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From 2012.igem.org

The golden bricks: A new, fast and reliable cloning technique for iGEM

Introduction

Context

Cloning is the most tedious and less rewarding work in engineering new living entities through synthetic biology. The first indisputable advance in the domain in undoubtedly the invention of the biobrick format that have enabled to assemble in a standard way almost all the genetic pieces, and the creation of a huge database containing up to now XXXX thousands of described and characterized biological parts.

If biobricks have opened new perspectives and has proven to be efficient over years, this technique only make possible the assembly of two parts at the time, and couldn't really be automatize. iGEMers today's spent a lot of month to master the subtlety of its protocol and spend month to assemble parts together, leaving little time for their characterization and testing their systems.

New and more efficient cloning techniques enabling the assembly of several parts at the time has been invented since then, amongst them we have to cite the Gibson method [] (up to 3-4 fragments at the time) the Golden Gate[] (>20 fragments at the time) and its new evolution, the MoClo [] (47 fragments in two times). If these technique speed up the cloning time dramatically, up to now, no work has been done in the sense of standardizing these methodology. Therefore, a new strategy and a new library has to be make for each different construct, which prevents them from be used in a single standardized biological parts library such as the registry.

The described work

The present page reports the invention and the development of a new technique invented by our iGEM team this year, that is a huge step towards the standardization of the fastest cloning technique known so far, the Golden Gate (also known as type II restriction enzyme technique) making them compatible with a database of biological parts such as the partsregistry, that also keep the compatibility with the known biobrick RFC 10 standard. This technique enable one-shot cassette construction with DNA coming either from the registry distributions or from PCR product, engaging up to seven different parts. This technique is compatible with eukaryotes and procaryotes DNA design in the very same way. A new "split construction" method based on standard plasmids also enable to assemble parts in a non classical way.

Perspectives

Such technique offers a great perspective. First, it would enable the fast and simple biobrick assembly of complete cassette with almost no equipment required, accessible for both researchers and real beginners such as high school iGEMers. The process is simple: re-suspend the parts from the distribution plates, mix them together, put them in the PCR machine and transform. Second, it goes a lot faster than synthesizing the construct, which would guaranty the interest for databases approach versus de-novo synthesis. This method is fully automatable. And last, the new standard is fully compatible with the previous BBF RFC 10 standard.

Theoretical context

Requirements for the development of the new standard

In this work, we are trying to develop a new standard for the partsregistry. This lead to several requirements imposed by the registry itself:

1. The new assembly method should be compatible with a database approach
2. It should keep the compatibility with the standard RFC 10
3. Use the standard registry plasmid pSB1C3 with the J04450 red negative control

In order to improve the format, we have to:

4. Enable one step golden-gate cassette cloning
5. Minimize the number of different prefix and suffix
6. Allow to check the construct with a single digestion
7. Improve the compatibility with Gibson
8. Provide a solution for non standard assembly

The next paragraph explains how we managed to fulfil these requirements.

Analysis of the classical synthetic biology constructions

Traditional construct in synthetic biology is made of a repetition of the following different elements:

On this work, we are going to focus on the assembly of a single cassette in one shot. The assembly of the different cassette will have to be conducted with the standard assembly of with gibson assembly, that is very efficient for assembling long fragments.

There are several different overhangs to engineer. The first part is to make an unique overhangs for ligating plasmid to the front of the promoter and to the back of the primer. The scar between the RBS and the gene has to be very well controlled and shouldn’t be changed in the new format. We are going to use the property of the Type II enzyme to make this possible. Finally, the overhang at the front of the RBS have to be compatible with the one in the back of the gene to enable RBS-Protein unit polymerization. This leads to the design of 4 different overhang that is to say 8 different biobrick site.

The proposed new set of Golden Bricks extension

One condition to fulfil to get a RFC 10 compatible brick is to leave no illegal restriction site after the construction of the cassette.

Demonstration of a correct assembly in a simulated experiment

Control of the polymerization

Experimental work

Creation of a simple cassette

Polymerization statistics

Split construction vector used

Material and methods

Construction of the library

Assembly protocol

Further perspectives in implementing the technique in the partsregistry database

Conclusion