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?
• When a desired biological part is not yet a part of the Plug'n'Play with DNA kit
• When seamless assembly is required (e.g. protein fusion)
• When the introduction of mutations are required.
• When the introduction of deletions are required.
• When short sequences such as linkers, RBSs, signal peptides etc. are required.
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 parts
Creating 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.
1. To create a new part, forward and reverse primers have to be designed for the DNA of interest. The free-ware netprimer is an excellent tool for finding suitable primers.
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 assembly
Seamless 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.
1. If you want to design a construct where all parts are seamless assembled go directly to step 2. Otherwise the standard linkers should be added to the parts that do not need to be fused seamless according to the linker table. For more details see the 'creating new parts' section.
2. To design the primers for the seamless assembly the software PHUSER can be used. This software was created by the DTU iGEM team from 2009.
3. The primers are ordered from your favourite 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, quality and delivery time for primers from Integrated DNA Technology.
4. To amplify the BioBricks the protocol Amplification of biobricks by PCR can be used.
5. Check the PCR reaction on an agarose-gel before assembly. The new BioBricks can be used directly or purified by agarose-gel purification.
6. The assembly of new BioBricks are executed according to the protocol USER cloning.
7. A scar free BioBrick device or plasmid have been assembled.
Point mutations
Point 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
Both primers have to carry the desired mutation to make sure that the codon and the resulting amino acid are generated by the mutation.
1. To introduce a point mutation in a vector, the mutation has to be defined first.
2. Primers are designed for the defined mutation site. For introduction of the desired mutation one nucleotide is changed. Two primers are used per mutation.
3. The primers are ordered from your favourite 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 primers from Integrated DNA Technology.
4. To amplify the BioBricks the protocol Amplification of biobricks by PCR is used.
5. Check the PCR reaction on an agarosegel before assembly. The new BioBricks can be used directly or purified by agarose-gel purification if needed.
6. The assembly of new BioBricks parts are performed according to the protocol USER cloning.
7. A BioBrick, device or plasmid with the desired point mutation has been assembled.
Introducing a degenerated point mutation, which can give different use of codons
Only one primer have to carry the desired point mutation. The procedure of assembly is the same as for not degenerated point mutation above.
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.
1. First the deletion site is defined.
2. The primers need to be designed so the fusion point will bridge two desired sections of the gene at the matching USER linkers.
3. The primers are ordered from your favourite 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 primers from Integrated DNA Technology.
4. To amplify the BioBricks the protocol Amplification of biobricks by PCR is used.
5. Check the PCR reaction on an agarosegel before assembly. The new BioBricks can be used directly or purified by agarose-gel purification if needed.
6. The assembly of new BioBricks parts are performed according to the protocol USER cloning.
7. A BioBrick, device or new plasmid with the deletion has been assembled.
Adding short sequences
Introducing 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.
2. Primers are designed for the defined mutation site. To introduce the desired mutation one nucleotide are changed. Four primers are used per mutation.
3. The primers are ordered from your favourite 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 primers from Integrated DNA Technology.
4. To amplify the BioBricks the protocol Amplification of biobricks by PCR is used.
5. Check the PCR reaction on an agarosegel before assembly. The new BioBricks can be used directly or purified by agarose-gel purification if needed.
6. The assembly of new BioBricks parts are performed according to the protocol USER cloning.
7. A BioBrick with a mutation in GOI has been assembled.
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.
Example
Synthetic 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 & Tricks
Design primers so their mutual Tm's are within 2-5°C.
Remember that the annealing temperature should normally be 3°C under the Tm. However, when the USER extension is add on the primer and the Tm is increased. We have experience that the best results are obtain when the final annealing temperature is above 62°C, if the primer was around the 59°C to begin with.
The X7 Phusion polymerase have in the PCR reaction a extension time of 1000bp per minut.
References
[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.
|
"
