Sauer:Plasmid-born λRed recombineering using dsDNA

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This protocol was taken (with minor modifications) from Thomason et. al. Current Protocols in Molecular Biology (2007) 1.16.1-1.16.24
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This protocol was taken (with minor modifications) from Thomason et. al. Current Protocols in Molecular Biology (2007) 1.16.1-1.16.24, the procedure can be found here: http://mrw.interscience.wiley.com/emrw/9780471142720/cp/cpmb/article/mb0116/current/pdf
 +
 
 +
the original reference can be found here:
 +
http://www.ncbi.nlm.nih.gov/pubmed/16750601
 +
 
Using short (35-40 bp) regions of homology, bacteriophage-encoded recombination machinery allows for the efficient insertion or deletions in gram-negative bacterial chromosomes without regard to restriction sites.  The Court lab has extended the "Wanner" method by placing the plasmid-born bacteriophage recombination functions under the control of a temperature sensitive allele lambda repressor (cI857).  At low temperatures (30-34 C) the downstream genes are tightly repressed however following a temperature shift to 42 C, these genes are expressed at high levels from the lambda pL promoter.  Following induction of the recombination functions, transformed dsDNA is efficiently recombined into the chromosome and recombinants identified by either selection or PCR based screening.  Some of the Court lab plasmids carry a temperature sensitive origin allowing for facile plasmid curing by simply shifting to the non-permissive temperature (37) and omitting the selection marker (chloramphenicol in the case of pSIM5).
Using short (35-40 bp) regions of homology, bacteriophage-encoded recombination machinery allows for the efficient insertion or deletions in gram-negative bacterial chromosomes without regard to restriction sites.  The Court lab has extended the "Wanner" method by placing the plasmid-born bacteriophage recombination functions under the control of a temperature sensitive allele lambda repressor (cI857).  At low temperatures (30-34 C) the downstream genes are tightly repressed however following a temperature shift to 42 C, these genes are expressed at high levels from the lambda pL promoter.  Following induction of the recombination functions, transformed dsDNA is efficiently recombined into the chromosome and recombinants identified by either selection or PCR based screening.  Some of the Court lab plasmids carry a temperature sensitive origin allowing for facile plasmid curing by simply shifting to the non-permissive temperature (37) and omitting the selection marker (chloramphenicol in the case of pSIM5).
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Primer Design:
+
'''Primer Design:'''
5' to 3':
5' to 3':
40 nt of homology targeting the chromosomal region of interest
40 nt of homology targeting the chromosomal region of interest
20 nt which bind the the cassette to be amplified (i've often use the b.b. prefix ans suffix for this region allowing me to knock in a variety of constructs to the same chromosomal locus)
20 nt which bind the the cassette to be amplified (i've often use the b.b. prefix ans suffix for this region allowing me to knock in a variety of constructs to the same chromosomal locus)
-
PCR:
+
'''PCR:'''
Due to the size of the primers i typically anneal at 57-60 although when this is problematic I will included 2 rounds with lower annealing tempatures at the beginning of the reaction.
Due to the size of the primers i typically anneal at 57-60 although when this is problematic I will included 2 rounds with lower annealing tempatures at the beginning of the reaction.
ex:
ex:
92
92
-
92-\
 
-
48---2x
 
-
72-/
 
-
92-\
+
92-
-
58---28x
+
{|
-
72-/
+
|-
 +
|92
 +
|-
 +
|48
 +
|2 rounds
 +
|-
 +
|72
 +
|
 +
|}
 +
 
 +
 
 +
{|
 +
|-
 +
|92
 +
|-
 +
|57
 +
|28 rounds
 +
|-
 +
|72
 +
|
 +
|}
 +
 
72
72
 +
 +
 +
'''Preparing Electrocompetant Cells:'''
 +
Grow a strain carrying one of the recombineering plasmids (pSIM5 for example) o/n at 30 C with antibiotic (chloramphenicol)
 +
 +
Diluted this at least 50 fold into a 50 mL culture and grow with antibiotic to an O.D. of 0.4-0.6 again at 30 C (you may want to also do this with a second sample that will not undergo the heat treatment). 
 +
 +
Induce the recombinase function by moving the flask to a 42 C shaker and shake for 15 min.
 +
 +
Rapidly cool the flask in an water/ice slurry for at least 5 mins.
 +
 +
Transfer the culture to a pre-cooled centrifuge tube and spin for 7 min at 4600xg.
 +
 +
Pour off supernatant and resuspend in 30 mL of cold distilled water.
 +
 +
Repeat this twice more (3 spins in total)
 +
 +
Resuspend final pellet in 250 uL ice-cold 10% glycerol (this should be enough for 4-5 transformations) and save at -80.  The freeze thaw cycle will reduce the transformation efficiency so if you are doing all of your transformations immediately you can resuspend in water.
 +
 +
'''Electroporation:'''
 +
You will likely want to electroporate both the induced and uninduced samples with your PCR product and select on appropriate plates.   
 +
If the uninduced and induced have roughly equal numbers of colonies the resistance may not be the result of recombination.  I have often had problems with contaminating plasmid from the PCR product gel extraction - a DPNI digest will help with this.
 +
 +
""Recovery and Plating""
 +
After electroporation, recover your cells in a supplemented, rich medium (such as SOC) for either 1 hour at 37 C or 2 hours at 30 C (ensures recombination system is off).  Harvest the cells, remove all but ~100 uL of medium, resuspend the culture, and plate half on selectable medium. Keep the remainder at room temp over night.  We usually recover the plates at 30 C over night, then transfer the plates to 37 for the remainder of the next day. Re-streak from a few colonies to obtain isolated colonies derived from it. Screen these re-streaks.  If there are no colonies, plate the second half of the culture.
 +
 +
'''Verification:'''
 +
PCR can be used to verify the correct insertion.  Use primers that flank the insertion/modification site so you can detect either wild-type or the recombinant.  We have found that mutating essential genes can give merodiploidy and both wt and recombinant versions will be present in the cells.

