Composite

Part:BBa_K2842666

Designed by: Catherine Fan, Stefanie Santamaria   Group: iGEM18_UCL   (2018-10-01)
Revision as of 17:42, 16 October 2018 by Stoffifee (Talk | contribs)


Golden Gate compatible system with blue-white screening

Flexible Cloning System
Function Standardised blue-white screening
Use in E. coli cells
Chassis Tested DH5α cells
Abstraction Hierarchy Composite Device
Related Device BBa_I732902
RFC standard RFC10,RFC12,RFC21,RFC23 & RFC25 compatible
Backbone pSB1C3
Submitted by [http://2018.igem.org/Team:UCL UCL iGEM 2018]

While developing our modular platform, we conceived a BioBrick-compatible standard with improved flexibility that enables the integration of conventional cloning methods into iGEM’s workflow. This construct, once inserted in a backbone allows cloning through Golden Gate assembly and Gibson assembly. At the same time, our construct has a LacZ reporter which can be used to screen plates for successful colonies. Our final brick is supposed to be a BiobrickV2.0 which could facilitate the cloning process for iGEM teams.


Blue-White Screening

The commercial E. coli we used have a LacZomega in their genomes that is not enough to metabolise galactose. Providing the alpha fragment through a plasmid enables the strains to metabolise galactose or structurally similar substrates like X-Gal. In the case of X-Gal, once the colourless compound is metabolised, it generates a blue compound that is easily detectable. The result is that colonies containing plasmids with LacZalpha (plated in media containing X-Gal) are blue. We used that as a screen to detect successful cloning of this block and for its displacement as we clone our bricks.


Figure 1: Final Brick and BBa_I732902 cloning comparison

(1) BsaI digested BBa_I732902 (Golden Gate)
(2) BsaI digested Final Brick (Golden Gate)
(3) EcoRI+PstI digested BBa_I732902 (BioBrick Assembly)
(4) EcoRI+PstI digested Final Brick (BioBrick Assembly)
(5) undigested BBa_I732902
(6) undigested Final Brick





























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Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 647
    Illegal BsaI.rc site found at 28


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