protocols

Lab protocols for the Lowe-Power lab (scroll down for table of contents Readme)

View the Project on GitHub lowepowerlab/protocols

Natural Transformation of Ralstonia

Written/Edited by: Tiffany Lowe-Power

Adapted from: Perrier A., Barberis P., Genin S. (2018) Introduction of Genetic Material in Ralstonia solanacearum Through Natural Transformation and Conjugation. In: Medina C., López-Baena F. (eds) Host-Pathogen Interactions. Methods in Molecular Biology, vol 1734. Humana Press, New York, NY

Natural transformation was optimized for GMI1000. Other Ralstonia strains have varying ability to be naturally transformed.
Methods for optimizing natural transformation for a new strain can be found in:

Protocol

Required media

  1. Liquid MM: quarter-strength M63 MM with 2% v/v glycerol as a carbon source
  2. Transformation plates
    • Option 1. CPG plates that lack glucose and lack TZC.
    • Option 2. Solid MM: quarter-strength M63 MM with 2% v/v glycerol and 10 mM glucose as a carbon source. Importantly, when preparing MM agar, the agar and MM components should be autoclaved separately and mixed before plates are poured. Otherwise the mineral components in MM can react with the agar to yield toxic products

Inducing competent state

  1. Grow Ralstonia strain on CPG (+antibiotics) for 2 d at 28 C.
  2. Inoculate a single, isolated colony into 10 ml of quarter-strength M63 MM with 2% v/v glycerol as a carbon source. Grow at 28 C for 2 day with 180 rpm shaking.
    • Glycerol is a very poor carbon source for Ralstonia. The nutrient-limiting conditions promotes natural competence.
    • For max competence, culture should be at OD 0.5 to OD 1.0. OD 0.8 is ideal. On 2019-11-04, OD was 0.338, 0.557, and 0.634 for 3 biological replicates.

Natural Transformation

  1. In 1.5 ml tube, mix 50 ul of competent cells with DNA:

    DNA type DNA volume (approx) DNA amount (ng)
    Plasmid 5 ul 300-500
    Genomic 10 ul 2,000-4,000
  2. Mix gently and spot the entire volume into the center of a solid MM plate.
    • Alternatively, first overlay a sterile 0.45 um pore nitrocellulose filter onto the plate, and spot bacteria+DNA mixture onto the filter. This will make it easier to harvest bacteria from the plate, but heresay suggests this will reduce transformation efficiency. XXX test this and report results at end of lab protocol.
  3. Incubate at 28C for 3 h-2d.

Select Transformants

  1. Add 1 ml CPG broth to 1.5 ml tube. Transfer the entire spot of bacteria into the tube.
    • Either using sterile forceps to transfer the filter, a sterile inoculating loop, or a sterile swab.
  2. Thoroughly mix suspension by low speed vortexing.
  3. Spread plate 100 ul cells onto CPG plates with appropriate selective antibiotics.
  4. Plate a 1:10 dilution: Mix 10 ul cells with 90 ul diH2O.
  5. Plate the remaining cells: Centrifuge remaining suspension at 13k-16k xg for 1 min. Discard supernatant, resuspend pellet in ~100 ul diH2O, and plate.
    • Hopefully this dilution range will result in a plate with isolated colonies. Some plates may be lawns if transformation was highly efficient.
  6. Incubate plates at 28 C for 2 days. Restreak isolated colonies as described in the gene_deletion protocol before confirming genotype with colony pcr.

Expected results