Difference between revisions of "Part:BBa K2842666"

Revision as of 00:01, 16 October 2018

Golden Gate compatible system with blue-white screening

Flexible Cloning System
Function	Standardised blue/white measurement
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

-

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

@@ Line 29: / Line 29: @@
 |[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.