Difference: ProtocolsGeneGorgingMarker (1 vs. 14)

Revision 142019-04-10 - JeffreyBarrick

Line: 1 to 1
 
META TOPICPARENT name="ProtocolList"

Gene Gorging Marker Mutations

Changed:
<
<
For competitive fitness assays, one must be able to distinguish two E. coli subpopulations in a mixed culture. One way this can be accomplished is by mutating genes required for sugar fermentation to create a genetic marker (Ara, Mal, Lac, etc.). Plating a version of these strains will produce red colonies and plating the + version will produce pinkish white colonies on tetrazolium indicator agar containing a rich media base plus the sugar of interest (TA, TM, etc).
>
>
For competitive fitness assays, one must be able to distinguish two E. coli subpopulations in a mixed culture. One way this can be accomplished is by mutating genes required for sugar fermentation to create a genetic marker (Ara, Mal, Lac, etc.). Plating a version of these strains will produce red colonies and plating the + version will produce pinkish-white colonies on tetrazolium indicator agar containing a rich media base plus the sugar of interest (TA, TM, etc).
  This procedure has been used to create defined Ara+ and Ara versions of Escherichia coli B (REL606) and Mal- derivatives of Escherichia coli K12 containing the malF in-frame deletion from the Keio collection strain with its KanR marker removed by FLP recombination.
Line: 16 to 16
 

Materials

Changed:
<
<
Plasmid Marker
pJEB11 →Ara
pJEB12 →Ara+
pJEB15 →Mal
>
>
Strain Plasmid Marker Resistance
JEB205 pJEB11 →Ara AmpR+KanR
JEB207 pJEB12 →Ara+ AmpR+KanR
JEB600 pJEB15 →Mal AmpR+KanR
JEB208 pACBSR for gene-gorging CamR
 

Changed:
<
<
    • Final concentrations of antibiotics in all media (liquid or plates) are 25 g/ml Cam and 100 g/ml Amp.
>
>
    • Final concentrations of antibiotics in all media (liquid or plates) are 20 g/ml Cam and 100 g/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.
Changed:
<
<
  • 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.
>
>
  • pACBSR (the gene-gorging plasmid) This plasmid is CamR.
  • Donor plasmid for desired mutation (see Table for TOP10 strains containing each plasmid).
    • 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 pCR4-derived plasmid. They are pBR322 type plasmids with AmpR and KanR markers.
 

Day 1: Transform Gene-Gorging and Donor Plasmid

Line: 85 to 86
 

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.
Changed:
<
<
  1. Sean Sleight's Detailed Procedure.
>
>
  1. Sean Sleight's Detailed Procedure from the Lenski Lab.
 

META TOPICMOVED by="JeffreyBarrick" date="1273244634" from="Lab.ProceduresGeneGorgingNeutralMarkers" to="Lab.ProtocolsGeneGorgingMarker"

Revision 132019-03-29 - JeffreyBarrick

Line: 1 to 1
 
META TOPICPARENT name="ProtocolList"

Gene Gorging Marker Mutations

For competitive fitness assays, one must be able to distinguish two E. coli subpopulations in a mixed culture. One way this can be accomplished is by mutating genes required for sugar fermentation to create a genetic marker (Ara, Mal, Lac, etc.). Plating a version of these strains will produce red colonies and plating the + version will produce pinkish white colonies on tetrazolium indicator agar containing a rich media base plus the sugar of interest (TA, TM, etc).

Changed:
<
<
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.
>
>
This procedure has been used to create defined Ara+ and Ara versions of Escherichia coli B (REL606) and Mal- derivatives of Escherichia coli K12 containing the malF in-frame deletion from the Keio collection strain with its KanR marker removed by FLP recombination.
  For a more detailed description of the Ara genetic marker, see also: Ara Marker.

