Coding

Part:BBa_K891234

Designed by: Ryan Muller   Group: iGEM12_Arizona_State   (2012-10-02)

D168A Double Cysteine Mutant of Variola Virus Topoisomerase

Wild type topoisomerase is involved in the DNA replication fork. When helicase causes super-coiling in the replication fork, topoisomerase nicks the DNA, revolves the nicked strand to relieve super-coiling, and re-ligates the nicked DNA. This mutant version of topoisomerase alters the active site, changing the 168th amino acid residue from aspartic acid to alanine. This decreases the re-ligation rate constant, holding the mutant topoisomerase in a covalently bound complex to the 3' phosphate. Based on the melting temperature of the ssDNA, topoisomerase can peel the ssDNA probe off a template plasmid.


Safety Concerns

This biobrick does not code for any virulence factors associated with Variola virus. Topoisomerase D168A codes only for a mutant version of topoisomerase and is only toxic to E.coli cells when expressed in high concentrations.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 895
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 895
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 895
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 895
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 895
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None