Difference between revisions of "Part:BBa K2842666"
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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.
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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===
 
===Blue-White Screening===
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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.
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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.  
  
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===Compatibility===
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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.
  
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[[File:T--UCL--FB gelfig.png|400px|thumb|left|
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<center>'''Figure 1: Final Brick and BBa_I732902 cloning comparison'''</center>
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[[File:T--UCL--UCL-FB-gelfig.png|400px|thumb|left|
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<center>'''Figure 1: BioBrickV2.0 in pSB1C3 Digestion'''</center>
  
 
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Revision as of 13:42, 17 October 2018


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.


Figure 1: BioBrickV2.0 in pSB1C3 Digestion

(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