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 and vectors 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.

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 standard can be found here (BBF RFC 80).

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

Plug 'n' Play benefits

Improvements of BBF RFC 39

What you get

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):
• 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 inflexible
• 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. For all the PCR reactions it is important to remove the template vector, to ensure assembly in USER reaction and avoid inauspicious transformation and false positive. The template can simply be removed by DpnI treatment (2). However, a gel band purification was instead performed, when preparing the vector to further ensure a high degree a purification of our BioBricks.

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.

Illustrated below, the sequence of linkers between the parts, which are generated from the overhangs(tail) on the PCR products.

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, B.G.; Salomonsen, B.; Nielsen, M. T.; Nielsen, J. B.; Hansen, N. B.; Nielsen, K. F.; Regueira, T. B.; Nielsen, J.; Patil, K. R.; Mortensen, U. F.; “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) Hansen, B. G.; Holm, D. K.; Nielsen M.T.; Mortensen, U.H. PCR based USER cloning for restriction enzyme and ligase-independent vector construction. Manuscript.

(3) Frandsen, R. J. N.; Andersson, J.A.; Kristensen, M. B.; Giese, H. 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).