Difference: ProceduresCompetenceAssays (1 vs. 10)

Revision 102021-05-22 - IsaacGifford

 
META TOPICPARENT name="ProtocolList"

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-△IS6::tdk-kan is commonly used for donor DNA.

Materials (for n samples)

  • 13n+9 sterile culture tubes
  • n+1 sterile 50 mL flasks
  • 18n+12 mL sterile LB broth
  • Antibiotic stock solution
  • 100n ng genomic DNA extract
  • 3n+2 Antibiotic LB plates
  • 3n+2 LB plates

Day 0: Inoculation

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

Day 1: Preconditioning

1. Prepare n+1 flasks with 10 mL LB (n samples and 1 uninoculated control)
2. Inoculate each flask with 10 µL of their respective culture
3. Incubate all flasks overnight at 30°C and 140 RPM

Day 2: Transformation

1. Prepare 3n+2 culture tubes with 1 mL LB (three replicates per strain, an uninoculated control, and –DNA control)
Changed:
<
<		2. Add the selected antibiotic stock to each tube to its final working concentration
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
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)
5. Add 70 µL of one of the strains to the –DNA control tube (used to confirm the antibiotic is effective)
>
>		2. Add 100 ng of antibiotic resistant gDNA (ex: "AB-KAN") into all tubes except the two designated for the blank and the –DNA control
3. 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)
4. Add 70 µL of one of the strains to the –DNA control tube (used to confirm the antibiotic is effective)
5. Incubate overnight at 30°C and 140 RPM
Deleted:
<
<		6. Incubate overnight at 30°C and 140 RPM
 

Day 3: Dilutions and Plating

1. Prepare 9n+6 culture tubes containing 10 mL of sterile saline solution (3x per tube incubated overnight)
2. Create a dilution series of 10-2, 10-4, and 10-6 (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)
3. Plate 100 µL of each 10-2 dilution onto LB+Antibiotic plates
4. Plate 100 µL of each 10-6 dilution onto LB plates (without antibiotics)
5. Incubate overnight at 30°C

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)
2. If antibiotic effectiveness is confirmed, count all colonies on each plate and record the results
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 10-3 to 10-6)

References

*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.
*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.

-- AurkoDasgupta - 06 Aug 2012
-- Edited by IsaacGifford - 09 Apr 2020

Revision 92021-05-21 - IsaacGifford

 
META TOPICPARENT name="ProtocolList"

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-△IS6::tdk-kan is commonly used for donor DNA.

Materials (for n samples)

Changed:
<
<
  • 13n+9 sterile tubes
>
>
  • 13n+9 sterile culture tubes
 
  • n+1 sterile 50 mL flasks
  • 18n+12 mL sterile LB broth
  • Antibiotic stock solution
  • 100n ng genomic DNA extract
  • 3n+2 Antibiotic LB plates
  • 3n+2 LB plates

Day 0: Inoculation

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

Day 1: Preconditioning

1. Prepare n+1 flasks with 10 mL LB (n samples and 1 uninoculated control)
2. Inoculate each flask with 10 µL of their respective culture
3. Incubate all flasks overnight at 30°C and 140 RPM

Day 2: Transformation

Changed:
<
<		1. Prepare 3n+2 test tubes with 1 mL LB (three replicates per strain, an uninoculated control, and –DNA control)
>
>		1. Prepare 3n+2 culture tubes with 1 mL LB (three replicates per strain, an uninoculated control, and –DNA control)
 2. Add the selected antibiotic stock to each tube to its final working concentration
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
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)
5. Add 70 µL of one of the strains to the –DNA control tube (used to confirm the antibiotic is effective)
6. Incubate overnight at 30°C and 140 RPM

Day 3: Dilutions and Plating

Changed:
<
<		1. Prepare 9n+6 test tubes containing 10 mL of sterile saline solution (3x per tube incubated overnight)
>
>		1. Prepare 9n+6 culture tubes containing 10 mL of sterile saline solution (3x per tube incubated overnight)
 2. Create a dilution series of 10-2, 10-4, and 10-6 (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)
3. Plate 100 µL of each 10-2 dilution onto LB+Antibiotic plates
4. Plate 100 µL of each 10-6 dilution onto LB plates (without antibiotics)
5. Incubate overnight at 30°C

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)
2. If antibiotic effectiveness is confirmed, count all colonies on each plate and record the results
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 10-3 to 10-6)

References

*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.
*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.

