Plasmid_Backbone

Part:BBa_K2429180:Design

Designed by: Tinna-Solveig F Kosoko-Thoroddsen   Group: iGEM17_MIT   (2017-10-25)


U6:LacZ Direct Repeat


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 800
    Illegal EcoRI site found at 1496
    Illegal XbaI site found at 1523
    Illegal PstI site found at 1535
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 800
    Illegal EcoRI site found at 1496
    Illegal PstI site found at 1535
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 800
    Illegal EcoRI site found at 1496
    Illegal BamHI site found at 1517
  • 23
    INCOMPATIBLE WITH RFC[23]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 800
    Illegal EcoRI site found at 1496
    Illegal XbaI site found at 1523
    Illegal PstI site found at 1535
  • 25
    INCOMPATIBLE WITH RFC[25]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 800
    Illegal EcoRI site found at 1496
    Illegal XbaI site found at 1523
    Illegal PstI site found at 1535
    Illegal NgoMIV site found at 2002
    Illegal AgeI site found at 794
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI site found at 1762
    Illegal BsaI.rc site found at 1142


Design Notes

We included the direct repeat portion in the LacZ backbone because including it in the backbone allowed us to reduce the length of the target specific portion we cloned in so it was less than 60bp (cheaper to synthesize).



Source

LacZ is an operon found in Escherichia coli that produces beta-galactosidase, which digests lactose into galactose and glucose. In the presence of beta-galactosidase, X-gal causes the production of an insoluble blue pigment, which makes the colonies blue. If a gene is successfully inserted into the LacZ gene, the LacZ gene is now non-functional and can no longer produce beta-galactosidase. This disruption of the gene means that the colonies containing inserted DNA no longer appear blue, but white. This change in color allows us to look at a golden gate transformation plate and know which colonies contain our desired sequence.


References