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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
Vectors for Plug’n’Play have been prepared by PCR ensuring 100% linear fragments. This means that the occurences of false positives due to the presence uncut vector can be completely avoided (2).
Drawbacks of the USER cassette (2):
• The USER cassette has to be inserted into the vector of choice, which has to be done by classical cloning.
• Fragments can only be inserted at the site of the USER cassette making it unflexible
• The USER cassette has to be digested by a restriction endonuclease and a nicking enzyme
The USER compatible backbone vectors were made by PCR amplification with primers containing the standardized linkers 6 and 1. The template vector can simply be removed by DpnI treatment (2), in preparing the vectors we did however skip this step without problems. Following gel band purification was performed.
Previous studies have demonstrated a high level of correct assembly and directionality of fragments (2,3) and although we did not make such analysis ourselves it was in general our perception that this was the case, of the more than 25 different Plug’n’Play assemblies we performed we encounterd no more than 2 false-positives.
It is well known that mutations can be introduced during PCR, so making vectors by PCR amplification might not be the smartest thing? To minimize mutations the X7 proof-reading polymerases (4) are used which are fully compatible with the Plug’n’Play standard, this polymerase helps lowering the risk of introducing mutations. The things to be most concerned about is the introduction of mutations into the gene of interest because mutations in the vector worst case scenario would mean that the vector would not be able to propagate.
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.
References
(1) Hansen, Bjarke G.; Bo Salomonsen; Morten T. Nielsen; Jakob B. Nielsen; Niels B. Hansen; Kristian F. Nielsen; Torsten B. Regueira; Jens Nielsen; Kiran R. Patil; and Uffe H. Mortensen; “Versatile enzyme expression and Characterization system for Aspergillus, with the Penicillium brevicompactum Polyketide Synthase Gene from the Mycophenolic Acid Gene Cluster as a Test Case.” American Society for Microbiology, 2011, 3044-3051.
(2) Bjarne Gram Hansen, Dorte Koefoed Holm, Morten Thrane Nielsen and Uffe Hasbro Mortensen. PCR based USER cloning for restriction enzyme and ligase-independent vector construction. Manuscript.
(3) Rasmus JN Frandsen, Jens A Andersson, Matilde B Kristensen and Henriette Giese. Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi.
BMC Molecular Biology 2008, 9:70.
(4) Nørholm, M. H. H. A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering. BMC Biotechnol. 10, 21 (2010).
