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The system is customizable!!
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Customization of the Plug 'n' Play with DNA assembly standard opens the door to a wide range of applications. When do you need to customize the system?
A guide for the most common design requests is given below. The guide is based on work conducted by Morten H. H. Nørholm and Hansen et. al. Creating new partsCreating new biological parts can be necessary, when they are not already included in the Plug 'n' Play kit and submitted to the Registry of Standard Biological Parts.
Seamless assemblySeamless assembly is often required when constructing fusion protein or if large genetic parts have to be assembled in pieces, e.g. when constructing complex enzymes and synthases for fungi.
Point mutationsThe introduction of point mutations can have different purposes, for instance the removal of undesired restriction site or for alteration of a proteins catalytic function. When introducing mutations it is only necessary for the primers to overlap in the complementary region (linker). Depending on whether the point mutation should be degenerate or not only one or both primers needs to carry the mutation. Degeneration means that more than one codon may code for the same amino acid. Thereby, a degenerate mutation can give different outcomes in use of tRNA's.
Introducing a point mutation with non-degenerate codons
Introducing a degenerate point mutation, which can give a different codon usage
It is also possible to introduce several point mutations in different regions by customizing the Plug 'n' Play assembly standard. This only requires one round of PCR and one round of cloning. The difference is that the PCR fragments made for each mutation will have to be fused together meaning that the introduction of two mutations would require the fusion of three PCR fragments, and so on. Deletions
With this guide a gene or part of a gene or any other desired sequence of a plasmid, device, or BioBrick can be deleted.
Adding short sequencesShort sequences such as ribosomal binding sites, kozak sequences etc. can be added by incorporating them into the primer that will be used to amplify the part it should be fused with. Introducing a short sequence can also be of interest when two proteins are wished to be connected by a linker. The linker can also carry a single point mutation, be degenerate or non-degenerate, which means that only one or both primers needs to carry the mutation. When creating a linker a short sequence of either random or known sequence is added by incorporating the sequence into the forward or reverse primers. Introducing a linker
The figure below shows the process of creating a degenerate linker (illustrated with a loop). Primers can be designed by using the standard Plug’n’Play linkers.
Illustrated below the process of creating a non-degenerate linker.
ExampleSynthetic biology is all about creativity, and sometimes the standard is just not enough, making it necessary to combine the above mentioned customizations. The figure below illustrates that "Plug 'n' Play with DNA" makes it possible to assemble a degenerate linker (red), a non-degenerate linker (yellow), introduce a non-degenerate point mutation (X) as well as a degenerate point mutation (N) into one plasmid in just ONE round of cloning.
Linker table
Tips & TricksReferences[1]Nørholm, M.H.H. A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering. BMC Biotechnol. 10, 21, 2010.[2]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. H.; “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. |
