Composite

Part:BBa_K2842666

Designed by: Catherine Fan, Stefanie Santamaria   Group: iGEM18_UCL   (2018-10-01)
Revision as of 13:53, 17 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]

This BioBrick-compatible standard with improved flexibility enables the integration of conventional cloning methods into iGEM’s workflow. Once inserted into a backbone, BioBrickV2.0 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. BioBrickV2.0 is a new standard which can facilitate the cloning process for iGEM teams.


Blue-White Screening

The lacZ reporter allows for blue-white screening. It can be displaced by compatible constructs through BioBrick assembly, Golden Gate or Gibson assembly. Blue-white screening is based on the inability of commercial E. coli to metabolise galactose or structurally similar substrates like X-Gal because of a lacZ deletion mutation. Once lacZα is provided through a plasmid, X-Gal can be metabolised to an easily detectable blue compound. This screening can also be used for subsequent cloning where the displacement of lacZ results in white colonies.

Compatibility

For Golden Gate assembly, BsaI cut sites and defined overhangs were added. The BsaI overhangs are based on Vladimir et al. 2018. CCAA was chosen for upstream and ACGG for downstream. For Gibson assembly, a defined area of homology was designed. BsaI can be used to open the plasmid, removing the reporter and exposing the Gibson overhangs.


<img class="okay"src="T--UCL--UCL-BB2-platefig.png" alt="oops! You can't load this image." width:"50vw">

Figure 1: BioBrickV2.0 in pSB1C3 Digestion

(1) BsaI digested BioBrickV2.0 (Golden Gate Assembly)
(2) EcoRI+PstI digested BioBrickV2.0 (BioBrick Assembly)
(3) undigested BioBrickV2.0

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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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