Revision 122018-11-03 - JeffreyBarrick

Line: 1 to 1
 
META TOPICPARENT name="ProtocolList"

Gene Gorging Marker Mutations

Line: 19 to 19
 
Plasmid Marker
pJEB11 →Ara
pJEB12 →Ara+
Changed:
<
<
pJEB15 →Mal Amp<sup</
>
>
pJEB15 →Mal
 

Line: 30 to 30
 
  • Electrocompetent cells of the parent strain.
  • pACBSR (the gene-gorging plasmid)
  • Donor plasmid for desired mutation (see Table).
Changed:
<
<
    • 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.
>
>
    • 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

Revision 112018-10-17 - JeffreyBarrick

Line: 1 to 1
 
META TOPICPARENT name="ProtocolList"

Gene Gorging Marker Mutations

Line: 19 to 19
 
Plasmid Marker
pJEB11 →Ara
pJEB12 →Ara+
Changed:
<
<
pJEB15 →Mal
>
>
pJEB15 →Mal Amp<sup</
 
Changed:
<
<
  • Antibiotic stock solutions of Chloramphenicol (Cam) and Kanamycin (Kan).
    • Final concentrations of antibiotics in all media (liquid or plates) are 25 g/ml Cam and 50 g/ml Kan.
  • LB and TA plates with antibiotic. You will need LB + Cam + Kan, TA + Cam, TA + Kan, and TA plates.
>
>
  • Antibiotic stock solutions of Chloramphenicol (Cam) and Ampicillin (Amp).
    • Final concentrations of antibiotics in all media (liquid or plates) are 25 g/ml Cam and 100 g/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.
Line: 41 to 41
 
  1. Electroporate
  2. Pipette sample out of cuvette into original microfuge tube on ice. Add 500 l of room temperature SOC medium.
  3. Grow at 37C for 1 hr in a shaking incubator to induce antiobiotic expression.
Changed:
<
<
  1. Plate 100 l and 10 l + 90 l saline on two separate LB + Cam + Kan plates.
>
>
  1. Plate 100 l and 10 l + 90 l saline on two separate LB + Cam + Amp plates.
 
  1. Grow plates overnight at 37C.

Day 2: Induce Gene-Gorging

Line: 70 to 70
 

Day 6: Patch for Plasmid Loss

Changed:
<
<
  1. Patch 6-12 colonies from each plate on three LB (or TA/TM) plates with 20 g/ml Chloramphenicol, with Kanamycin, and - last - with no antibiotic.
>
>
  1. Patch 6-12 colonies from each plate on three LB (or TA/TM) plates with Chloramphenicol, with Ampicillin, and - last - with no antibiotic.
 
  1. Grow plates overnight at 37C.

Day 7: Save a Freezer Stock of the New Strain

Revision 102013-02-20 - JeffreyBarrick

Line: 1 to 1
 
META TOPICPARENT name="ProtocolList"
Changed:
<
<

Gene Gorging Marker Mutations

>
>

Gene Gorging Marker Mutations

  For competitive fitness assays, one must be able to distinguish two E. coli subpopulations in a mixed culture. One way this can be accomplished is by mutating genes required for sugar fermentation to create a genetic marker (Ara, Mal, Lac, etc.). Plating a version of these strains will produce red colonies and plating the + version will produce pinkish white colonies on tetrazolium indicator agar containing a rich media base plus the sugar of interest (TA, TM, etc).
Line: 9 to 9
  For a more detailed description of the Ara genetic marker, see also: Ara Marker.
Added:
>
>

Table of Contents

 

Materials

Revision 92010-05-11 - JeffreyBarrick

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META TOPICPARENT name="ProtocolList"

Gene Gorging Marker Mutations

Line: 36 to 36
 
  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 l of pACBSR and 1 l 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.
Changed:
<
<
  1. 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).
>
>
  1. Electroporate
 
  1. Pipette sample out of cuvette into original microfuge tube on ice. Add 500 l of room temperature SOC medium.
  2. Grow at 37C for 1 hr in a shaking incubator to induce antiobiotic expression.
  3. Plate 100 l and 10 l + 90 l saline on two separate LB + Cam + Kan plates.
Line: 44 to 44
 

Day 2: Induce Gene-Gorging

Changed:
<
<
  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 g/ml Chloramphenicol (Cam). The arabinose is to induce expression of the λ Red genes from the gene-gorging plasmid.
>
>
  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 g/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.
 
