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Plug `n´ Play with DNA – making molecular biology easier
We imagine that iGEM and synthetic biology should be about assembling BioBricks fast. It should be easy to combine any thinkable part, device or existing BioBrick. Unfortunately, classical cloning techniques can cause problems and even PCR can be cumbersome, if you have little or no laboratory experience.
Therefore, we introduce a simple and fast way of building new devices, vectors or whatever preferred with ready to use PCR products. All what is needed is to select the favourite BioBricks and one of the ready to use destination vector, and mix it with USER enzyme.The assembled vector is ready for transformation in competent E. coli cells only 70 minutes later.
No need to worry if the destination vector has been fully linearized, no need to perform site-directed mutagenesis to remove unwished restriction sites, just simple, fast, and easy.
The new assembly system
Successful assembly of up to six biological parts in one reaction is possible, 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.
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.
The Plug `n´ Play assembly - How it works
We have demonstrated successful assembly of up to six biological parts in one reaction is possible (links). The entire process of combining 6 BioBricks are illustrated in the figure below. The number of BioBricks can be adjusted according to preferred as well as introducing mutation see customization (link).
The Procedure of assembly
2. USER enzyme mix and buffer is added. This will remove the uracil, making sticky end overhangs on all BioBricks. All BioBricks and backbone plasmid have matching sticky ends, and the BioBricks will self-assemble in the plasmid.
3. Transformation in E. coli. Reduced incubation of the transformation mix is obtained when using ampicillin as antibiotic resistance marker.
If a part is not incluede in the Plug `n´ Play kit a simple PCR amplification can performed on the part or BioBricks intended for the assembly. The PCR product should subsequently be gel band purified.
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
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 encountered 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).
