Team:DTU-Denmark-2/Project/PlugnplayAssembly
From 2011.igem.org
Plug 'n' Play with DNA – making molecular biology easier
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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. The new Plug 'n' play 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. The Plug `n´ Play assembly - How it works We have demonstrated successful assembly of up to six biological parts in one reaction is possible e.g. (BBa_K678068). 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.
The Procedure of assembly 1. Pre-produced PCR products are picked from the Plug `n´ Play kit. 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
Vectors for Plug `n´ Play have been prepared by PCR ensuring 100% linear fragments. This means that the occurrences of false positives due to the presence uncut vector can be completely avoided (2).
Drawbacks of the USER cassette (2): 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. To minimize mutations the X7 proof-reading polymerases (4) are used which are fully compatible with the Plug `n´ Play standard. The x7 proof-reading polymerase helps lowering the risk of introducing mutations. The concern for introduction mutations is specially regarding the gene of interest (GOI), because mutations occurring in the vector backbone or resistance cassette would in worst case scenario mean the vector might not be able to propagate. The Design of The System
The system is designed in order to assemble BioBricks from the categories promotor, a gene of interest (GOI), a targeting signal (TS), a terminator, a marker cassette and a vector backbone. The marker cassette consist of a promoter, a resistance gene, and a terminator. Ribosome binding site (RBS) is integrated in the promoter. However, a RBS can if needed be integrated in the reverse primer of the promoter when amplifying by PCR. This was the case when constructing the BioBricks and devices for the iGEM team Copenhagen.
The system is depending on custom-made overhangs on each part to be assembled. The parts with overhangs is formed by PCR amplifying the target DNA using primers designed with an upstream extension of 8-9 additional nucleotides that ends in a single deoxyuridine residue. In order to assembled multiple parts and having a flexible system the final DNA extension is preferred to consist of a adenine in the 5' end and a thymine in the 3' end.
Furthermore, the it is important that the overhangs (tails) of the different parts are not identical to ensure directionality and correct order of the biobricks. The linkers are making the whole system flexible in that it is possible use any promoter, since it will all ways end up have the same overhang generated in the PCR reaction. In the Plug `n´ Play system we are distinguishing between having a gene of interest where a targeting signal or reporter is wanted or not. Therefore, a GOI is a a gene of interest without any reporter or targeting signal, which can be assembled with a TS targeting signal or reporter. However, if the gene of interest is wanted without any reporter or targeting signal, we call it a module. A module can also include a gene of interest where the a targeting signal is included already or if it is a reporter by itself. 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. |