  1. Grow cultures overnight at 37C, 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 l 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.
Changed:
<
<
  1. If you are gene gorging the version of the the marker you must screen for successful mutations. Plate 200 l of a 104 dilution of each culture on tetrazolium arabinose (TA), tetrazolium maltose (TM), or other suitable indicator agar containing 20 g/ml Chloramphenicol (Cam). Successful mutants will be red.
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 l of a 102 dilution on minimal arabinose (MA) or minimal maltose (MM).
>
>
  1. A. If you are gene gorging the version of the the marker you must screen for successful mutations. Plate 200 l of a 104 dilution of each culture on tetrazolium arabinose (TA), tetrazolium maltose (TM), or other suitable indicator agar containing 20 g/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 l of a 102 dilution on minimal arabinose (MA) or minimal maltose (MM).
 
  1. Grow plates overnight at 37C.

Day 4: Screen for Gene-Gorging Plasmid Loss

Revision 82010-05-09 - JeffreyBarrick

Line: 1 to 1
 
META TOPICPARENT name="ProtocolList"

Gene Gorging Marker Mutations

Line: 22 to 22
 
  • Antibiotic stock solutions of Chloramphenicol (Cam) and Kanamycin (Kan).
    • Final concentrations of antibiotics in all media (liquid or plates) are 25 g/ml Cam and 50 g/ml Kan.
Changed:
<
<
  • [[ProtocolsMediaRecipes][LB and TA plates] with antibiotic. You will need LB+Cam+Kan, TA+Cam, TA+Kan, and TA plates.
>
>
  • 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.

Revision 72010-05-09 - JeffreyBarrick

Line: 1 to 1
 
META TOPICPARENT name="ProtocolList"

Gene Gorging Marker Mutations

Line: 20 to 20
 
pJEB15 →Mal
Changed:
<
<
  • Concentrations of antibiotics in all media are 34 g/L Chloramphenicol (Cam) and ???? Kanamycin (Kan). See also Antibiotic Stock Solutions
>
>
  • Antibiotic stock solutions of Chloramphenicol (Cam) and Kanamycin (Kan).
    • Final concentrations of antibiotics in all media (liquid or plates) are 25 g/ml Cam and 50 g/ml Kan.
  • [[ProtocolsMediaRecipes][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.
 
Changed:
<
<
  • Donor Plasmid (contains desired mutation). See table.
>
>
  • 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

Changed:
<
<
  1. Transform electrocompetent cells of your parent strain with 1 l of pACBSR and 1 l of the donor plasmid for the desired marker change.
  2. Plate on LB + Cam + Kan.
>
>
  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 l of pACBSR and 1 l 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 l of room temperature SOC medium.
  6. Grow at 37C for 1 hr in a shaking incubator to induce antiobiotic expression.
  7. Plate 100 l and 10 l + 90 l saline on two separate LB + Cam + Kan plates.
 
  1. Grow plates overnight at 37C.

Day 2: Induce Gene-Gorging

Revision 62010-05-08 - JeffreyBarrick

Line: 1 to 1
 
META TOPICPARENT name="ProtocolList"
Added:
>
>
 

Gene Gorging Marker Mutations

Changed:
<
<
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.
>
>
For competitive fitness assays, one must be able to distinguish two E. coli subpopulations in a mixed culture. One way this can be accomplished is by mutating genes required for sugar fermentation to create a genetic marker (Ara, Mal, Lac, etc.). Plating a version of these strains will produce red colonies and plating the + version will produce pinkish white colonies on tetrazolium indicator agar containing a rich media base plus the sugar of interest (TA, TM, etc).
 
Changed:
<
<

General Notes

>
>
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.
 
Changed:
<
<
  • Concentrations of antibiotics in all media are 34 g/L Chloramphenicol (Cam) and Kanamycin (Kan). See also Antibiotic Stock Solutions
  • For a more detailed description of genetic markers see also: Ara Marker.
>
>
For a more detailed description of the Ara genetic marker, see also: Ara Marker.
 