Current revision

This protocol was taken (with minor modifications) from Thomason et. al. Current Protocols in Molecular Biology (2007) 1.16.1-1.16.24, the procedure can be found here: http://mrw.interscience.wiley.com/emrw/9780471142720/cp/cpmb/article/mb0116/current/pdf

the original reference can be found here: http://www.ncbi.nlm.nih.gov/pubmed/16750601


Using short (35-40 bp) regions of homology, bacteriophage-encoded recombination machinery allows for the efficient insertion or deletions in gram-negative bacterial chromosomes without regard to restriction sites. The Court lab has extended the "Wanner" method by placing the plasmid-born bacteriophage recombination functions under the control of a temperature sensitive allele lambda repressor (cI857). At low temperatures (30-34 C) the downstream genes are tightly repressed however following a temperature shift to 42 C, these genes are expressed at high levels from the lambda pL promoter. Following induction of the recombination functions, transformed dsDNA is efficiently recombined into the chromosome and recombinants identified by either selection or PCR based screening. Some of the Court lab plasmids carry a temperature sensitive origin allowing for facile plasmid curing by simply shifting to the non-permissive temperature (37) and omitting the selection marker (chloramphenicol in the case of pSIM5).

Primer Design: 5' to 3': 40 nt of homology targeting the chromosomal region of interest 20 nt which bind the the cassette to be amplified (i've often use the b.b. prefix ans suffix for this region allowing me to knock in a variety of constructs to the same chromosomal locus)

PCR: Due to the size of the primers i typically anneal at 57-60 although when this is problematic I will included 2 rounds with lower annealing tempatures at the beginning of the reaction. ex: 92


92-

92
48 2 rounds
72


92
57 28 rounds
72


72


Preparing Electrocompetant Cells: Grow a strain carrying one of the recombineering plasmids (pSIM5 for example) o/n at 30 C with antibiotic (chloramphenicol)

Diluted this at least 50 fold into a 50 mL culture and grow with antibiotic to an O.D. of 0.4-0.6 again at 30 C (you may want to also do this with a second sample that will not undergo the heat treatment).

Induce the recombinase function by moving the flask to a 42 C shaker and shake for 15 min.

Rapidly cool the flask in an water/ice slurry for at least 5 mins.

Transfer the culture to a pre-cooled centrifuge tube and spin for 7 min at 4600xg.

Pour off supernatant and resuspend in 30 mL of cold distilled water.

Repeat this twice more (3 spins in total)

Resuspend final pellet in 250 uL ice-cold 10% glycerol (this should be enough for 4-5 transformations) and save at -80. The freeze thaw cycle will reduce the transformation efficiency so if you are doing all of your transformations immediately you can resuspend in water.

Electroporation: You will likely want to electroporate both the induced and uninduced samples with your PCR product and select on appropriate plates. If the uninduced and induced have roughly equal numbers of colonies the resistance may not be the result of recombination. I have often had problems with contaminating plasmid from the PCR product gel extraction - a DPNI digest will help with this.

""Recovery and Plating"" After electroporation, recover your cells in a supplemented, rich medium (such as SOC) for either 1 hour at 37 C or 2 hours at 30 C (ensures recombination system is off). Harvest the cells, remove all but ~100 uL of medium, resuspend the culture, and plate half on selectable medium. Keep the remainder at room temp over night. We usually recover the plates at 30 C over night, then transfer the plates to 37 for the remainder of the next day. Re-streak from a few colonies to obtain isolated colonies derived from it. Screen these re-streaks. If there are no colonies, plate the second half of the culture.

Verification: PCR can be used to verify the correct insertion. Use primers that flank the insertion/modification site so you can detect either wild-type or the recombinant. We have found that mutating essential genes can give merodiploidy and both wt and recombinant versions will be present in the cells.

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