-- AurkoDasgupta - 06 Aug 2012
-- Edited by IsaacGifford - 09 Apr 2020

Revision 82021-05-18 - IsaacGifford

 
META TOPICPARENT name="ProtocolList"

Competence Assays

Changed:
<
<		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 IS-x 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-△IS6::tdk-kan is commonly used for donor DNA.
>
>		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-△IS6::tdk-kan is commonly used for donor DNA.
 

Materials (for n samples)

  • 13n+9 sterile tubes
  • n+1 sterile 50 mL flasks
  • 18n+12 mL sterile LB broth
  • Antibiotic stock solution
  • 100n ng genomic DNA extract
  • 3n+2 Antibiotic LB plates
  • 3n+2 LB plates

Day 0: Inoculation

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

Day 1: Preconditioning

1. Prepare n+1 flasks with 10 mL LB (n samples and 1 uninoculated control)
2. Inoculate each flask with 10 µL of their respective culture
3. Incubate all flasks overnight at 30°C and 140 RPM

Day 2: Transformation

1. Prepare 3n+2 test tubes with 1 mL LB (three replicates per strain, an uninoculated control, and –DNA control)
2. Add the selected antibiotic stock to each tube to its final working concentration
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
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)
5. Add 70 µL of one of the strains to the –DNA control tube (used to confirm the antibiotic is effective)
6. Incubate overnight at 30°C and 140 RPM

Day 3: Dilutions and Plating

1. Prepare 9n+6 test tubes containing 10 mL of sterile saline solution (3x per tube incubated overnight)
2. Create a dilution series of 10-2, 10-4, and 10-6 (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)
3. Plate 100 µL of each 10-2 dilution onto LB+Antibiotic plates
4. Plate 100 µL of each 10-6 dilution onto LB plates (without antibiotics)
5. Incubate overnight at 30°C

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)
2. If antibiotic effectiveness is confirmed, count all colonies on each plate and record the results
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 10-3 to 10-6)

References

Changed:
<
<		Renda BA, Dasgupta A, Leon D, Barrick JE. (2015) Genome instability mediates the loss of key traits by Acinetobacter baylyi ADP1 during laboratory evolution. J. Bacteriol. 197: 872-881.
Renda BA, Chan C, Parent KN, and Barrick JE. (2016) Emergence of a competence reducing filamentous phage from the genome of Acinetobacter baylyi ADP1. J. Bacteriol. 198: 3209-3219.
>
>		*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.
*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.
  -- AurkoDasgupta - 06 Aug 2012
-- Edited by IsaacGifford - 09 Apr 2020

Revision 72020-04-09 - IsaacGifford

 
META TOPICPARENT name="ProtocolList"

Competence Assays

Changed:
<
<		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 IS-x 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.
>
>		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 IS-x 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-△IS6::tdk-kan is commonly used for donor DNA.
 
