Plasmid

Part:BBa_K617000:Design

Designed by: Will Jones   Group: iGEM11_UIUC-Illinois   (2011-09-27)

Biobrick Compatible Lambda Chromosomal Insertion Plasmid


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 2398
    Illegal suffix found in sequence at 2420
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2398
    Illegal NheI site found at 273
    Illegal SpeI site found at 2421
    Illegal PstI site found at 2435
    Illegal NotI site found at 1102
    Illegal NotI site found at 2404
    Illegal NotI site found at 2428
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2398
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 2398
    Illegal suffix found in sequence at 2421
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 2398
    Illegal XbaI site found at 2413
    Illegal SpeI site found at 2421
    Illegal PstI site found at 2435
    Illegal NgoMIV site found at 1318
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2129
    Illegal SapI site found at 1258
    Illegal SapI site found at 1468


Design Notes

This plasmid allows the user to integrate his/her synthetic circuit, constructed via biobricks, into the Lambda attB site of an E. coli pir- strain. The integration will be at a single copy number.

This part is a biobrick modified Lambda CRIM Vector. It is based off the Lambda CRIM vector pAH125 from Barry Wanner's laboratory at Purdue University (Haldimann, Wanner 2001). See http://www.ncbi.nlm.nih.gov/nuccore/16209194 for the sequence of pAH125.

K617000 is analogous to pAH125 except it's MCS is replaced with a biobrick cloning site and the lacZ ORF is deleted.

The first reference describes the protocol for use of this plasmid, it can also be found on our website.

NOTE: K617000 can only be propagated in a pir+ or pir-116 strain.

Source

K617000 is the pcr product of a pAH125 template. The pAH125 template is originally from Dr.Haldimann and Professor Wanner (2001) at Purdue University. Primers were used to amplify the functional region of pAH125 and excluded the original multiple cloning site and lacZ ORF. The primers contained overlaps, which formed a biobrick site upon SpeI digestion and an intramolecular ligation reaction. See our website for complete details of how this part was made.

References

1. Haldimann, A., Wanner, B. L. 2001. Conditional-Replication, Integration, Excision, and Retrieval Plasmid-Host Systems for Gene Structure-Function Studies of Bacteria. Journal of Bacteriology. 183:21 6384-6393. 2. Metcalf, W. W., Weihong, J., Wanner, B. L. 1994. Use of the rep technique for allele replacement to construct new Escherichia coli hosts for maintenance of R6Kϒ origin plasmids at different copy numbers. Gene. 138: 1-7.