Team:Paris Bettencourt/Assembly Standard


We propose an easy way to expand the compatibility of the BioBrick assembly standard. The BBG (BioBrick-Gibson) adapter method links existing BioBrick standards with emerging Gibson-assembly-based technologies. The method was successful in our hands and expanded the cloning options of our iGEM team while keeping our work compatible with the rest of our community.

   Engineering demands standards. Synthetic Biology as an engineering science is no exception. Standard biological parts make biological systems easier to engineer, a fact that motivated the early adoption in iGEM of the BioBrick cloning conventions. But with the benefits of standardization come also some disadvantages. A given standard will generally not be universally adopted, and may not be compatible with all possible assembly methods. Therefore a standards-based engineering community, confronted with emerging technologies, must weigh some inherent trade-offs. Should standards be maintained, abandoned, or perhaps adapted and extended?

The BioBrick standard allows cheap and easy sequential DNA assembly up to the level of gene circuits. But the BioBrick standard does not support parallel assembly.

Because parallel assembly is often faster, and time is of the essence especially during iGEM, many teams supplement or replace BioBrick cloning with Gibson assembly. Yet intra-team part compatibility still requires BioBrick conventions. The result is additional cloning steps and lost time.

An ideal new standard would allow teams access to the widest range of possible assembly methods while preserving the exiting iGEM registry as a resource. We believe that the BBG adapter method can fill this role. It was originally developed in-house to meet our team's demand for Gibson assembly with BioBrick overhangs. We present it here in the hope that the rest of iGEM can benefit from the experience.


   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.

Figure 1: Generation of the backbone

By using only the BioBrick standard longer time periods are needed for cloning, 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

Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France
+33 1 44 41 25 22/25
Hit Counter by Digits
Copyright (c) 2013 All rights reserved.