Difference between revisions of "Part:BBa K2842666"
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| − | ===Blue | + | ===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. | 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. | ||
Revision as of 17:42, 16 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] |
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.
(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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 647
Illegal BsaI.rc site found at 28