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The system is customizable!!
We know how important flexibility within molecular biology is, however setting up a standard often entails rigidity. Therefore we here provide a guide on how to customize the Plug’n’Play assembly standard. All procedures only require one round of PCR and one round of USER cloning ensuring a fast construction time. The customization of the system is based on the papers by Morten Nørholm and Hansen et. al. and the work performed by the iGEM team DTU Denmark from 2009.
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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 you want to introduce mutations
• When you want to introduce deletions
• When you want to add short sequences such as an RBS, signal peptide etc.
Creating new parts
1. To create a new part you first have to design primers as you normally do it for the DNA of interest. The free-ware netprimer can assist you in finding suitable primers.
2. To make the part compatible with the Plug’n’Play assembly standard you have to determine the category of the part i.e. promoter, GOI, TS, module, or marker cassette.
3. Once you have done this you have to identify the category in the linker table to find the appropriate Plug’n’Play tails for your part. In the last column of the table, the tails that you should put in front of your forward and reverse primers can be found.
4. You simply take these 8-9 base sequences at place them at the 5’-end of your primers.
5. Next you order the primers from your favorite company. You should notice that ordering primers with a uracil incorporated increases the price of your primers, Integrated DNA Technology offers a fair price for such primers.
6. When you receive your primers you set up a PCR reaction, we recommend you to use our protocol Amplification of biobricks by PCR.
7. After checking the PCR reaction on an agarosegel you can use your new biobrick directly or if needed you can purify the PCR product by agarosegelpurification.
8. Now you are ready to assemble your favorite parts by following the protocol USER cloning.
9. Congratulations you should now have your desired construct!
Seamless assembly
1. Decide how many parts you will have to assemble scar-free. Depending on if this part constitutes a single part, several parts, or an entire device select the corresponding linkers from the linker table and put these linkers on the forward and reverse primers corresponding to your final construct.
2. To design the primers for the scar-free assembly the software PHUSER developed by the DTU iGEM team from 2009 can be used.
3. Next you order the primers from your favorite company. You should notice that ordering primers with a uracil incorporated increases the price of your primers, Integrated DNA Technology offers a fair price for such primers.
4. When you receive your primers you set up a PCR reaction, we recommend you to use our protocol Amplification of biobricks by PCR.
5. After checking the PCR reaction on an agarosegel you can use your new biobrick directly or if needed you can purify the PCR product by agarosegelpurification.
6. Now you are ready to assemble your favorite parts by following the protocol USER cloning.
7. Congratulations you should now have joined your biobricks without leaving a scar construct!
Point mutations
When introducing the mutation it is only neccessary for the primers to overlap in the complementary region (linker) and only one primer needs to carry the mutation.
Introducing point mutations in vectors
1. Define the mutation you want to introduce in your vector
2. Design primers to anneal at the site where you want to introduce the mutation by using the software PHUSER developed by the DTU iGEM team from 2009. In the primers you should introduce the desired mutation simply by changing a nucleotide to the one you wish. For one mutation you in total need two primers.
3. Next you order the primers from your favorite company. You should notice that ordering primers with a uracil incorporated increases the price of your primers, Integrated DNA Technology offers a fair price for such primers.
4. When you receive your primers you set up a PCR reaction, we recommend you to use our protocol Amplification of biobricks by PCR.
5. After checking the PCR reaction on an agarosegel you can use your new biobrick directly or if needed you can purify the PCR product by agarose gel purification.
6. Now you are ready to assemble your vector by following the protocol USER cloning.
7. Congratulations you should now have introduced a point mutation into your vector.
Introducing point mutations in a gene of interest
1. Define the mutation you want to introduce in the gene of interest (GOI)
2. Design primers to anneal at the site where you want to introduce the mutation by using the software PHUSER developed by the DTU iGEM team from 2009. In the primers you should introduce the desired mutation simply by changing a nucleotide to the one you wish. For one mutation you in total need four primers.
3. Next you order the primers from your favorite company. You should notice that ordering primers with a uracil incorporated increases the price of your primers, Integrated DNA Technology offers a fair price for such primers.
4. When you receive your primers you set up the two PCR reactions, we recommend you to use our protocol Amplification of biobricks by PCR.
5. After checking the PCR reactions on an agarosegel you can use your new biobricks directly or if needed you can purify the PCR products by agarose gel purification.
6. Now you are ready to assemble your biobricks by following the protocol USER cloning.
7. Congratulations you should now have introduced a point mutation into your GOI.
Introducing several point mutations
It is possible to introduce several point mutations in different regions of interest by customizing the Plug’n’Play assembly standard. This still only requires one round of PCR and one round of cloning, the only difference is that more PCR fragments will have to be fused. This means that the introduction of two mutations would require the fusion of three PCR fragments, and so on.
1. Define the mutations you want to introduce.
2. Design primers to anneal at the sites where you want to introduce mutations by using the software PHUSER developed by the DTU iGEM team from 2009. In the primers you should introduce the desired mutations simply by changing the nucleotides to the ones you wish.
3. Next you order the primers from your favorite company. You should notice that ordering primers with a uracil incorporated increases the price of your primers, Integrated DNA Technology offers a fair price for such primers.
4. When you receive your primers you set up the PCR reactions. We recommend you to use our protocol Amplification of biobricks by PCR.
5. After checking the PCR reactions on an agarosegel you can use your new biobricks directly or if needed you can purify the PCR products by agarose gel purification.
6. Now you are ready to assemble your biobricks by following the protocol USER cloning.
7. Congratulations you should now have introduced several point mutations.
Deletions
1. Define where you want to introduce a deletion.
2. Design primers to anneal at the sites where you want to introduce the deletion by using the software PHUSER developed by the DTU iGEM team from 2009. The primers should be designed in such a way that on the fusion point will bridge two desired sections of the gene at the matching USER linkers.
3. Next you order the primers from your favorite company. You should notice that ordering primers with a uracil incorporated increases the price of your primers, Integrated DNA Technology offers a fair price for such primers.
4. When you receive your primers you set up the PCR reactions. We recommend you to use our protocol Amplification of biobricks by PCR.
5. After checking the PCR reactions on an agarosegel you can use your new biobricks directly or if needed you can purify the PCR products by agarose gel purification.
6. Now you are ready to assemble your biobricks by following the protocol USER cloning.
7. Congratulations you should now have introduced a deletion.
Adding short sequences
A short sequence is simply added by incorporating the sequence into the forward or reverse primer. This has been illustrated with a loop in the figure below. Primers can be designed by using the standard Plug’n’Play linkers or by using the software PHUSER.
Example
Synthetic biology is all about creativity, here we show an example of what can be done when using the standard is just not enough for you! The figure below shows that you in one cloning round can assemble a degenerate linker with a non-degenerate linker (yellow), introduce a point mutation non-degenerate with a point mutation degenerate.
Linker table
Tips & Tricks
Try to design your primers such that their Tm's are within 2 degrees celicus of each other
References
Nørholm, M. H. H. A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering. BMC Biotechnol. 10, 21 (2010).
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.
https://2009.igem.org/Team:DTU_Denmark
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