Changed:
<
<

Materials (for n samples)

  • 13n+9 sterile tubes
  • n+1 sterile 50mL flasks
  • 18n+12 mL sterile LB broth
>
>

Materials (for n samples)

  • 13n+9 sterile tubes
  • n+1 sterile 50 mL flasks
  • 18n+12 mL sterile LB broth
 
  • Antibiotic stock solution
Changed:
<
<
  • 100n ng genomic DNA extract
  • 3n+2 Antibiotic LB plates
  • 3n+2 LB plates
>
>
  • 100n ng genomic DNA extract
  • 3n+2 Antibiotic LB plates
  • 3n+2 LB plates
 

Day 0: Inoculation

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

Day 1: Preconditioning

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

Day 2: Transformation

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

Day 3: Dilutions and Plating

Changed:
<
<		1. Prepare 9n+6 test tubes containing 10mL of sterile saline solution (3x per tube incubated overnight)
2. Create a dilution series of 1:100, 1:10000, and 1:1000000 (this is done by transferring 100uL of the transformed culture to the first saline tube, vortexing, transferring 100uL of this into the second saline tube, vortexing again, then transferring 100uL of this into the third saline tube, and vortexing)
3. Plate 100uL of each 1:100 dilution onto LB+Antibiotic plates
4. Plate 100uL of each 1:1000000 dilution onto LB plates (without antibiotics)
>
>		1. Prepare 9n+6 test tubes containing 10 mL of sterile saline solution (3x per tube incubated overnight)
2. Create a dilution series of 10-2, 10-4, and 10-6 (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)
3. Plate 100 µL of each 10-2 dilution onto LB+Antibiotic plates
4. Plate 100 µL of each 10-6 dilution onto LB plates (without antibiotics)
 5. Incubate overnight at 30°C

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)
2. If antibiotic effectiveness is confirmed, count all colonies on each plate and record the results
Changed:
<
<		3. Transformation frequencies can be calculated by dividing the number of CFUs on selective media by the number of CFUs on non-selective media
>
>		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 10-3 to 10-6)
 

References

Changed:
<
<		Renda BA, Dasgupta A, Leon D, Barrick JE. (2015) Genome instability mediates the loss of key traits by Acinetobacter baylyi ADP1 during laboratory evolution. J. Bacteriol. 197: 872-881.
Renda BA, Chan C, Parent KN, and Barrick JE. (2016) Emergence of a competence reducing filamentous phage from the genome of Acinetobacter baylyi ADP1. J. Bacteriol. 198: 3209-3219.
>
>		Renda BA, Dasgupta A, Leon D, Barrick JE. (2015) Genome instability mediates the loss of key traits by Acinetobacter baylyi ADP1 during laboratory evolution. J. Bacteriol. 197: 872-881.
Renda BA, Chan C, Parent KN, and Barrick JE. (2016) Emergence of a competence reducing filamentous phage from the genome of Acinetobacter baylyi ADP1. J. Bacteriol. 198: 3209-3219.
  -- AurkoDasgupta - 06 Aug 2012
-- Edited by IsaacGifford - 09 Apr 2020

Revision 62020-04-09 - IsaacGifford

 
META TOPICPARENT name="ProtocolList"

Competence Assays

Changed:
<
<		A competence 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 IS-x an ADP1 strain with a tdk-KanR cassette inserted into the genome ("AB-KAN") is routinely used with kanamycin for selection as described below.
>
>		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 IS-x 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.
 

Materials (for n samples)

  • 13n+9 sterile tubes
  • n+1 sterile 50mL flasks
  • 18n+12 mL sterile LB broth
  • Antibiotic stock solution
  • 100n ng genomic DNA extract
  • 3n+2 Antibiotic LB plates
  • 3n+2 LB plates

Day 0: Inoculation

1. Prepare n+1 tubes with 5ml LB (n samples and 1 uninoculated control)
2. Inoculate sample tubes with either 2uL of liquid culture, a pipette tip with frozen culture, or an isolated colony
3. Incubate overnight at 30°C and 140 RPM

Day 1: Preconditioning

1. Prepare n+1 flasks with 10mL LB (n samples and 1 uninoculated control)
2. Inoculate each flask with 10uL of their respective culture
3. Incubate all flasks overnight at 30°C and 140 RPM

Day 2: Transformation

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

Day 3: Dilutions and Plating

1. Prepare 9n+6 test tubes containing 10mL of sterile saline solution (3x per tube incubated overnight)
2. Create a dilution series of 1:100, 1:10000, and 1:1000000 (this is done by transferring 100uL of the transformed culture to the first saline tube, vortexing, transferring 100uL of this into the second saline tube, vortexing again, then transferring 100uL of this into the third saline tube, and vortexing)
Changed:
<
<		3. Plate 100uL of each 1:100 dilution onto LB + Antibiotic plates
>
>		3. Plate 100uL of each 1:100 dilution onto LB+Antibiotic plates
 4. Plate 100uL of each 1:1000000 dilution onto LB plates (without antibiotics)
5. Incubate overnight at 30°C
Changed:
<
<

Day 4: Plate counting

1. Start by ensuring that the antibiotic is effective (there should be no growth on the –DNA control plate that contains LB+Kan, but growth on the LB only plate is expected)
>
>

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)
 2. If antibiotic effectiveness is confirmed, count all colonies on each plate and record the results
3. Transformation frequencies can be calculated by dividing the number of CFUs on selective media by the number of CFUs on non-selective media

References

Renda BA, Dasgupta A, Leon D, Barrick JE. (2015) Genome instability mediates the loss of key traits by Acinetobacter baylyi ADP1 during laboratory evolution. J. Bacteriol. 197: 872-881.
Renda BA, Chan C, Parent KN, and Barrick JE. (2016) Emergence of a competence reducing filamentous phage from the genome of Acinetobacter baylyi ADP1. J. Bacteriol. 198: 3209-3219.

-- AurkoDasgupta - 06 Aug 2012

Changed:
<
<		-- Edited by IsaacGifford - 08 Apr 2020
>
>		-- Edited by IsaacGifford - 09 Apr 2020

Revision 52020-04-08 - IsaacGifford

 
META TOPICPARENT name="ProtocolList"
Changed:
<
<

Competence Assays

>
>

Competence Assays

 
Changed:
<
<		A competence assay compares the ability of different 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 IS-x an ADP1 strain with a tdk-KanR cassette inserted into the genome ("AB-KAN") is routinely used as described below.
>
>		A competence 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 IS-x an ADP1 strain with a tdk-KanR cassette inserted into the genome ("AB-KAN") is routinely used with kanamycin for selection as described below.
 
Added:
>
>

Materials (for n samples)

  • 13n+9 sterile tubes
  • n+1 sterile 50mL flasks
  • 18n+12 mL sterile LB broth
  • Antibiotic stock solution
  • 100n ng genomic DNA extract
  • 3n+2 Antibiotic LB plates
  • 3n+2 LB plates
 

Day 0: Inoculation

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

Day 1: Preconditioning

Changed:
<
<		• Prepare n+1 flasks with 10mL LB (n samples and 1 uninoculated control)
• Transfer 10uL of each inoculated culture into their respective flask
• Incubate all flasks overnight at 30°C and 140 RPM.
>
>		1. Prepare n+1 flasks with 10mL LB (n samples and 1 uninoculated control)
2. Inoculate each flask with 10uL of their respective culture
3. Incubate all flasks overnight at 30°C and 140 RPM
 

Day 2: Transformation

Changed:
<
<		• Prepare 3n+2 test tubes with 1mL LB for three replicates per strain, as well as an uninoculated control and –DNA control
• Put 100ng of antibiotic resistant A. baylyi gDNA ("AB-KAN") into all tubes except the two designated for the blank and the –DNA control
• For each preconditioned flask from Day 1, transfer 70uL of each flask into three separate tubes containing 1mL of LB and the DNA (this will give us three trials for each strain we are trying to measure competence for)
• Add 70uL of one of the strains to the –DNA control tube (this ensures that the antibiotic is effective, and can be determined the day after plating)
• Incubate overnight at 30°C and 140 RPM
>
>		1. Prepare 3n+2 test tubes with 1mL LB for three replicates per strain, as well as an uninoculated control and –DNA control
2. Add the selected antibiotic stock to each tube to its final working concentration
3. Put 100ng of antibiotic resistant A. baylyi gDNA ("AB-KAN") into all tubes except the two designated for the blank and the –DNA control
4. For each preconditioned flask from Day 1, transfer 70uL of each flask into three separate tubes containing 1mL of LB and the DNA (this gives three trials for each strain we are measuring competence for)
5. Add 70uL of one of the strains to the –DNA control tube (this ensures that the antibiotic is effective, and can be determined the day after plating)
Added:
>
>		6. Incubate overnight at 30°C and 140 RPM
 

Day 3: Dilutions and Plating

Changed:
<
<		• Obtain test tubes containing 10mL of sterile saline solution (get three times as many tubes as there are test tubes undergoing transformation in the incubator)
• Create a dilution series of 1:100, 1:10000, and 1:1000000 (this is done by transferring 100uL of the transformed culture to the first saline tube, vortexing, transferring 100uL of this into the second saline tube, vortexing again, then transferring 100uL of this into the third saline tube, and vortexing)
• Plate 100uL of each 1:100 dilution on LB + Kanamycin plates
• Plate 100uL of each 1:1000000 dilution on plates of LB plates (without antibiotics)
• Incubate overnight at 30°C
>
>		1. Prepare 9n+6 test tubes containing 10mL of sterile saline solution (3x per tube incubated overnight)
2. Create a dilution series of 1:100, 1:10000, and 1:1000000 (this is done by transferring 100uL of the transformed culture to the first saline tube, vortexing, transferring 100uL of this into the second saline tube, vortexing again, then transferring 100uL of this into the third saline tube, and vortexing)
3. Plate 100uL of each 1:100 dilution onto LB + Antibiotic plates
4. Plate 100uL of each 1:1000000 dilution onto LB plates (without antibiotics)
5. Incubate overnight at 30°C
 

Day 4: Plate counting

Changed:
<
<		• Start by ensuring that the antibiotic is effective (there should be no growth on the –DNA control plate that contains LB+Kan, but growth on the LB only plate is expected)
• If antibiotic effectiveness is confirmed, count all colonies on each plate and record the results
• Transformation frequencies can be calculated by dividing the number of CFUs on selective media by the number of CFUs on non-selective media
>
>		1. Start by ensuring that the antibiotic is effective (there should be no growth on the –DNA control plate that contains LB+Kan, but growth on the LB only plate is expected)
2. If antibiotic effectiveness is confirmed, count all colonies on each plate and record the results
3. Transformation frequencies can be calculated by dividing the number of CFUs on selective media by the number of CFUs on non-selective media
 
Changed:
<
<		References Renda BA, Dasgupta A, Leon D, Barrick JE. (2015) Genome instability mediates the loss of key traits by Acinetobacter baylyi ADP1 during laboratory evolution. J. Bacteriol. 197: 872-881. Renda BA, Chan C, Parent KN, and Barrick JE. (2016) Emergence of a competence reducing filamentous phage from the genome of Acinetobacter baylyi ADP1. J. Bacteriol. 198: 3209-3219.
>
>

References

Renda BA, Dasgupta A, Leon D, Barrick JE. (2015) Genome instability mediates the loss of key traits by Acinetobacter baylyi ADP1 during laboratory evolution. J. Bacteriol. 197: 872-881.
Renda BA, Chan C, Parent KN, and Barrick JE. (2016) Emergence of a competence reducing filamentous phage from the genome of Acinetobacter baylyi ADP1. J. Bacteriol. 198: 3209-3219.
 
Deleted:
<
<
 -- AurkoDasgupta - 06 Aug 2012
Changed:
<
<		-- Edited by IsaacGifford - 07 Apr 2020
>
>		-- Edited by IsaacGifford - 08 Apr 2020

Revision 42020-04-07 - IsaacGifford

 
META TOPICPARENT name="ProtocolList"

Competence Assays

Added:
>
>		A competence assay compares the ability of different 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 IS-x an ADP1 strain with a tdk-KanR cassette inserted into the genome ("AB-KAN") is routinely used as described below.
 

Day 0: Inoculation

• Prepare n+1 tubes with 5ml LB (n samples and 1 uninoculated control)
Changed:
<
<		• Inoculate tubes with either 2uL of liquid culture or a pipette tip with frozen culture.
>
>		• Inoculate sample tubes with either 2uL of liquid culture or a pipette tip with frozen culture
 • Incubate overnight at 30°C and 140 RPM

Day 1: Preconditioning

• Prepare n+1 flasks with 10mL LB (n samples and 1 uninoculated control)
• Transfer 10uL of each inoculated culture into their respective flask
Changed:
<
<		• Put all flasks in the incubator and incubate overnight at 30°C and 140 RPM.
>
>		• Incubate all flasks overnight at 30°C and 140 RPM.
 

Day 2: Transformation

Changed:
<
<		• Prepare 3n+2 test tubes with 1mL LB for three replicates per strain, as well as a blank and –DNA.
• Put 100ng of the Kanamycin resistant AB gDNA into all tubes except the two designated for the blank and the –DNA control
>
>		• Prepare 3n+2 test tubes with 1mL LB for three replicates per strain, as well as an uninoculated control and –DNA control
• Put 100ng of antibiotic resistant A. baylyi gDNA ("AB-KAN") into all tubes except the two designated for the blank and the –DNA control
 • For each preconditioned flask from Day 1, transfer 70uL of each flask into three separate tubes containing 1mL of LB and the DNA (this will give us three trials for each strain we are trying to measure competence for)
• Add 70uL of one of the strains to the –DNA control tube (this ensures that the antibiotic is effective, and can be determined the day after plating)
• Incubate overnight at 30°C and 140 RPM

Day 3: Dilutions and Plating

• Obtain test tubes containing 10mL of sterile saline solution (get three times as many tubes as there are test tubes undergoing transformation in the incubator)
• Create a dilution series of 1:100, 1:10000, and 1:1000000 (this is done by transferring 100uL of the transformed culture to the first saline tube, vortexing, transferring 100uL of this into the second saline tube, vortexing again, then transferring 100uL of this into the third saline tube, and vortexing)
Changed:
<
<		• Plate 100uL of each 1:100 dilution on plates of LB + Kanamycin
• Plate 100uL of each 1:1000000 dilution on plates of LB only
>
>		• Plate 100uL of each 1:100 dilution on LB + Kanamycin plates
• Plate 100uL of each 1:1000000 dilution on plates of LB plates (without antibiotics)
 • Incubate overnight at 30°C

Day 4: Plate counting

• Start by ensuring that the antibiotic is effective (there should be no growth on the –DNA control plate that contains LB+Kan, but growth on the LB only plate is expected)
Changed:
<
<		• If antibiotic effectiveness is confirmed, count all colonies on each plate and record the results
>
>		• If antibiotic effectiveness is confirmed, count all colonies on each plate and record the results
Added:
>
>		• Transformation frequencies can be calculated by dividing the number of CFUs on selective media by the number of CFUs on non-selective media
 
Added:
>
>		References Renda BA, Dasgupta A, Leon D, Barrick JE. (2015) Genome instability mediates the loss of key traits by Acinetobacter baylyi ADP1 during laboratory evolution. J. Bacteriol. 197: 872-881. Renda BA, Chan C, Parent KN, and Barrick JE. (2016) Emergence of a competence reducing filamentous phage from the genome of Acinetobacter baylyi ADP1. J. Bacteriol. 198: 3209-3219.
 

-- AurkoDasgupta - 06 Aug 2012

Changed:
<
<		-- Edited by IsaacGifford - 13 Feb 2020
>
>		-- Edited by IsaacGifford - 07 Apr 2020

Revision 32020-02-20 - IsaacGifford

 
META TOPICPARENT name="ProtocolList"

Competence Assays

Day 0: Inoculation

• Prepare n+1 tubes with 5ml LB (n samples and 1 uninoculated control)
• Inoculate tubes with either 2uL of liquid culture or a pipette tip with frozen culture.
Changed:
<
<		• Incubate overnight at 30° C and 140 RPM
>
>		• Incubate overnight at 30°C and 140 RPM
 

Day 1: Preconditioning

• Prepare n+1 flasks with 10mL LB (n samples and 1 uninoculated control)
• Transfer 10uL of each inoculated culture into their respective flask
• Put all flasks in the incubator and incubate overnight at 30°C and 140 RPM.

Day 2: Transformation

• Prepare 3n+2 test tubes with 1mL LB for three replicates per strain, as well as a blank and –DNA.
• Put 100ng of the Kanamycin resistant AB gDNA into all tubes except the two designated for the blank and the –DNA control
• For each preconditioned flask from Day 1, transfer 70uL of each flask into three separate tubes containing 1mL of LB and the DNA (this will give us three trials for each strain we are trying to measure competence for)
• Add 70uL of one of the strains to the –DNA control tube (this ensures that the antibiotic is effective, and can be determined the day after plating)
• Incubate overnight at 30°C and 140 RPM

Day 3: Dilutions and Plating

• Obtain test tubes containing 10mL of sterile saline solution (get three times as many tubes as there are test tubes undergoing transformation in the incubator)
• Create a dilution series of 1:100, 1:10000, and 1:1000000 (this is done by transferring 100uL of the transformed culture to the first saline tube, vortexing, transferring 100uL of this into the second saline tube, vortexing again, then transferring 100uL of this into the third saline tube, and vortexing)
• Plate 100uL of each 1:100 dilution on plates of LB + Kanamycin
• Plate 100uL of each 1:1000000 dilution on plates of LB only
• Incubate overnight at 30°C

Day 4: Plate counting

• Start by ensuring that the antibiotic is effective (there should be no growth on the –DNA control plate that contains LB+Kan, but growth on the LB only plate is expected)
• If antibiotic effectiveness is confirmed, count all colonies on each plate and record the results

-- AurkoDasgupta - 06 Aug 2012
-- Edited by IsaacGifford - 13 Feb 2020

Revision 22020-02-13 - IsaacGifford

 
META TOPICPARENT name="ProtocolList"

Competence Assays

Day 0: Inoculation

Changed:
<
<		• Prepare n+1 tubes with 5ml LB
• Inoculate tubes with either 2ul of liquid culture or just get pipette tip with frozen culture in there.
>
>		• Prepare n+1 tubes with 5ml LB (n samples and 1 uninoculated control)
• Inoculate tubes with either 2uL of liquid culture or a pipette tip with frozen culture.
 • Incubate overnight at 30° C and 140 RPM

Day 1: Preconditioning

Changed:
<
<		• Prepare n+1 flasks with 10ml LB
>
>		• Prepare n+1 flasks with 10mL LB (n samples and 1 uninoculated control)
 • Transfer 10uL of each inoculated culture into their respective flask
Changed:
<
<		• Put all flasks in the incubator and incubate overnight at 30° C and 140 RPM.
>
>		• Put all flasks in the incubator and incubate overnight at 30°C and 140 RPM.
 

Day 2: Transformation

Changed:
<
<		• Prepare 3n+2 test tubes with 1ml LB for three replicates per strain, as well as blank and –DNA.
>
>		• Prepare 3n+2 test tubes with 1mL LB for three replicates per strain, as well as a blank and –DNA.
 • Put 100ng of the Kanamycin resistant AB gDNA into all tubes except the two designated for the blank and the –DNA control
• For each preconditioned flask from Day 1, transfer 70uL of each flask into three separate tubes containing 1mL of LB and the DNA (this will give us three trials for each strain we are trying to measure competence for)
• Add 70uL of one of the strains to the –DNA control tube (this ensures that the antibiotic is effective, and can be determined the day after plating)
Changed:
<
<		• Incubate overnight at 30° C and 140 RPM
>
>		• Incubate overnight at 30°C and 140 RPM
 

