Difference between revisions of "Part:BBa K592201"

Revision as of 18:03, 21 September 2011

Low to medium copy Lambda Red recombineering compatible plasmid

BBa_K592201 is a BioBrick friendly vector with low to medium copy p15A replication origin (BBa_I50032) and chloramphenicol antibiotic resistance marker, usable for Lambda Red recombineering in E coli. The E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22, and the chloramphenicol resistance marker is flanked by FRT sites so that the resistance marker can be removed after chromosomal integration using the FLP recombinase.

Usage and Biology

Recombineering (RECOMBInation-mediated genetic engiNEERING) is a method to knock out, replace, modify or insert genetic material on the bacterial chromosome or on a plasmid in vivo. This method can often be a quick and powerful alternative to traditional in vitro cloning techiques. The λ Red recombineering method uses homologous recombination proteins from phage Lambda, produced inside the bacterial cell. This method allows the integration of genetic inserts using short (40-50 bp) flanking homologies that can be added using PCR primers.

To avoid unpredictable chromosomal integration, this plasmid carries no homologies to the E coli chromosome on the region between the Lambda Red primer binding sites. When amplifying this plasmid using primers binding to these sites and with 40 nt overhangs with homologies to the chromosomal locus where the gene should be integrated, the resulting PCR product can be used for linear transformation. Any genetic construct cloned into the BioBrick site will be part of the PCR product, and will subsequently be integrated on the chromosome after transformation. Larger constructs will however lower the recombination efficiency. For more information and protocols on λ Red recombineering, see the referenced articles.

To be able to remove the antibiotic resistance marker after chromosomal integration, the resistance marker from the pKD3 plasmid (Datsenko and Wanner, 2000) was used. The resistance marker can be removed after chromosomal integration using the FLP recombinase, leaving only the plasmid insert and a scar behind. Since the FRT recombination sites contain XbaI sites, this plasmid does not comply to the RFC10 BioBrick standard, and can not be used for assembly. Instead, the assembly should be done in standard BioBrick plasmids, and then the final construct can be moved into this plasmid prior to recombineering using the EcoRI and PstI restriction enzymes.

References

[http://www.pnas.org/content/97/12/6640.short] Datsenko, K. A. and B. L. Wanner (2000). "One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products." Proc Natl Acad Sci U S A 97(12): 6640-5.

[http://www.nature.com/nprot/journal/v4/n2/full/nprot.2008.227.html] Sharan, S. K., L. C. Thomason, et al. (2009). "Recombineering: a homologous recombination-based method of genetic engineering." Nat Protoc 4(2): 206-23.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal XbaI site found at 1806
Illegal XbaI site found at 2736
12
INCOMPATIBLE WITH RFC[12]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2946
Illegal NheI site found at 1403
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NotI site found at 9
Illegal NotI site found at 2952
21
INCOMPATIBLE WITH RFC[21]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2946
23
INCOMPATIBLE WITH RFC[23]
Illegal prefix found at 2946
Illegal suffix found at 2
Illegal XbaI site found at 1806
Illegal XbaI site found at 2736
25
INCOMPATIBLE WITH RFC[25]
Illegal prefix found at 2946
Plasmid lacks a suffix.
Illegal XbaI site found at 1806
Illegal XbaI site found at 2736
Illegal XbaI site found at 2961
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal AgeI site found at 994
Illegal AgeI site found at 1317
1000
INCOMPATIBLE WITH RFC[1000]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal BsaI site found at 289

@@ Line 12: / Line 12: @@
 To avoid unpredictable chromosomal integration, this plasmid carries no homologies to the ''E coli'' chromosome on the region between the Lambda Red primer binding sites. When amplifying this plasmid using primers binding to these sites and with 40 nt overhangs with homologies to the chromosomal locus where the gene should be integrated, the resulting PCR product can be used for linear transformation. Any genetic construct cloned into the BioBrick site will be part of the PCR product, and will subsequently be integrated on the chromosome after transformation. Larger constructs will however lower the recombination efficiency. For more information and protocols on λ Red recombineering, see the referenced articles.
+To be able to remove the antibiotic resistance marker after chromosomal integration, the resistance marker from the pKD3 plasmid (Datsenko and Wanner, 2000) was used. The resistance marker can be removed after chromosomal integration using the FLP recombinase, leaving only the plasmid insert and a scar behind. Since the FRT recombination sites contain XbaI sites, this plasmid does not comply to the RFC10 BioBrick standard, and can not be used for assembly. Instead, the assembly should be done in standard BioBrick plasmids, and then the final construct can be moved into this plasmid prior to recombineering using the EcoRI and PstI restriction enzymes.
 ===References===