ProtocolsWorkingWithPseudomonasSyringae < Lab

---+ Working with _Pseuodmonas syringae_ (PSY)

_Pseudomonas syringae_ (PSY) comprises many species of bacteria that live on or within different plant species, as noted at [[http://www.pseudomonas-syringae.org/][PseudomonasSyringae.org]]. Our lab works with the strains Psy508 and Psy642, which non-pathogenically colonize the leaf surfaces of several species of plant, including tomato, tobacco, and bean (see [[https://apsjournals.apsnet.org/doi/pdf/10.1094/MPMI-23-2-0198][Vinatzer et al. 2009]] for more information). 

---++ _In vitro_ culture conditions
---+++++ Plate and Liquid Media Growth
Both Psy508 and Psy642 typically take one day to sufficiently grow in liquid LB media. Colonies can be seen on LB agar plates after one day, however optimal colony size may be reached on the second day.

*Known Culture Media:*
   * LB
   * TSB
   * KB (King's Broth)

*Known Antibiotic Resistance:*

(For reference: a 1x concentration is 5 microliters in a 5 mL culture)

Psy508
   * CRB: Resistant up to 2x concentration
   * CAM: Not resistant 
   * SPEC: Not resistant 
   * TET: Not resistant 
   * KAN: Not resistant 

Psy642
   * CRB: Resistant up to 3x concentration 
   * CAM: Resistant up to 2x concentration
   * SPEC: Resistant up to 2x concentration 
   * TET: Not resistant 
   * KAN: Not resistant

*Temperature:*
   * PSY is typically grown at room temperature (~25°C) and will die after prolonged exposure to 37°C

---++ Transformation
---+++++ Making electrocompetent PSY

This protocol has been adapted from both the [[https://academic.oup.com/femsec/article/93/5/fix057/3778241#87888168][Blanchard et al. 2017]] and [[http://barricklab.org/twiki/bin/view/Lab/ProtocolsElectrocompetentCells][Barrick lab _E. coli_ protocol]]. This can be applied to either Psy508 or Psy642. A 50 mL competent cell culture should yield enough cells for ~10 electroporations. 

*Day 1*
   1 Grow an overnight culture in liquid LB media.
      * Our lab typically inoculates a 5 mL culture

*Day 2*
   1 Grow up 50 mL of competent cells (in a flat-bottomed flask for maximum aeration), innolcuated from the overnight culture. In general, inoculate to OD<sub>600</sub> of ~0.1. 
      * To calculate how much of overnight culture to add: 
         * Use (c<sub>1</sub>)(v<sub>1</sub>) = (c<sub>2</sub>)(v<sub>2</sub>), where v<sub>2</sub> is 50 mL, c<sub>2</sub> is 0.1, and c<sub>1</sub> as the overnight culture's current OD<sub>600</sub> value. 
         * V<sub>overnight culture</sub> + V<sub>LB media</sub> = 50 mL (subtract v<sub>1</sub> from 50 to see how much LB to add).
   1 Shake the 50 mL flask at room temperature, until they reach mid-exponential phase. . 
      * Wait 3+ hours for the 50 mL flask to get to an OD600 of 0.4 - 0.6. 
      * After 2 hours have passed, check the OD<sub>600</sub> every 30 minutes or so. Psy642 and Psy508 do not grow at the exact the same rate. 
         * If you are making electrocompetent cells for both PSY strains and one strain reaches the correct OD<sub>600</sub> before the other, put the flask on ice to stall its growth.
   1 Chill the flask on ice for at least 20 minutes. 
   1 Chill all reagents on ice and set the centrifuge to 4°C.
      * Reagents:
         * 0.5 M sucrose
         * Sterile water
         * Centrifuge tubes
   1 Transfer the 50 mL into a chilled centrifuge tube and spin at 5,500 rcf for 10 minutes at 4°C.
   1 Discard the supernatant and resuspend the pellet in 40 microliters of chilled 0.5 M sucrose. 
   1 Repeat this process 4 times, each time with decreasing amounts of sucrose (40, 30, 20, and 10 microliters). 
   1 Resuspend in 500 microliters of 0.5 M sucrose.
   1 Make 50 microliter aliquots in normal microcentrifuge tubes and store at -80°C.

---+++++ Electroporation

   1 Thaw the electrocompetent cells on ice.
   1 To the electrocompetent cells, add 2 &mu;l of DNA (&lt;100 ng of DNA).
   1 Mix by gently pipetting up and down
   1 Pipette the mixture into a chilled cuvette, making sure that the mixture is at the bottom of the cuvette by gently tapping the cuvette on a flat surface. 
      * Be sure to wipe any condensation off the sides of the cuvette before electroporation.
   1 Place the cuvette in the pulser and press the "Pulse" button. 
      * Use the Ec 2 setting (12.5 kC/cm)).
   1 After electroporation, add 950 &mu;l of LB to the cuvette to recover the cells. 
      *Pipette up and down to mix. 
   1 Transfer the mixture to a 1.5 mL microcentrifuge tube.
   1 Incubate for 2 hours at room temperature (~25°C) in a shaking incubator. 
   1 Plate the cells (~ 50 &mu;l) on an LB plate containing the appropriate antibiotic.

-- Main.ElizabethRobinson - 07 Jun 2018
Barrick Lab > ProtocolList > ProtocolsWorkingWithPseudomonasSyringae
Topic revision: r1 - 2018-06-07 - 20:58:34 - Main.ElizabethRobinson