Team:DTU-Denmark-2/Project/PlugnplayAssembly/customization

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Revision as of 23:38, 19 September 2011

The system is customizable!!

When do you need to customize the system?

intro text!!

• 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

Creating new parts biological part can be necessary, when they are not included in the Plug 'n' Play kit and submitted to the Registry of Standard Biological Parts. This will also be the case when expanding the Plug 'n' Play system.

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 plasmid or device have been assembled.

Seamless assembly

Seamless assembly is often needed when working with fusion protein or if a part have to be assembled in pieces if it is of a large size. This can be the case when have working with complex enzymes and synthases from fungi.

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 agarose-gel 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 executed with the protocol USER cloning.
7. Now, one new scar free BioBrick device or plasmid have been assembled.

Point mutations

Point mutation can be of request if a undesired restriction site is present or if alteration of a proteins catalytic function is desired.
When introducing mutations it is only necessary for the primers to overlap in the complementary region (linker). Depending on 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 giving degeneration of codons
Both primers have to carry the desired mutation, which make sure the codon and the amino acid generated by the mutation.

1. To introduce a point 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, device or plasmid with the point mutation in vector have been assembled.

Introducing a degenerate 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 generated point mutation above.

It is also possible to introduce several point mutations in different regions of interest 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. This means 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 containing a undesired parts.

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. The assembly of new BioBricks parts are executed with the protocol USER cloning.
7. Now, one new BioBrick, device or new plasmid with the deletion have 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. For creatin a linker a short sequence of either random or known sequence is simply 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. To introduce linker in the conection of a gene of interest (GOI), the mutation are first defined.
2. The software PHUSER developed by the DTU iGEM team from 2009 can be used.
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 agarose gel 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 executed with the protocol USER cloning.
7. Now, one new BioBrick with mutation in GOI have been assembled.

Illustrated below the process of creating a degenerated linker is shown with a loop in the figure. Primers can be designed by using the standard Plug’n’Play linkers or by using the software PHUSER.

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 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) into one plasmid.

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, 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.

[3]https://2009.igem.org/Team:DTU_Denmark (Website, accessed 19.09.2011)

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