Part:BBa_K592200

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 backbone sequence is based on pSB3T5, but theE 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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE 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
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3116
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 3116
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE 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
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 289