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, Hansen et. al. (1). 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.
2. In accordance with the Plug’n’Play assembly system the category of the part i.e. promoter, GOI, TS, module, or marker cassette has to be determined. 3. Subsequently the linkers belonging to the category have to be identified. The appropriate linkers and Plug'n'Play tail for the part can be found in the linker table. The forward and reverse primer linkers are 8-9 bp long, and are placed at the 5’-end of the primers. 5. The primers are ordered from your favourite oligo company. Notice that the price and delivery time can be increased, when ordering primers with a uracil incorporated. DTU-Denmark-2 has had great experience with the price, delivery time and quality from Integrated DNA Technology. 6. After receiving the primers, you are ready to perform the PCR (see protocol Amplification of biobricks by PCR ). Be aware that the addition of the linker to the primer may increase the melting temperature of the primer. 7. The size of the PCR reaction are checked and compared to the expected band size on an agarose-gel. The new BioBrick can be used directly or purified by agarose-gel purification first. 8. The assembly of the new BioBricks should be performed according to the protocol USER cloning. 9. One new plasmid or linear device has now been assembled. Seamless assemblySeamless assembly is often required when constructing genes for fusion protein or if large genetic parts have to be assembled in pieces, e.g. when constructing complex enzymes and synthases in fungi.
Point mutationsPoint mutation can be used for getting rid off 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 degenerated 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 degenerated mutation can give different outcome in use of tRNA's.
Introducing a point mutation not without degeneration of codons
1. To introduce a point mutation in a vector, the mutation has to be defined first.
Introducing a degenerated point mutation, which can give different use of codons
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 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
In some cases whole deletion in parts of a gene or a whole gene can be of interest. This can be the case for creating new BioBricks or if a plasmid contains undesired parts.
Adding short sequencesIntroducing a short sequence can be of interest if two proteins is desired to be close to each other by a linker. This can increase speed of one or more coupled enzymatic reactions. The linker can as a single point mutation be degenerated or not, which means 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 primer. For both typer of linker the below process in creating the assembled plasmid is overall the same. Introducing a linker
1. When introducing a linker in the connection of a gene of interest (GOI), the first step is to define the mutation. The figure below shows the process of creating a degenerated linker (illustrated with a loop). Primers can be designed by using the standard Plug’n’Play linkers.
Illustrated below the process of creating a not degenerated 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. |