Changed:
<
<

Transform Gene-Gorging and Donor Plasmid

>
>

Materials

 
Plasmid Marker
Line: 18 to 20
 
pJEB15 →Mal
Changed:
<
<
  1. Prepare electrocompetent cells of the strain in which you wish to change the genetic marker.
  2. Transform with 1 l of pACBSR (the gene-gorging plasmid) and 1 l of the donor plasmid for the desired marker change (see table).
>
>
  • Concentrations of antibiotics in all media are 34 g/L Chloramphenicol (Cam) and ???? Kanamycin (Kan). See also Antibiotic Stock Solutions
  • Electrocompetent cells of the parent strain.
  • pACBSR (the gene-gorging plasmid)
  • Donor Plasmid (contains desired mutation). See table.

Day 1: Transform Gene-Gorging and Donor Plasmid

  1. Transform electrocompetent cells of your parent strain with 1 l of pACBSR and 1 l of the donor plasmid for the desired marker change.
 
  1. Plate on LB + Cam + Kan.
  2. Grow plates overnight at 37C.
Deleted:
<
<

Induce Gene-Gorging

 
Changed:
<
<
  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 g/ml Chloramphenicol (Cam).
>
>

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 g/ml Chloramphenicol (Cam). The arabinose is to induce expression of the λ Red genes from the gene-gorging plasmid.
 
  1. Grow cultures overnight at 37C, shaking at 120 rpm.
Changed:
<
<

Screen for Desired Mutation

>
>

Day 3: Screen or Select for Desired Mutation

 
Changed:
<
<
  1. Plate 200 l of a 104 dilution of each culture on tetrazolium arabinose (TA, blue stripe) or tetrazolium maltose (TM, purple stripe) containing 20 g/ml Chloramphenicol (Cam). The dilution can be made with two 100 l transfers through dilution tubes containing 10 ml of saline. Alternately, plate 100 l of a 102 dilution on minimal arabinose (MA) or minimal maltose (MM).
>
>
  1. Make a 104-fold dilution of each overnight culture with two 100 l 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 l of a 104 dilution of each culture on tetrazolium arabinose (TA), tetrazolium maltose (TM), or other suitable indicator agar containing 20 g/ml Chloramphenicol (Cam). Successful mutants will be red.
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 l of a 102 dilution on minimal arabinose (MA) or minimal maltose (MM).
 
  1. Grow plates overnight at 37C.
Changed:
<
<

Select for Gene-Gorging Plasmid Loss

>
>

Day 4: Screen for Gene-Gorging Plasmid Loss

 
Changed:
<
<
Expected results are 0-10 white (Ara+) or red (Mal-) colonies per 200 colonies of the other color.
>
>
Expected results are 0-10 colonies with the marker change per 200 colonies of the other color.
 
Changed:
<
<
  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).
>
>
  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
 
  1. Grow cultures overnight at 37C, shaking at 120 rpm.
  2. Grow plates overnight at 37C.
Changed:
<
<

Plate to Single Colonies

>
>

Day 5: Plate to Single Colonies

 
  1. Plate 100 l 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 l transfers through dilution tubes containing 10 ml of saline.
  2. Grow plates overnight at 37C.
Changed:
<
<

Patch for Plasmid Loss

>
>

Day 6: Patch for Plasmid Loss

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

Save a Freezer Stock of the New Strain

>
>

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 37C and archive 2 × 1 ml frozen copies in 10% glycerol.
Changed:
<
<
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 in our experience.
>
>

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.
Changed:
<
<
>
>
 
META TOPICMOVED by="JeffreyBarrick" date="1273244634" from="Lab.ProceduresGeneGorgingNeutralMarkers" to="Lab.ProtocolsGeneGorgingMarker"

Revision 52010-05-07 - JeffreyBarrick

Line: 1 to 1
 
META TOPICPARENT name="ProtocolList"

Gene Gorging Marker Mutations

Line: 6 to 6
 

General Notes

Changed:
<
<
  • Concentrations of antibiotics in all media are 34 g/L Chloramphenicol (Cam) and Kanamycin (Kan)
    see also Antibiotic Stock Solutions
>
>
  • Concentrations of antibiotics in all media are 34 g/L Chloramphenicol (Cam) and Kanamycin (Kan). See also Antibiotic Stock Solutions
 
  • For a more detailed description of genetic markers see also: Ara Marker.

