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Team:DTU-Denmark-2/Project/PlugnplayAssembly
From 2011.igem.org
Making Molecular Biology Easier
In 2009 the first team from DTU to participate in iGEM introduced the USER fusion Assembly standard (BBF RFC 39). This year we introduce a more standardized version allowing easier use of the system.
We think that iGEM should be about assembling biobricks fast and combining them in any thinkable way with existing or new parts. Unfortunately classical cloning techniques can cause problems and even PCR can be cumbersome if you have little or no laboratory experience.
We therefore introduce a simple and fast way of building new devices or whatever you want with ready to use PCR products. All you have to do is to select your favorite bricks, mix them with the ready to use destination vector and 70 minutes later you are ready to transform your competent E. coli cells. No need to worry if your destination vector has been fully linearized, no need to perform site-directed mutagenesis to remove unwished restriction sites, just simple, fast, and easy. We have demonstrated the successful assembly of up to six biological parts in one reaction, reducing the plasmid construction time significantly. The upper limit of fragments that efficiently can be assembled has not been delineated (1). Due to the 8-9 basepair overhangs the parts are ligated without the use of DNA-ligases.
Plug'n'Play benefits
Improvements of BBF RFC 39
• No need to use a USER cassette, thereby completely avoiding the use of restriction enzymes.
• Standardized linkers
What you get
• Rapid assembly of biological parts
• A well documented system
• Assembly of multiple biobricks in one step
• No illegal restrictionsites
• 100% restriction enzyme free
• 100% DNA ligase free
• High fidelity ensured by PfuTurbo® 7x Hotstart DNA polymerase
• Directionality of inserts are supported
• Any vector can be made Plug’n’Play compatible
• Suitable for large scale high throughput projects
Designing the system
When designing the system, the first thing we considered was which types of biobricks a device or other construct would consist of. We decided that the most systems would at least consist of a vector backbone, promoter, a gene of interest (GOI), a terminater, and a marker cassette.
For all these parts to be assembled by USER cloning it was important that the overhangs (tails) of the different parts were not identical. Designing a different tail for each end of a part was important to ensure directionality and correct order of the biobricks.
