protocols

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

View the Project on GitHub lowepowerlab/protocols

Gene deletion in Ralstonia

Writing/editing credits: Tiffany Lowe-Power

This protocol describes the unmarked gene deletion strategy in Ralstonia. If you are making a marked deletion (e.g. replacing a gene with a GmR marker), pUFR80 is not the best choice. Unmarked gene deletion involves

  1. a negative selection where antibiotic selection drives a vector to integrate into the genome next to the gene-of-interest followed by a
  2. positive selection where sucrose sensitivity (conferred by SacB levanosucrase expressed from the vector backbone) drives the vector to recombine out of the genome resulting in ~50% wildtype and 50% knockout genotype clones that must be genotyped by (colony) PCR

Unmarked gene deletion (sacB)

Overview of how clean deletions with sacB works

Video by David Baltrus. Note: Baltrus lab uses a slightly different vector, but the molecular mechanisms are the same. Both sacB and the kanR genes in pUFR80 work in Ralstonia.

Clone the knockout vector:

Resources:

Clone approximately 0.9-1.2 kb upstream DNA and 0.9-1.2 kb downstream DNA adjacent to each other. Aim for similar sized upstream and downstream fragments so that crossovers on either flanking side are equally likely. Try to have the up/downstream regions immediately up/downstream from the start and stop codons, respectively. Take care to avoid disrupting neighboring or overlapping genes.

Suggested method: Gibson Assembly cloning in pUFR80. The following protocol assumes a vector containing kanamycin resistance was used, the CPG-Kan concentration was 25 μg/ml unless otherwise stated, and all solid CPG contains 0.5 % w/v TZC.

Introduce knockout vector into Ralstonia and select on kanamycin

Electroporation, natural transformation, and conjugation are all possible strategies for introducing the vector into Ralstonia

Note: The pUFR80 knockout vector must be circular. Do not digest the vector before transformation.

Counter-select on 5% w/v sucrose plates

*Note: If having issues with getting individual colonies, increase concentration to 10% w/v sucrose plates.

  1. Once single colonies appear (2 days up to 4 days), grow 2 KanR colonies for ~6 hrs or overnight in CPG broth.
    • Optional: Create a temporary freezer stock in cheap 1.5 ml tubes in case the counter-selection fails
    • Optional: Streak directly from CPG+KAN plate onto sucrose plate
  2. Wash each in 3x in CPG without antibiotic. Dilute and plate 200 μl of the 10-1, 10-2, 10-3 dilutions on CPG + 5% w/v sucrose plates. Note – we need to update this protocol with better advice about the dilutions. We might need to dilute further if we do the overnight incubation.
    • Looping out of the plasmid is a much more frequent genetic event than the original integration of the plasmid into the genome
    • Optional: plate the dilutions on CPG without sucrose to verify sacB is conferring sucrose susceptibility.
  3. Once single colonies appear (2 days up to 4 days), select approximately 24 isolated colonies (transformants) and restreak on CPG for single colonies. Grow for 2 days at 28C or 3 days at RT.
    • Tip: Streaking one colony per plate is the best way to avoid mixing up colonies from different streaks. If streaking more than one colony per plate, leave a couple centimeters between streaks to avoid mixing up colonies from each streak.
    • Tip 2: Patch colonies onto both a CPG plate and CPG + Antibiotic plate to test for antibiotic sensitivity. Reference image below.

  1. Grow double recombinants overnight in 1.5 ml CPG in 24-well plate (28C with shaking) and perform colony PCR with 3 μl culture.
    • Note: colony PCR directly from colonies on the plate tends to yield false-negatives in EPS+ Ralstonia strains.
    • Note 2: if short on time, add glycerol to the plate to create a temporary freezer stock until you have time to confirm the strains.*

Re-use: