For a more detailed description of the Ara genetic marker, see also: Ara Marker.

Table of Contents

Materials

• Antibiotic stock solutions of Chloramphenicol (Cam) and Ampicillin (Amp).

• LB and TA plates with antibiotic. You will need LB + Cam + Amp, TA + Cam, TA + Amp, and TA plates.
  • Note: The protocol refers to TA plates throughout. If you are not using the Ara marker, substitute the proper sugar, e.g. make TM plates if using the maltose marker.
• 20% L-Arabinose stock. Dissolve 10 g in 50 ml of ddH₂0 and filter sterilize or autoclave.
• Electrocompetent cells of the parent strain.

Day 1: Transform Gene-Gorging and Donor Plasmid

1. Thaw one 1.7 ml microfuge tube of electrocompetent cells for each parent strain on ice.
2. While waiting for this to thaw, place one electroporation cuvette on ice for each parent strain.
3. Add 1 µl of pACBSR and 1 µl of the donor plasmid to each tube of electrocompetent cells on ice. Mix by flicking the tube with your finger. Do not pipette up and down or vortex to mix.
4. Electroporate
5. Pipette sample out of cuvette into original microfuge tube on ice. Add 500 µl of room temperature SOC medium.
6. Grow at 37°C for 1 hr in a shaking incubator to induce antiobiotic expression.
7. Plate 100 µl and 10 µl + 90 µl saline on two separate LB + Cam + Amp plates.
8. Grow plates overnight at 37°C.

Day 2: Induce Gene-Gorging

1. Pick three colonies from each successful transformation into separate test tubes containing 5 ml of LB medium supplemented with 0.2% L-Arabinose and 20 µg/ml Chloramphenicol (Cam). The arabinose is to induce expression of the λ Red genes from the gene-gorging plasmid. Use arabinose regardless of which sugar marker you are changing.
2. Grow cultures overnight at 37°C, shaking at 120 rpm.

Day 3: Screen or Select for Desired Mutation

1. Make a 10⁴-fold dilution of each overnight culture with two 100 µl transfers through dilution tubes containing 10 ml of saline. Note that the cell density achieved after induction is considerably lower than that usually achieved during growth in LB.
2. A. If you are gene gorging the – version of the the marker you must screen for successful mutations. Plate 200 µl of a 10⁴ dilution of each culture on tetrazolium arabinose (TA), tetrazolium maltose (TM), or other suitable indicator agar containing 20 µg/ml Chloramphenicol (Cam). Successful mutants will be red.
   B. If you are gene gorging the + version of the the marker you could screen for successful mutations as above, but it is easier to select for them by plating 100 µl of a 10² dilution on minimal arabinose (MA) or minimal maltose (MM).
3. Grow plates overnight at 37°C.

Day 4: Screen for Gene-Gorging Plasmid Loss

Expected results are 0-10 colonies with the marker change per 200 colonies of the other color.

1. Pick one colony from each plate into 5 ml of LB medium in a test tube. Give each picked colony an isolate designation. Colonies from the same plate are not necessarily independent isolates, they may share undesired second-dite mutations. Picking individual isolated
2. Grow cultures overnight at 37°C, shaking at 120 rpm.
3. Grow plates overnight at 37°C.

Day 5: Plate to Single Colonies

1. Plate 100 µl of a 10⁶ dilution of each culture on tetrazolium arabinose (TA, blue stripe) or tetrazolium maltose (TM, purple stripe). The dilution can be made with three 100 µl transfers through dilution tubes containing 10 ml of saline.
2. Grow plates overnight at 37°C.

Day 6: Patch for Plasmid Loss

1. Patch 6-12 colonies from each plate on three LB (or TA/TM) plates with Chloramphenicol, with Ampicillin, and - last - with no antibiotic.
2. Grow plates overnight at 37°C.

Day 7: Save a Freezer Stock of the New Strain

1. Pick from a patch that grows without antibiotic and not with either antibiotic present. Usually a majority of patched colonies have successfully lost both plasmids.
2. Grow cultures overnight at 37°C and archive 2 × 1 ml frozen copies in 10% glycerol.

Test for Marker Neutrality

Competitive fitness assays should be used to test for non-neutral mutations that sometimes occur during strain construction by this method. Testing three independent isolates usually assures one success.

References

1. Herring, C.D., Glasner, J.D., and Blattner, F.R. (2003) Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli. Gene 311, 153-163.

Table of Contents

Materials

• Antibiotic stock solutions of Chloramphenicol (Cam) and Ampicillin (Amp).
  • Final concentrations of antibiotics in all media (liquid or plates) are 25 µg/ml Cam and 100 µg/ml Amp.
• LB and TA plates with antibiotic. You will need LB + Cam + Amp, TA + Cam, TA + Amp, and TA plates.
  • Note: The protocol refers to TA plates throughout. If you are not using the Ara marker, substitute the proper sugar, e.g. make TM plates if using the maltose marker.
• 20% L-Arabinose stock. Dissolve 10 g in 50 ml of ddH₂0 and filter sterilize or autoclave.
• Electrocompetent cells of the parent strain.
• pACBSR (the gene-gorging plasmid)
• Donor plasmid for desired mutation (see Table).
  • These plasmids were generated by TOPO cloning a ~1000 bp DNA fragment PCR amplified from a strain containing the marker mutation with a meganucelase I-Sce I site added in the primer on one end of the homology region (5′-TAGGGATAACAGGGTAAT) into the vector pCR3.1-derived plasmid. They are pBR322 type plasmids with AmpR.

Day 1: Transform Gene-Gorging and Donor Plasmid

1. Thaw one 1.7 ml microfuge tube of electrocompetent cells for each parent strain on ice.
2. While waiting for this to thaw, place one electroporation cuvette on ice for each parent strain.
3. Add 1 µl of pACBSR and 1 µl of the donor plasmid to each tube of electrocompetent cells on ice. Mix by flicking the tube with your finger. Do not pipette up and down or vortex to mix.
4. Electroporate
5. Pipette sample out of cuvette into original microfuge tube on ice. Add 500 µl of room temperature SOC medium.
6. Grow at 37°C for 1 hr in a shaking incubator to induce antiobiotic expression.
7. Plate 100 µl and 10 µl + 90 µl saline on two separate LB + Cam + Amp plates.
8. Grow plates overnight at 37°C.

Day 2: Induce Gene-Gorging

1. Pick three colonies from each successful transformation into separate test tubes containing 5 ml of LB medium supplemented with 0.2% L-Arabinose and 20 µg/ml Chloramphenicol (Cam). The arabinose is to induce expression of the λ Red genes from the gene-gorging plasmid. Use arabinose regardless of which sugar marker you are changing.
2. Grow cultures overnight at 37°C, shaking at 120 rpm.

Day 3: Screen or Select for Desired Mutation

1. Make a 10⁴-fold dilution of each overnight culture with two 100 µl transfers through dilution tubes containing 10 ml of saline. Note that the cell density achieved after induction is considerably lower than that usually achieved during growth in LB.
2. A. If you are gene gorging the – version of the the marker you must screen for successful mutations. Plate 200 µl of a 10⁴ dilution of each culture on tetrazolium arabinose (TA), tetrazolium maltose (TM), or other suitable indicator agar containing 20 µg/ml Chloramphenicol (Cam). Successful mutants will be red.
   B. If you are gene gorging the + version of the the marker you could screen for successful mutations as above, but it is easier to select for them by plating 100 µl of a 10² dilution on minimal arabinose (MA) or minimal maltose (MM).
3. Grow plates overnight at 37°C.

Day 4: Screen for Gene-Gorging Plasmid Loss

Expected results are 0-10 colonies with the marker change per 200 colonies of the other color.

1. Pick one colony from each plate into 5 ml of LB medium in a test tube. Give each picked colony an isolate designation. Colonies from the same plate are not necessarily independent isolates, they may share undesired second-dite mutations. Picking individual isolated
2. Grow cultures overnight at 37°C, shaking at 120 rpm.
3. Grow plates overnight at 37°C.

Day 5: Plate to Single Colonies

1. Plate 100 µl of a 10⁶ dilution of each culture on tetrazolium arabinose (TA, blue stripe) or tetrazolium maltose (TM, purple stripe). The dilution can be made with three 100 µl transfers through dilution tubes containing 10 ml of saline.
2. Grow plates overnight at 37°C.

Day 6: Patch for Plasmid Loss

1. Patch 6-12 colonies from each plate on three LB (or TA/TM) plates with Chloramphenicol, with Ampicillin, and - last - with no antibiotic.
2. Grow plates overnight at 37°C.

Day 7: Save a Freezer Stock of the New Strain

1. Pick from a patch that grows without antibiotic and not with either antibiotic present. Usually a majority of patched colonies have successfully lost both plasmids.
2. Grow cultures overnight at 37°C and archive 2 × 1 ml frozen copies in 10% glycerol.

Test for Marker Neutrality

Competitive fitness assays should be used to test for non-neutral mutations that sometimes occur during strain construction by this method. Testing three independent isolates usually assures one success.

References

1. Herring, C.D., Glasner, J.D., and Blattner, F.R. (2003) Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli. Gene 311, 153-163.
2. Sean Sleight's Detailed Procedure.

• Antibiotic stock solutions of Chloramphenicol (Cam) and Ampicillin (Amp).
  • Final concentrations of antibiotics in all media (liquid or plates) are 25 µg/ml Cam and 100 µg/ml Amp.
• LB and TA plates with antibiotic. You will need LB + Cam + Amp, TA + Cam, TA + Amp, and TA plates.
  • Note: The protocol refers to TA plates throughout. If you are not using the Ara marker, substitute the proper sugar, e.g. make TM plates if using the maltose marker.
• 20% L-Arabinose stock. Dissolve 10 g in 50 ml of ddH₂0 and filter sterilize or autoclave.
• Electrocompetent cells of the parent strain.
• pACBSR (the gene-gorging plasmid)
• Donor plasmid for desired mutation (see Table).

Day 1: Transform Gene-Gorging and Donor Plasmid

1. Thaw one 1.7 ml microfuge tube of electrocompetent cells for each parent strain on ice.
2. While waiting for this to thaw, place one electroporation cuvette on ice for each parent strain.
3. Add 1 µl of pACBSR and 1 µl of the donor plasmid to each tube of electrocompetent cells on ice. Mix by flicking the tube with your finger. Do not pipette up and down or vortex to mix.
4. Electroporate
5. Pipette sample out of cuvette into original microfuge tube on ice. Add 500 µl of room temperature SOC medium.
6. Grow at 37°C for 1 hr in a shaking incubator to induce antiobiotic expression.
7. Plate 100 µl and 10 µl + 90 µl saline on two separate LB + Cam + Amp plates.
8. Grow plates overnight at 37°C.

Day 2: Induce Gene-Gorging

1. Pick three colonies from each successful transformation into separate test tubes containing 5 ml of LB medium supplemented with 0.2% L-Arabinose and 20 µg/ml Chloramphenicol (Cam). The arabinose is to induce expression of the λ Red genes from the gene-gorging plasmid. Use arabinose regardless of which sugar marker you are changing.
2. Grow cultures overnight at 37°C, shaking at 120 rpm.

Day 3: Screen or Select for Desired Mutation

1. Make a 10⁴-fold dilution of each overnight culture with two 100 µl transfers through dilution tubes containing 10 ml of saline. Note that the cell density achieved after induction is considerably lower than that usually achieved during growth in LB.
2. A. If you are gene gorging the – version of the the marker you must screen for successful mutations. Plate 200 µl of a 10⁴ dilution of each culture on tetrazolium arabinose (TA), tetrazolium maltose (TM), or other suitable indicator agar containing 20 µg/ml Chloramphenicol (Cam). Successful mutants will be red.
   B. If you are gene gorging the + version of the the marker you could screen for successful mutations as above, but it is easier to select for them by plating 100 µl of a 10² dilution on minimal arabinose (MA) or minimal maltose (MM).
3. Grow plates overnight at 37°C.

Day 4: Screen for Gene-Gorging Plasmid Loss

Expected results are 0-10 colonies with the marker change per 200 colonies of the other color.

1. Pick one colony from each plate into 5 ml of LB medium in a test tube. Give each picked colony an isolate designation. Colonies from the same plate are not necessarily independent isolates, they may share undesired second-dite mutations. Picking individual isolated
2. Grow cultures overnight at 37°C, shaking at 120 rpm.
3. Grow plates overnight at 37°C.

Day 5: Plate to Single Colonies

1. Plate 100 µl of a 10⁶ dilution of each culture on tetrazolium arabinose (TA, blue stripe) or tetrazolium maltose (TM, purple stripe). The dilution can be made with three 100 µl transfers through dilution tubes containing 10 ml of saline.
2. Grow plates overnight at 37°C.

Day 6: Patch for Plasmid Loss

1. Patch 6-12 colonies from each plate on three LB (or TA/TM) plates with Chloramphenicol, with Ampicillin, and - last - with no antibiotic.
2. Grow plates overnight at 37°C.

Day 7: Save a Freezer Stock of the New Strain

1. Pick from a patch that grows without antibiotic and not with either antibiotic present. Usually a majority of patched colonies have successfully lost both plasmids.
2. Grow cultures overnight at 37°C and archive 2 × 1 ml frozen copies in 10% glycerol.

Test for Marker Neutrality

Competitive fitness assays should be used to test for non-neutral mutations that sometimes occur during strain construction by this method. Testing three independent isolates usually assures one success.

References

1. Herring, C.D., Glasner, J.D., and Blattner, F.R. (2003) Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli. Gene 311, 153-163.
2. Sean Sleight's Detailed Procedure.

• • Note: The protocol refers to TA plates throughout. If you are not using the Ara marker, substitute the proper sugar, e.g. make TM plates if using the maltose marker.
• 20% L-Arabinose stock. Dissolve 10 g in 50 ml of ddH₂0 and filter sterilize or autoclave.
• Electrocompetent cells of the parent strain.
• pACBSR (the gene-gorging plasmid)
• Donor plasmid for desired mutation (see Table).
  • Typically this is a pCR3.1-derived plasmid generated by TOPO cloning a ~1000 bp DNA fragment PCR amplified from a strain containing the marker mutation.

Day 1: Transform Gene-Gorging and Donor Plasmid

1. Thaw one 1.7 ml microfuge tube of electrocompetent cells for each parent strain on ice.
2. While waiting for this to thaw, place one electroporation cuvette on ice for each parent strain.
3. Add 1 µl of pACBSR and 1 µl of the donor plasmid to each tube of electrocompetent cells on ice. Mix by flicking the tube with your finger. Do not pipette up and down or vortex to mix.
4. Electroporate
5. Pipette sample out of cuvette into original microfuge tube on ice. Add 500 µl of room temperature SOC medium.
6. Grow at 37°C for 1 hr in a shaking incubator to induce antiobiotic expression.

1. Grow plates overnight at 37°C.

Day 2: Induce Gene-Gorging

1. Pick three colonies from each successful transformation into separate test tubes containing 5 ml of LB medium supplemented with 0.2% L-Arabinose and 20 µg/ml Chloramphenicol (Cam). The arabinose is to induce expression of the λ Red genes from the gene-gorging plasmid. Use arabinose regardless of which sugar marker you are changing.
2. Grow cultures overnight at 37°C, shaking at 120 rpm.

Day 3: Screen or Select for Desired Mutation

1. Make a 10⁴-fold dilution of each overnight culture with two 100 µl transfers through dilution tubes containing 10 ml of saline. Note that the cell density achieved after induction is considerably lower than that usually achieved during growth in LB.
2. A. If you are gene gorging the – version of the the marker you must screen for successful mutations. Plate 200 µl of a 10⁴ dilution of each culture on tetrazolium arabinose (TA), tetrazolium maltose (TM), or other suitable indicator agar containing 20 µg/ml Chloramphenicol (Cam). Successful mutants will be red.
   B. If you are gene gorging the + version of the the marker you could screen for successful mutations as above, but it is easier to select for them by plating 100 µl of a 10² dilution on minimal arabinose (MA) or minimal maltose (MM).
3. Grow plates overnight at 37°C.

Day 4: Screen for Gene-Gorging Plasmid Loss

Expected results are 0-10 colonies with the marker change per 200 colonies of the other color.

1. Pick one colony from each plate into 5 ml of LB medium in a test tube. Give each picked colony an isolate designation. Colonies from the same plate are not necessarily independent isolates, they may share undesired second-dite mutations. Picking individual isolated
2. Grow cultures overnight at 37°C, shaking at 120 rpm.
3. Grow plates overnight at 37°C.

Day 5: Plate to Single Colonies

1. Plate 100 µl of a 10⁶ dilution of each culture on tetrazolium arabinose (TA, blue stripe) or tetrazolium maltose (TM, purple stripe). The dilution can be made with three 100 µl transfers through dilution tubes containing 10 ml of saline.
2. Grow plates overnight at 37°C.

Day 6: Patch for Plasmid Loss

1. Grow plates overnight at 37°C.

Day 7: Save a Freezer Stock of the New Strain

1. Pick from a patch that grows without antibiotic and not with either antibiotic present. Usually a majority of patched colonies have successfully lost both plasmids.
2. Grow cultures overnight at 37°C and archive 2 × 1 ml frozen copies in 10% glycerol.

Test for Marker Neutrality

Competitive fitness assays should be used to test for non-neutral mutations that sometimes occur during strain construction by this method. Testing three independent isolates usually assures one success.

References

1. Herring, C.D., Glasner, J.D., and Blattner, F.R. (2003) Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli. Gene 311, 153-163.
2. Sean Sleight's Detailed Procedure.

This procedure has been used to create defined Ara⁺ and Ara^– versions of Escherichia coli B (REL606) and Mal^- derivatives of Escherichia coli K12 (Keio collection strains) with the malF Keio deletion.

For a more detailed description of the Ara genetic marker, see also: Ara Marker.

Materials

• Antibiotic stock solutions of Chloramphenicol (Cam) and Kanamycin (Kan).
  • Final concentrations of antibiotics in all media (liquid or plates) are 25 µg/ml Cam and 50 µg/ml Kan.
• LB and TA plates with antibiotic. You will need LB + Cam + Kan, TA + Cam, TA + Kan, and TA plates.
  • Note: The protocol refers to TA plates throughout. If you are not using the Ara marker, substitute the proper sugar, e.g. make TM plates if using the maltose marker.
• 20% L-Arabinose stock. Dissolve 10 g in 50 ml of ddH₂0 and filter sterilize or autoclave.
• Electrocompetent cells of the parent strain.
• pACBSR (the gene-gorging plasmid)
• Donor plasmid for desired mutation (see Table).
  • Typically this is a pCR3.1-derived plasmid generated by TOPO cloning a ~1000 bp DNA fragment PCR amplified from a strain containing the marker mutation.

Day 1: Transform Gene-Gorging and Donor Plasmid

1. Thaw one 1.7 ml microfuge tube of electrocompetent cells for each parent strain on ice.
2. While waiting for this to thaw, place one electroporation cuvette on ice for each parent strain.
3. Add 1 µl of pACBSR and 1 µl of the donor plasmid to each tube of electrocompetent cells on ice. Mix by flicking the tube with your finger. Do not pipette up and down or vortex to mix.
4. Electroporate
5. Pipette sample out of cuvette into original microfuge tube on ice. Add 500 µl of room temperature SOC medium.
6. Grow at 37°C for 1 hr in a shaking incubator to induce antiobiotic expression.
7. Plate 100 µl and 10 µl + 90 µl saline on two separate LB + Cam + Kan plates.
8. Grow plates overnight at 37°C.

Day 2: Induce Gene-Gorging

1. Pick three colonies from each successful transformation into separate test tubes containing 5 ml of LB medium supplemented with 0.2% L-Arabinose and 20 µg/ml Chloramphenicol (Cam). The arabinose is to induce expression of the λ Red genes from the gene-gorging plasmid. Use arabinose regardless of which sugar marker you are changing.
2. Grow cultures overnight at 37°C, shaking at 120 rpm.

Day 3: Screen or Select for Desired Mutation

1. Make a 10⁴-fold dilution of each overnight culture with two 100 µl transfers through dilution tubes containing 10 ml of saline. Note that the cell density achieved after induction is considerably lower than that usually achieved during growth in LB.
2. A. If you are gene gorging the – version of the the marker you must screen for successful mutations. Plate 200 µl of a 10⁴ dilution of each culture on tetrazolium arabinose (TA), tetrazolium maltose (TM), or other suitable indicator agar containing 20 µg/ml Chloramphenicol (Cam). Successful mutants will be red.
   B. If you are gene gorging the + version of the the marker you could screen for successful mutations as above, but it is easier to select for them by plating 100 µl of a 10² dilution on minimal arabinose (MA) or minimal maltose (MM).
3. Grow plates overnight at 37°C.

Day 4: Screen for Gene-Gorging Plasmid Loss

Expected results are 0-10 colonies with the marker change per 200 colonies of the other color.

1. Pick one colony from each plate into 5 ml of LB medium in a test tube. Give each picked colony an isolate designation. Colonies from the same plate are not necessarily independent isolates, they may share undesired second-dite mutations. Picking individual isolated
2. Grow cultures overnight at 37°C, shaking at 120 rpm.
3. Grow plates overnight at 37°C.

Day 5: Plate to Single Colonies

1. Plate 100 µl of a 10⁶ dilution of each culture on tetrazolium arabinose (TA, blue stripe) or tetrazolium maltose (TM, purple stripe). The dilution can be made with three 100 µl transfers through dilution tubes containing 10 ml of saline.
2. Grow plates overnight at 37°C.

Day 6: Patch for Plasmid Loss

1. Patch 6-12 colonies from each plate on three LB (or TA/TM) plates with 20 µg/ml Chloramphenicol, with Kanamycin, and - last - with no antibiotic.
2. Grow plates overnight at 37°C.

Day 7: Save a Freezer Stock of the New Strain

1. Pick from a patch that grows without antibiotic and not with either antibiotic present. Usually a majority of patched colonies have successfully lost both plasmids.
2. Grow cultures overnight at 37°C and archive 2 × 1 ml frozen copies in 10% glycerol.

Test for Marker Neutrality

Competitive fitness assays should be used to test for non-neutral mutations that sometimes occur during strain construction by this method. Testing three independent isolates usually assures one success.

References

1. Herring, C.D., Glasner, J.D., and Blattner, F.R. (2003) Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli. Gene 311, 153-163.
2. Sean Sleight's Detailed Procedure.

1. Pipette sample out of cuvette into original microfuge tube on ice. Add 500 µl of room temperature SOC medium.
2. Grow at 37°C for 1 hr in a shaking incubator to induce antiobiotic expression.
3. Plate 100 µl and 10 µl + 90 µl saline on two separate LB + Cam + Kan plates.
4. Grow plates overnight at 37°C.

Day 2: Induce Gene-Gorging

1. Grow cultures overnight at 37°C, shaking at 120 rpm.

Day 3: Screen or Select for Desired Mutation

1. Make a 10⁴-fold dilution of each overnight culture with two 100 µl transfers through dilution tubes containing 10 ml of saline. Note that the cell density achieved after induction is considerably lower than that usually achieved during growth in LB.

1. Grow plates overnight at 37°C.

Day 4: Screen for Gene-Gorging Plasmid Loss

Expected results are 0-10 colonies with the marker change per 200 colonies of the other color.

1. Pick one colony from each plate into 5 ml of LB medium in a test tube. Give each picked colony an isolate designation. Colonies from the same plate are not necessarily independent isolates, they may share undesired second-dite mutations. Picking individual isolated
2. Grow cultures overnight at 37°C, shaking at 120 rpm.
3. Grow plates overnight at 37°C.

Day 5: Plate to Single Colonies

1. Plate 100 µl of a 10⁶ dilution of each culture on tetrazolium arabinose (TA, blue stripe) or tetrazolium maltose (TM, purple stripe). The dilution can be made with three 100 µl transfers through dilution tubes containing 10 ml of saline.
2. Grow plates overnight at 37°C.

Day 6: Patch for Plasmid Loss

1. Patch 6-12 colonies from each plate on three LB (or TA/TM) plates with 20 µg/ml Chloramphenicol, with Kanamycin, and - last - with no antibiotic.
2. Grow plates overnight at 37°C.

Day 7: Save a Freezer Stock of the New Strain

1. Pick from a patch that grows without antibiotic and not with either antibiotic present. Usually a majority of patched colonies have successfully lost both plasmids.
2. Grow cultures overnight at 37°C and archive 2 × 1 ml frozen copies in 10% glycerol.

Test for Marker Neutrality

Competitive fitness assays should be used to test for non-neutral mutations that sometimes occur during strain construction by this method. Testing three independent isolates usually assures one success.

References

1. Herring, C.D., Glasner, J.D., and Blattner, F.R. (2003) Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli. Gene 311, 153-163.
2. Sean Sleight's Detailed Procedure.

• • Note: The protocol refers to TA plates throughout. If you are not using the Ara marker, substitute the proper sugar, e.g. make TM plates if using the maltose marker.
• 20% L-Arabinose stock. Dissolve 10 g in 50 ml of ddH₂0 and filter sterilize or autoclave.
• Electrocompetent cells of the parent strain.
• pACBSR (the gene-gorging plasmid)
• Donor plasmid for desired mutation (see Table).
  • Typically this is a pCR3.1-derived plasmid generated by TOPO cloning a ~1000 bp DNA fragment PCR amplified from a strain containing the marker mutation.

Day 1: Transform Gene-Gorging and Donor Plasmid

1. Thaw one 1.7 ml microfuge tube of electrocompetent cells for each parent strain on ice.
2. While waiting for this to thaw, place one electroporation cuvette on ice for each parent strain.
3. Add 1 µl of pACBSR and 1 µl of the donor plasmid to each tube of electrocompetent cells on ice. Mix by flicking the tube with your finger. Do not pipette up and down or vortex to mix.
4. Electroporate (pipette sample into chilled cuvette on ice, place cuvette in electroporator, run program "bacteria EC2", typical results are 6 ms and 2.5 mV).
5. Pipette sample out of cuvette into original microfuge tube on ice. Add 500 µl of room temperature SOC medium.
6. Grow at 37°C for 1 hr in a shaking incubator to induce antiobiotic expression.
7. Plate 100 µl and 10 µl + 90 µl saline on two separate LB + Cam + Kan plates.
8. Grow plates overnight at 37°C.

Day 2: Induce Gene-Gorging

1. Pick three colonies from each successful transformation into separate test tubes containing 5 ml of LB medium supplemented with 0.2% L-Arabinose and 20 µg/ml Chloramphenicol (Cam). The arabinose is to induce expression of the λ Red genes from the gene-gorging plasmid.
2. Grow cultures overnight at 37°C, shaking at 120 rpm.

Day 3: Screen or Select for Desired Mutation

1. Make a 10⁴-fold dilution of each overnight culture with two 100 µl transfers through dilution tubes containing 10 ml of saline. Note that the cell density achieved after induction is considerably lower than that usually achieved during growth in LB.
2. If you are gene gorging the – version of the the marker you must screen for successful mutations. Plate 200 µl of a 10⁴ dilution of each culture on tetrazolium arabinose (TA), tetrazolium maltose (TM), or other suitable indicator agar containing 20 µg/ml Chloramphenicol (Cam). Successful mutants will be red.
   

1. Grow plates overnight at 37°C.

Day 4: Screen for Gene-Gorging Plasmid Loss

Expected results are 0-10 colonies with the marker change per 200 colonies of the other color.

1. Pick one colony from each plate into 5 ml of LB medium in a test tube. Give each picked colony an isolate designation. Colonies from the same plate are not necessarily independent isolates, they may share undesired second-dite mutations. Picking individual isolated
2. Grow cultures overnight at 37°C, shaking at 120 rpm.
3. Grow plates overnight at 37°C.

Day 5: Plate to Single Colonies

1. Plate 100 µl of a 10⁶ dilution of each culture on tetrazolium arabinose (TA, blue stripe) or tetrazolium maltose (TM, purple stripe). The dilution can be made with three 100 µl transfers through dilution tubes containing 10 ml of saline.
2. Grow plates overnight at 37°C.

Day 6: Patch for Plasmid Loss

1. Patch 6-12 colonies from each plate on three LB (or TA/TM) plates with 20 µg/ml Chloramphenicol, with Kanamycin, and - last - with no antibiotic.
2. Grow plates overnight at 37°C.

Day 7: Save a Freezer Stock of the New Strain

1. Pick from a patch that grows without antibiotic and not with either antibiotic present. Usually a majority of patched colonies have successfully lost both plasmids.
2. Grow cultures overnight at 37°C and archive 2 × 1 ml frozen copies in 10% glycerol.

Test for Marker Neutrality

Competitive fitness assays should be used to test for non-neutral mutations that sometimes occur during strain construction by this method. Testing three independent isolates usually assures one success.

References

1. Herring, C.D., Glasner, J.D., and Blattner, F.R. (2003) Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli. Gene 311, 153-163.
2. Sean Sleight's Detailed Procedure.

• Electrocompetent cells of the parent strain.
• pACBSR (the gene-gorging plasmid)

Day 1: Transform Gene-Gorging and Donor Plasmid

1. Grow plates overnight at 37°C.

Day 2: Induce Gene-Gorging

1. Pick three colonies from each successful transformation into separate test tubes containing 5 ml of LB medium supplemented with 0.2% L-Arabinose and 20 µg/ml Chloramphenicol (Cam). The arabinose is to induce expression of the λ Red genes from the gene-gorging plasmid.
2. Grow cultures overnight at 37°C, shaking at 120 rpm.

Day 3: Screen or Select for Desired Mutation

1. Make a 10⁴-fold dilution of each overnight culture with two 100 µl transfers through dilution tubes containing 10 ml of saline. Note that the cell density achieved after induction is considerably lower than that usually achieved during growth in LB.
2. If you are gene gorging the – version of the the marker you must screen for successful mutations. Plate 200 µl of a 10⁴ dilution of each culture on tetrazolium arabinose (TA), tetrazolium maltose (TM), or other suitable indicator agar containing 20 µg/ml Chloramphenicol (Cam). Successful mutants will be red.
   

1. Grow plates overnight at 37°C.

Day 4: Screen for Gene-Gorging Plasmid Loss

Expected results are 0-10 colonies with the marker change per 200 colonies of the other color.

1. Pick one colony from each plate into 5 ml of LB medium in a test tube. Give each picked colony an isolate designation. Colonies from the same plate are not necessarily independent isolates, they may share undesired second-dite mutations. Picking individual isolated
2. Grow cultures overnight at 37°C, shaking at 120 rpm.
3. Grow plates overnight at 37°C.

Day 5: Plate to Single Colonies

1. Plate 100 µl of a 10⁶ dilution of each culture on tetrazolium arabinose (TA, blue stripe) or tetrazolium maltose (TM, purple stripe). The dilution can be made with three 100 µl transfers through dilution tubes containing 10 ml of saline.
2. Grow plates overnight at 37°C.

Day 6: Patch for Plasmid Loss

1. Patch 6-12 colonies from each plate on three LB (or TA/TM) plates with 20 µg/ml Chloramphenicol, with Kanamycin, and - last - with no antibiotic.
2. Grow plates overnight at 37°C.

Day 7: Save a Freezer Stock of the New Strain

1. Pick from a patch that grows without antibiotic and not with either antibiotic present. Usually a majority of patched colonies have successfully lost both plasmids.
2. Grow cultures overnight at 37°C and archive 2 × 1 ml frozen copies in 10% glycerol.

Test for Marker Neutrality

Competitive fitness assays should be used to test for non-neutral mutations that sometimes occur during strain construction by this method. Testing three independent isolates usually assures one success.

References

1. Herring, C.D., Glasner, J.D., and Blattner, F.R. (2003) Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli. Gene 311, 153-163.
2. Sean Sleight's Detailed Procedure.

Gene Gorging Marker Mutations

1. Plate on LB + Cam + Kan.
2. Grow plates overnight at 37°C.

1. Grow cultures overnight at 37°C, shaking at 120 rpm.

1. Grow plates overnight at 37°C.

1. Grow cultures overnight at 37°C, shaking at 120 rpm.
2. Grow plates overnight at 37°C.

1. Plate 100 µl of a 10⁶ dilution of each culture on tetrazolium arabinose (TA, blue stripe) or tetrazolium maltose (TM, purple stripe). The dilution can be made with three 100 µl transfers through dilution tubes containing 10 ml of saline.
2. Grow plates overnight at 37°C.

1. Patch 6-12 colonies from each plate on three LB (or TA/TM) plates with 20 µg/ml Chloramphenicol, with Kanamycin, and - last - with no antibiotic.
2. Grow plates overnight at 37°C.

1. Pick from a patch that grows without antibiotic and not with either antibiotic present. Usually a majority of patched colonies have successfully lost both plasmids.
2. Grow cultures overnight at 37°C and archive 2 × 1 ml frozen copies in 10% glycerol.

References

1. Herring, C.D., Glasner, J.D., and Blattner, F.R. (2003) Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli. Gene 311, 153-163.
2. Sean Sleight's Detailed Procedure.

Gene Gorging Marker Mutations

This procedure has been used to create defined Ara⁺ revertants of Escherichia coli B (REL606) and Mal^- derivatives of Escherichia coli K12 (Keio collection strains) with the malF Keio deletion.

General Notes

• For a more detailed description of genetic markers see also: Ara Marker.

1. Prepare electrocompetent cells of the strain in which you wish to change the genetic marker.

1. Plate on LB + Cam + Kan.
2. Grow plates overnight at 37°C.

1. Pick three colonies from each successful transformation into separate test tubes containing 5 ml of LB medium supplemented with 0.2% L-Arabinose and 20 µg/ml Chloramphenicol (Cam).
2. Grow cultures overnight at 37°C, shaking at 120 rpm.

1. Plate 200 µl of a 10⁴ dilution of each culture on tetrazolium arabinose (TA, blue stripe) or tetrazolium maltose (TM, purple stripe) containing 20 µg/ml Chloramphenicol (Cam). The dilution can be made with two 100 µl transfers through dilution tubes containing 10 ml of saline. Alternately, plate 100 µl of a 10² dilution on minimal arabinose (MA) or minimal maltose (MM).
2. Grow plates overnight at 37°C.

1. Pick one colony from each plate into 5 ml of LB medium. Give each picked colony an isolate designation. (Colonies from the same plate are not independent isolates).
2. Grow cultures overnight at 37°C, shaking at 120 rpm.
3. Grow plates overnight at 37°C.

1. Plate 100 µl of a 10⁶ dilution of each culture on tetrazolium arabinose (TA, blue stripe) or tetrazolium maltose (TM, purple stripe). The dilution can be made with three 100 µl transfers through dilution tubes containing 10 ml of saline.
2. Grow plates overnight at 37°C.

1. Patch 6-12 colonies from each plate on three LB (or TA/TM) plates with 20 µg/ml Chloramphenicol, with Kanamycin, and - last - with no antibiotic.
2. Grow plates overnight at 37°C.

1. Pick from a patch that grows without antibiotic and not with either antibiotic present. Usually a majority of patched colonies have successfully lost both plasmids.

1. Herring, C.D., Glasner, J.D., and Blattner, F.R. (2003) Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli. Gene 311, 153-163.
2. Sean Sleight's Detailed Procedure.

1. Transform with 1 µl of pACBSR (the gene-gorging plasmid) and 1 µl of pJEB12 (for Ara⁺) or pJEB for Mal^-(the donor plasmid).
2. Plate on LB + Cam + Kan.
3. Grow plates overnight at 37°C.

Induce Gene-Gorging

1. Pick three colonies from each successful transformation into separate test tubes containing 5 ml of LB medium supplemented with 0.2% L-Arabinose and 20 µg/ml Chloramphenicol (Cam).
2. Grow cultures overnight at 37°C, shaking at 120 rpm.

Screen for Desired Mutation

1. Plate 200 µl of a 10⁴ dilution of each culture on tetrazolium arabinose (TA, blue stripe) or tetrazolium maltose (TM, purple stripe) containing 20 µg/ml Chloramphenicol (Cam). The dilution can be made with two 100 µl transfers through dilution tubes containing 10 ml of saline. Alternately, plate 100 µl of a 10² dilution on minimal arabinose (MA) or minimal maltose (MM).
2. Grow plates overnight at 37°C.

Select for Gene-Gorging Plasmid Loss

Expected results are 0-10 white (Ara+) or red (Mal-) colonies per 200 colonies of the other color.

1. Pick one colony from each plate into 5 ml of LB medium. Give each picked colony an isolate designation. (Colonies from the same plate are not independent isolates).
2. Grow cultures overnight at 37°C, shaking at 120 rpm.
3. Grow plates overnight at 37°C.

Plate to Single Colonies

1. Plate 100 µl of a 10⁶ dilution of each culture on tetrazolium arabinose (TA, blue stripe) or tetrazolium maltose (TM, purple stripe). The dilution can be made with three 100 µl transfers through dilution tubes containing 10 ml of saline.
2. Grow plates overnight at 37°C.

Patch for Plasmid Loss

1. Patch 6-12 colonies from each plate on three LB (or TA/TM) plates with 20 µg/ml Chloramphenicol, with Kanamycin, and - last - with no antibiotic.
2. Grow plates overnight at 37°C.

Patch for Plasmid Loss

1. Pick from a patch that grows without antibiotic and not with either antibiotic present. Usually a majority of patched colonies have successfully lost both plasmids.
2. Grow cultures overnight at 37°C and archive 2 x 1 ml frozen copies in 10% glycerol.

Competitive fitness assays should be used to test for non-neutral mutations that sometimes occur during strain construction by this method. Testing three independent isolates usually assures success.

Sources

1. Herring, C.D., Glasner, J.D., and Blattner, F.R. (2003) Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli. Gene 311, 153-163.
2. Sean Sleight's Detailed Procedure.

Gene Gorging Neutral Markers

This procedure has been used to create defined Ara⁺ revertants of Escherichia coli B (REL606) and Mal^-derivatives of Escherichia coli K12 (Keio collection strains) with the_malF_ Keio deletion.

Transform Gene-Gorging and Donor Plasmid

1. Prepare electrocompetent cells of the strain in which you wish to change the sugar marker.
2. Transform with 1 µl of pACBSR (the gene-gorging plasmid) and 1 µl of pJEB12 (for Ara⁺) or pJEB for Mal^-(the donor plasmid).
3. Plate on LB + Cam + Kan.
4. Grow plates overnight at 37°C.

Induce Gene-Gorging

1. Pick three colonies from each successful transformation into separate test tubes containing 5 ml of LB medium supplemented with 0.2% L-Arabinose and 20 µg/ml Chloramphenicol (Cam).
2. Grow cultures overnight at 37°C, shaking at 120 rpm.

1. Plate 200 µl of a 10⁴ dilution of each culture on tetrazolium arabinose (TA, blue stripe) or tetrazolium maltose (TM, purple stripe) containing 20 µg/ml Chloramphenicol (Cam). The dilution can be made with two 100 µl transfers through dilution tubes containing 10 ml of saline. Alternately, plate 100 µl of a 10² dilution on minimal arabinose (MA) or minimal maltose (MM).
2. Grow plates overnight at 37°C.

Select for Gene-Gorging Plasmid Loss

Expected results are 0-10 white (Ara+) or red (Mal-) colonies per 200 colonies of the other color.

1. Pick one colony from each plate into 5 ml of LB medium. Give each picked colony an isolate designation. (Colonies from the same plate are not independent isolates).
2. Grow cultures overnight at 37°C, shaking at 120 rpm.
3. Grow plates overnight at 37°C.

Plate to Single Colonies

1. Plate 100 µl of a 10⁶ dilution of each culture on tetrazolium arabinose (TA, blue stripe) or tetrazolium maltose (TM, purple stripe). The dilution can be made with three 100 µl transfers through dilution tubes containing 10 ml of saline.
2. Grow plates overnight at 37°C.

Patch for Plasmid Loss

1. Patch 6-12 colonies from each plate on three LB (or TA/TM) plates with 20 µg/ml Chloramphenicol, with Kanamycin, and - last - with no antibiotic.
2. Grow plates overnight at 37°C.

Patch for Plasmid Loss

1. Pick from a patch that grows without antibiotic and not with either antibiotic present. Usually a majority of patched colonies have successfully lost both plasmids.
2. Grow cultures overnight at 37°C and archive 2 x 1 ml frozen copies in 10% glycerol.

Competitive fitness assays should be used to test for non-neutral mutations that sometimes occur during strain construction by this method. Testing three independent isolates usually assures success.

Sources

1. Herring, C.D., Glasner, J.D., and Blattner, F.R. (2003) Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli. Gene 311, 153-163.
2. Sean Sleight's Detailed Procedure.

Sources

1. Herring, C.D., Glasner, J.D., and Blattner, F.R. (2003) Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli. Gene 311, 153-163.
2. Sean Sleight's Detailed Procedure.

Sources

1. Herring, C.D., Glasner, J.D., and Blattner, F.R. (2003) Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli. Gene 311, 153-163.
2. Sean Sleight's Detailed Procedure.