Plasmid_Backbone
K592200

Part:BBa_K592200

Designed by: Erik Gullberg, Antonio Ascue Avalos, Erik Lundin   Group: iGEM11_Uppsala-Sweden   (2011-09-21)
Revision as of 17:50, 21 September 2011 by Antonio (Talk | contribs)

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.


K592200-J04450.png

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.

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


[edit]
Categories
//dna/chromosomalintegration
//plasmidbackbone/assembly
//plasmidbackbone/copynumber/medium
//plasmidbackbone/operation
Parameters
chassis
copies10-12
functionLambda Red recombineering
mcsBioBrick
originp15A
resistanceS