry the broad host range plasmid. Confirm the presence of the plasmid within these colonies by isolating plasmid DNA and checking it on agarose gels.

   CLICK HERE FOR Instructor's Note 6

OBSERVATIONS:

Calculate the electroporation frequency for each strain at each electroporation condition. This is reported as transformants/µg DNA.

Resistance

 Voltage

 Efficiency (transformants/µg DNA)

 200 Ω

 1.8 kV/cm

 

 400 Ω

 1.8 kV/cm

 

 600 Ω

 1.8 kV/cm

 

 800 Ω

 1.8 kV/cm

 
     

 200 Ω

 1.2 kV/cm

 

 400 Ω

 1.2 kV/cm

 

 600 Ω

 1.2 kV/cm

 

 800 Ω

 1.2 kV/cm

 

Efficiencies of transformation can vary but generally an electroporation efficiency of 1 x 105 transformants/µg DNA is attained for pUCP26, and an electroporation efficiency of 1 x 104  transformants/µg DNA is attained for pRK415 when electroporated into P. syringae pv. syringae strain B301D with a voltage of 1.8 kV/cm and a resistance of 200 Ω. The electroporation efficiencies attained for strain B728a are usually very similar to those of B301D at these conditions; however, the electroporation efficiencies for strain HS191 are usually 10 to 100 fold lower at these conditions. The electroporation efficiencies for strain HS191 tend to increase as the capacitance increases and the voltage is lower.

QUESTIONS FOR DISCUSSION:

  1. What were the most effective conditions for electroporation of each strain of P. syringae pv. syringae?
  2. Which variable, the resistance or the voltage, exhibits the greater effect on the efficiency of electroporation in P. syringae pv. syringae strains?
  3. How does plasmid size affect the electroporation efficiency in each strain?

Instructors can find suggested answers to these discussion questions at: http://admin.apsnet.org/edcenter/instcomm/restricted/Pages/default.aspx

   CLICK HERE FOR Instructor's Note 7