Day 3: Dilutions and Plating

• Obtain test tubes containing 10mL of sterile saline solution (get three times as many tubes as there are test tubes undergoing transformation in the incubator)
Changed:
<
<		• Create a dilution series of 1:100, 1:10000, and 1:1000000 (this is done by transferring 100uL of the transformed culture to the first saline tube, vortex to ensure mixture, transfer 100uL of this into the second saline tube, vortex, then transfer 100uL of this into the third saline tube, vortex)
>
>		• Create a dilution series of 1:100, 1:10000, and 1:1000000 (this is done by transferring 100uL of the transformed culture to the first saline tube, vortexing, transferring 100uL of this into the second saline tube, vortexing again, then transferring 100uL of this into the third saline tube, and vortexing)
 • Plate 100uL of each 1:100 dilution on plates of LB + Kanamycin
• Plate 100uL of each 1:1000000 dilution on plates of LB only
• Incubate overnight at 30°C

Day 4: Plate counting

Changed:
<
<		• Start by ensuring that the antibiotic is effective (there should be no growth on the –DNA control plate that contains LB+Kan, but growth on the LB only plate is expected
>
>		• Start by ensuring that the antibiotic is effective (there should be no growth on the –DNA control plate that contains LB+Kan, but growth on the LB only plate is expected)
 • If antibiotic effectiveness is confirmed, count all colonies on each plate and record the results

Changed:
<
<		-- AurkoDasgupta - 06 Aug 2012
>
>		-- AurkoDasgupta - 06 Aug 2012
Added:
>
>		-- Edited by IsaacGifford - 13 Feb 2020

Revision 12012-08-06 - AurkoDasgupta

 
META TOPICPARENT name="ProtocolList"

Competence Assays

Day 0: Inoculation

• Prepare n+1 tubes with 5ml LB
• Inoculate tubes with either 2ul of liquid culture or just get pipette tip with frozen culture in there.
• Incubate overnight at 30° C and 140 RPM

Day 1: Preconditioning

• Prepare n+1 flasks with 10ml LB
• Transfer 10uL of each inoculated culture into their respective flask
• Put all flasks in the incubator and incubate overnight at 30° C and 140 RPM.

Day 2: Transformation

• Prepare 3n+2 test tubes with 1ml LB for three replicates per strain, as well as blank and –DNA.
• Put 100ng of the Kanamycin resistant AB gDNA into all tubes except the two designated for the blank and the –DNA control
• For each preconditioned flask from Day 1, transfer 70uL of each flask into three separate tubes containing 1mL of LB and the DNA (this will give us three trials for each strain we are trying to measure competence for)
• Add 70uL of one of the strains to the –DNA control tube (this ensures that the antibiotic is effective, and can be determined the day after plating)
• Incubate overnight at 30° C and 140 RPM

Day 3: Dilutions and Plating

• Obtain test tubes containing 10mL of sterile saline solution (get three times as many tubes as there are test tubes undergoing transformation in the incubator)
• Create a dilution series of 1:100, 1:10000, and 1:1000000 (this is done by transferring 100uL of the transformed culture to the first saline tube, vortex to ensure mixture, transfer 100uL of this into the second saline tube, vortex, then transfer 100uL of this into the third saline tube, vortex)
• Plate 100uL of each 1:100 dilution on plates of LB + Kanamycin
• Plate 100uL of each 1:1000000 dilution on plates of LB only
• Incubate overnight at 30°C

Day 4: Plate counting

• Start by ensuring that the antibiotic is effective (there should be no growth on the –DNA control plate that contains LB+Kan, but growth on the LB only plate is expected
• If antibiotic effectiveness is confirmed, count all colonies on each plate and record the results

-- AurkoDasgupta - 06 Aug 2012

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