Difference between revisions of "Part:BBa K592200"
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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. | 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 | + | 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 lower the recombination efficiency. For more information and protocols on λ Red recombineering, see the referenced articles below. |
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| + | Using this plasmid for chromosomal integration of BioBrick constructs will leave a spectinomycin resistance cassette on the chromosome. In most circumstances this is of little importance, since BioBrick plasmids normally do not use spectinomycin resistance as a selection marker. If this should be a problem, there are two other Lambda Red recombineering compatible plasmids, <partinfo>BBa_K592201</partinfo> and <partinfo>BBa_K592202</partinfo>, carrying chloramphenicol or kanamycin markers flanked by FRT sites. These resistance cassettes can be removed after chromosomal integration using the FLP recombinase, leaving only the plasmid insert and a scar behind. | ||
===References=== | ===References=== | ||
Revision as of 18:19, 21 September 2011
Low to medium copy BioBrick standard vector
BBa_K592200 is a BioBrick standard vector with low to medium copy p15A replication origin (BBa_I50032) and spectinomycin 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.
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 lower the recombination efficiency. For more information and protocols on λ Red recombineering, see the referenced articles below.
Using this plasmid for chromosomal integration of BioBrick constructs will leave a spectinomycin resistance cassette on the chromosome. In most circumstances this is of little importance, since BioBrick plasmids normally do not use spectinomycin resistance as a selection marker. If this should be a problem, there are two other Lambda Red recombineering compatible plasmids, BBa_K592201 and BBa_K592202, carrying chloramphenicol or kanamycin markers flanked by FRT sites. These resistance cassettes can be removed after chromosomal integration using the FLP recombinase, leaving only the plasmid insert and a scar behind.
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 3116
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 3122 - 21INCOMPATIBLE WITH RFC[21]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 3116 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found at 3116
Illegal suffix found at 2 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found at 3116
Plasmid lacks a suffix.
Illegal XbaI site found at 3131
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NgoMIV site found at 1969
Illegal AgeI site found at 994
Illegal AgeI site found at 1317 - 1000INCOMPATIBLE WITH RFC[1000]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal BsaI site found at 289