(r8) ProceduresCompetenceAssays < Lab

---+Competence Assays

This assay quantifies the ability of bacterial strains to uptake DNA in culture. The protocol below utilizes genomic DNA extracts obtained using a PureLink genomic DNA minikit (Life Technologies). The DNA should come from a strain with a genome-encoded antibiotic resistance gene against an antibiotic the recipient strain is not resistant to. For transformation into _Acinetobacter baylyi_ ADP1 or ISx genomic DNA from an ADP1 strain with a tdk-KanR cassette inserted into the genome ("AB-KAN") is routinely used with kanamycin for selection as described below. In the Barrick Lab _A. baylyi_ ISx-&#9651;IS6::tdk-kan is commonly used for donor DNA.

---+++Materials (for <i>n</i> samples)<br>
   * 13<i>n</i>+9 sterile tubes<br>
   * <i>n</i>+1 sterile 50 mL flasks<br>
   * 18<i>n</i>+12 mL sterile LB broth<br>
   * Antibiotic stock solution<br>
   * 100<i>n</i> ng genomic DNA extract<br>
   * 3<i>n</i>+2 Antibiotic LB plates<br>
   * 3<i>n</i>+2 LB plates<br>

---+++Day 0: Inoculation
1. Prepare <i>n</i>+1 tubes with 5 ml LB (n samples and 1 uninoculated control)<br>
2. Inoculate sample tubes with either 2 µL of liquid culture, a pipette tip with frozen culture, or an isolated colony<br>
3. Incubate overnight at 30°C and 140 RPM

---+++Day 1: Preconditioning
1. Prepare <i>n</i>+1 flasks with 10 mL LB (n samples and 1 uninoculated control)<br>
2. Inoculate each flask with 10 µL of their respective culture<br>
3. Incubate all flasks overnight at 30°C and 140 RPM<br>

---+++Day 2: Transformation
1. Prepare 3<i>n</i>+2 test tubes with 1 mL LB (three replicates per strain, an uninoculated control, and –DNA control)<br>
2. Add the selected antibiotic stock to each tube to its final working concentration<br>
3. Add 100 ng of antibiotic resistant gDNA (ex: "AB-KAN") into all tubes except the two designated for the blank and the –DNA control<br>
4. For each preconditioned flask from Day 1, transfer 70 µL of each flask into three separate tubes containing 1 mL of LB and the DNA (this gives three trials for each strain we are measuring competence for)<br>
5. Add 70 µL of one of the strains to the –DNA control tube (used to confirm the antibiotic is effective)<br>
6. Incubate overnight at 30°C and 140 RPM<br>

---+++Day 3: Dilutions and Plating
1. Prepare 9<i>n</i>+6 test tubes containing 10 mL of sterile saline solution (3x per tube incubated overnight)<br>
2. Create a dilution series of 10<sup>-2</sup>, 10<sup>-4</sup>, and 10<sup>-6</sup> (this is done by transferring 100 µL of the transformed culture to the first saline tube, vortexing, transferring 100 uL of this into the second saline tube, vortexing again, then transferring 100 µL of this into the third saline tube, and vortexing)<br>
3. Plate 100 µL of each 10<sup>-2</sup> dilution onto LB+Antibiotic plates<br>
4. Plate 100 µL of each 10<sup>-6</sup> dilution onto LB plates (without antibiotics)<br>
5. Incubate overnight at 30°C<br>

---+++Day 4: Colony Counting
1. Start by confirming there is no growth on the -DNA LB+Antibiotic plate but there is growth on the -DNA LB plate (this ensures the antibiotic is effective)<br>
2. If antibiotic effectiveness is confirmed, count all colonies on each plate and record the results<br>
3. Transformation frequencies can be calculated by dividing the number of CFUs on selective media by the number of CFUs on non-selective media (expected values can range from 10<sup>-3</sup> to 10<sup>-6</sup>)<br>

---+++References<br>
*Renda B.A., Dasgupta A., Leon D., Barrick J.E. (2015). Genome instability mediates the loss of key traits by _Acinetobacter baylyi_ ADP1 during laboratory evolution. J. Bacteriol. 197: 872-881.<br>
*Renda B.A., Chan C., Parent K.N., and Barrick J.E. (2016). Emergence of a competence reducing filamentous phage from the genome of _Acinetobacter baylyi_ ADP1. J. Bacteriol. 198: 3209-3219.<br>

-- Main.AurkoDasgupta - 06 Aug 2012<br>
-- Edited by IsaacGifford - 09 Apr 2020
Barrick Lab > ProtocolList > ProceduresCompetenceAssays
Topic revision: r8 - 2021-05-18 - 16:05:56 - Main.IsaacGifford