Transform Gene-Gorging and Donor Plasmid

Added:
>
>
Plasmid Marker
pJEB11 →Ara
pJEB12 →Ara+
pJEB15 →Mal
 
  1. Prepare electrocompetent cells of the strain in which you wish to change the genetic marker.
Changed:
<
<
  1. Transform with 1 l of pACBSR (the gene-gorging plasmid) and 1 l of pJEB12 (for Ara+) or pJEB for Mal-(the donor plasmid).
>
>
  1. Transform with 1 l of pACBSR (the gene-gorging plasmid) and 1 l of the donor plasmid for the desired marker change (see table).
 
  1. Plate on LB + Cam + Kan.
  2. Grow plates overnight at 37C.
Changed:
<
<

Induce Gene-Gorging

>
>

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 g/ml Chloramphenicol (Cam).
  2. Grow cultures overnight at 37C, shaking at 120 rpm.
Changed:
<
<

Screen for Desired Mutation

>
>

Screen for Desired Mutation

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

Select for Gene-Gorging Plasmid Loss

>
>

Select for Gene-Gorging Plasmid Loss

  Expected results are 0-10 white (Ara+) or red (Mal-) colonies per 200 colonies of the other color.
Line: 34 to 41
 
  1. Grow cultures overnight at 37C, shaking at 120 rpm.
  2. Grow plates overnight at 37C.
Changed:
<
<

Plate to Single Colonies

>
>

Plate to Single Colonies

 
  1. Plate 100 l 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 l transfers through dilution tubes containing 10 ml of saline.
  2. Grow plates overnight at 37C.
Changed:
<
<

Patch for Plasmid Loss

>
>

Patch for Plasmid Loss

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

Patch for Plasmid Loss

>
>

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.
Changed:
<
<
  1. Grow cultures overnight at 37C and archive 2 x 1 ml frozen copies in 10% glycerol.
>
>
  1. Grow cultures overnight at 37C and archive 2 × 1 ml frozen copies in 10% glycerol.
 
Changed:
<
<
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.
>
>
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 in our experience.
 
Changed:
<
<

Sources

>
>

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.

Revision 42010-05-07 - JeffreyBarrick

Line: 1 to 1
 
META TOPICPARENT name="ProtocolList"
Changed:
<
<

Gene Gorging Neutral Markers

>
>

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.
Changed:
<
<

Transform Gene-Gorging and Donor Plasmid

>
>

General Notes

 
Changed:
<
<
  1. Prepare electrocompetent cells of the strain in which you wish to change the sugar marker.
>
>
  • Concentrations of antibiotics in all media are 34 g/L Chloramphenicol (Cam) and Kanamycin (Kan)
    see also Antibiotic Stock Solutions
  • For a more detailed description of genetic markers see also: Ara Marker.

Transform Gene-Gorging and Donor Plasmid

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

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META TOPICMOVED by="JeffreyBarrick" date="1273244634" from="Lab.ProceduresGeneGorgingNeutralMarkers" to="Lab.ProtocolsGeneGorgingMarker"

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Gene Gorging Neutral Markers

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  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 g/ml Chloramphenicol (Cam).
  2. Grow cultures overnight at 37C, shaking at 120 rpm.
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Induce Gene-Gorging

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Screen for Desired Mutation

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

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Gene Gorging Neutral Markers

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

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 g/ml Chloramphenicol (Cam).
  2. Grow cultures overnight at 37C, shaking at 120 rpm.

Induce Gene-Gorging

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

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 37C, shaking at 120 rpm.
  3. Grow plates overnight at 37C.

Plate to Single Colonies

  1. Plate 100 l 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 l transfers through dilution tubes containing 10 ml of saline.
  2. Grow plates overnight at 37C.

Patch for Plasmid Loss

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

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 37C 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

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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.

 
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