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Team:Paris Bettencourt/Assembly Standard
From 2013.igem.org
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Here, we show a novel suggestion for a standard. This suggestion opens up the possibility of keeping the BioBrick standard and adds at the same time the needed tools to perform the assembly of several parts in one step. BBG is a fusion out of the BioBrick standard cloning and Gibson isothermal assembly. The method is highly successful and was used by our team during iGEM.
To use this new method, only one PCR reaction is necessary. Overall, this new standard builds a bridge between BioBrick cloning and modern DNA assembly techniques and can be extended for more methods in the future.
Standardization is a necessary process of any engineering discipline. Synthetic Biology as a bioengineering science is not exempt from that. Standardizing biological parts is essential to make biological systems easier to engineer. A first step taken by the Synthetic Biology community was to set the BioBrick Cloning as a standard. We all know the benefits of this standardization but there are also some disadvantages. One is when such a standard is not yet everywhere adapted (e.g. sea level) and the other one is when new technologies and methods are limited by this standard. Very often decisions need to be made either to use a new standard, which supports a new method, or not to develop a new method. Both possibilities are not a good solution, as for example a new standard implies that all old parts need to be rebuilt.
The BioBrick standard was a big benefit for the Synthetic Biology community and the young scientist like us, who are doing iGEM. To flank DNA (BioBricks) with a standard sequence that includes standard restriction/ligation sites pushed forward the BioBrick standard. This allows a cheap and easy assembly of genes up to the level of gene circuits. But time is a limiting factor for most iGEM teams and their projects.
With BioBrick cloning you cannot clone several parts in one step, only sequential assembly is possible. This often takes a lot of time and hence many teams today are using Gibson assembly to overcome the problem. But to submit their parts they have to insert them into the BioBrick backbones, which is another step that could be avoided if there was a new/modified standard.
The iGEM community built over the years hundreds of new parts using the BioBrick standard, therefore it is important to think of a solution that allows using new methods and keeping the standard the same time. To provide a compromise we are introducing a new standard that opens up the possibility of using a new method and on the same time preserving the old standard. This novel standard is a fusion of BioBrick cloning and Gibson assembly called BBG (BioBrickGibson assembly). It was originally developed to be able to do Gibson assembly but still has restriction cut sites of the Biobrick enzymes to perform standard cloning as a backup. The iGEM Paris Bettencourt 2013 team tested and used this method successfully.
Methods
To combine Gibson assembly and BioBrick cloning we developed primers containing the BioBrick cut sites as well as an overlap of 45 bp that can be used for Gibson assembly. To be able to assemble several parts together we developed 4 different primers containing four different overlaps. As we want to use these primers to add the overhangs of the Gibson assembly to the BioBrick Backbones, there are 20 bp that match the ends of the Biobrick backbones with the Biobrick cut sites. As PCR is a quite simple method, it is easy to add the overhangs and then to be able to use new and existing parts to perform Gibson Assembly or BioBrick Cloning. This novel approach allows the implementation of new Cloning Methods within the already existing cloning standard. This method keeps the BioBrick standard but involves a variable, as new Gibson overhangs can be designed to allow the assembly of more than 4 parts in one step.
To catch up with new methods that facilitate cloning but are not the BioBrick standard, the BBG method can bridge the gap of keeping established methods and integrate new innovative ones. Especially the Gibson cloning allows us to do more cloning steps in one, which is becoming more and more necessary, as iGEM projects have become more complex over the years.
By using only the BioBrick standard longer time periods for cloning are needed, which can be time limiting for summer-long iGEM projects. In order to keep the existing database of Biobricks, a compromise like the introduced BBG cloning is a good alternative to open up to new methods and standards.
An important aspect of the Gibson overhang is to prevent the insertion of additional cut sites. With online available Gibson assembly overhang generators, reliable new overhangs can be generated. By setting the BBG as a new standard, students and other people that use BioBricks can use more cloning methods as before. The method of adding overhangs by PCR to use other cloning/assembly methods like Gibson can also be used to add overhangs for Golden Gate and MoClo as well as assembly PCR.
We hope to have highlighted the importance of integrating new assembly methods into the existing standard and that this proposal opens new ways for the standard of the BioBrick registry and the Synthetic Biology community.
Figure 2: Parallel assembly of genes of interest
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