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From 2011.igem.org

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• The Project
  • Abstract
  • Introduction
  • The Plug 'n' Play
    • Assembly system
    • Customization
  • Other assembly systems
  • Achievements
  • Attributions
• Results
  • Background
  • Characterization
  • Proof of concept
    • Mammalian Cells
    • Fungi
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	1 When do you need to customize the system? 2 Seamless assembly 3 Point mutations 4 Deletions 5 Adding short sequences 6 Example 7 Linker table 8 Tips & Tricks 9 References		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 introducing of mutation is required. • When the introducing of deletion is required. • When a short sequences such as an linker, RBS, signal peptide etc are required. Creating new parts 1. To create a new part, primers have to be designed as normally performed for the DNA of interest. The free-ware netprimer can assist in finding suitable primers. 2. To make the part compatible with the standard Plug’n’Play assembly system, the category of the part i.e. promoter, GOI, TS, module, or marker cassette have to be determined. 3. Subsequently the linker of the category have to be identified. The appropriate linker and Plug 'n' Play tails for the part can be found in the Linker table. The forward and reverse primer tail is listed, and just have to be added in front of the primers found in the 1. point. 4. These 8-9 base sequences are simply placed at the 5’-end of the primers. 5. The primers are ordered from the favourite company of oligos. Notice that ordering primers with a uracil incorporated increases the price of primers and longer delivery time. Integrated DNA Technology offers a fair price for such primers and deliver in two days. 6. When primers are received the PCR reaction can be set up. Usage of the protocol Amplification of biobricks by PCR should ensure results. Furthermore, be aware that the primer extension will increase the annealing temperature. 7. Check the PCR reaction on an agarose-gel according to expected size. The new BioBrick can be used directly or purified by agarose-gel purification before usage. 8. The assembly of new BioBricks parts are executed with the protocol USER cloning. 9. One new vector have been assembled. Seamless assembly 1. Depending on how many scar free parts wished to be assembled, the correspondent linkers can be found in the Linker table. The linkers are placed as extension on the forward and reverse primers for the parts. 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. The primers are ordered from the favourite company of oligos. Notice that ordering primers with a uracil incorporated increases the price of primers and longer delivery time. Integrated DNA Technology offers a fair price for such primers and deliver in two days. 4. To amplify the BioBricks the protocol Amplification of biobricks by PCR are used. 5. Check the PCR reaction on an agarosegel before assembly. The new BioBricks can be used directly or purified by agarosegelpurification if needed. 6. The assembly of new BioBricks parts are executed with the protocol USER cloning. 7. Now, one new scar free BioBrick have been assembled. 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. To introduce a mutation in a vector, the mutation has to be defined first. 2. The software PHUSER developed by the DTU iGEM team 2009, can be used to easily design primers for the defined mutation site. To introduce the desired mutation one nucleotide are changed. Two primers are used per mutation. 3. The primers are ordered from the favourite company of oligos. Notice that ordering primers with a uracil incorporated increases the price of primers and longer delivery time. Integrated DNA Technology offers a fair price for such primers and deliver in two days 4. To amplify the BioBricks the protocol Amplification of biobricks by PCR are used. 5. Check the PCR reaction on an agarosegel before assembly. The new BioBricks can be used directly or purified by agarosegelpurification if needed. 6. The assembly of new BioBricks parts are executed with the protocol USER cloning. 7. Now, one new BioBrick with mutation in vector have been assembled. Introducing point mutations in a gene of interest 1. To introduce mutation in a gene of interest (GOI), the mutation are first defined. 2. The software PHUSER developed by the DTU iGEM team 2009, can be used to easily design primers 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 the favourite company of oligos. Notice that ordering primers with a uracil incorporated increases the price of primers and longer delivery time. Integrated DNA Technology offers a fair price for such primers and deliver in two days. 4. To amplify the BioBricks the protocol Amplification of biobricks by PCR are used. 5. Check the PCR reaction on an agarosegel before assembly. The new BioBricks can be used directly or purified by agarosegelpurification if needed. 6. The assembly of new BioBricks parts are executed with the protocol USER cloning. 7. Now, one new BioBrick with mutation in GOI have been assembled. 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. To introduce more than one mutation in different regions, the mutations are first defined. 2. The software PHUSER developed by the DTU iGEM team 2009, can be used to easily design primers for the pre defined mutations site. To introduce the desired mutations one nucleotide are changed in the primer for each mutation site. 3. The primers are ordered from the favourite company of oligos. Notice that ordering primers with a uracil incorporated increases the price of primers and longer delivery time. Integrated DNA Technology offers a fair price for such primers and deliver in two days. 4.To amplify the BioBricks the protocol Amplification of biobricks by PCR are used. 5. Check the PCR reaction on an agarosegel before assembly. The new BioBricks can be used directly or purified by agarosegelpurification if needed. 6. The assembly of new BioBricks parts are executed with the protocol USER cloning. 7. Now, one new BioBrick with mutations in several regions have been assembled. Deletions 1. To introduce deletion, first define the deletion site. 2. The software PHUSER developed by the DTU iGEM team from 2009. The design of primers has to be done so the fusion point will bridge two desired sections of the gene at the matching USER linkers. 3. The primers are ordered from the favourite company of oligos. Notice that ordering primers with a uracil incorporated increases the price of primers and longer delivery time. Integrated DNA Technology offers a fair price for such primers and deliver in two days. 4.To amplify the BioBricks the protocol Amplification of biobricks by PCR are used. 5. Check the PCR reaction on an agarosegel before assembly. The new BioBricks can be used directly or purified by agarosegelpurification if needed. 6. To amplify the BioBricks the protocol Amplification of biobricks by PCR are used. 7. Now, one new BioBrick with deletion have been assembled. 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, and sometimes the standard is just not enough making it necessary to combine the above mentioned customizations. The figure below illustrates, that it is possible in only one cloning round to assemble a degenerate linker (green), a non-degenerate linker (yellow), introduce a non-degenerate point mutation (green) as well as a degenerate point mutation(yellow) to one plasmid. Linker table Tips & Tricks Design primers so their mutual Tm's are within 2°C. 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