What is iGEM?

iGEM (international Genetically Engineered Machine) competition is the world’s largest competition within Synthetic Biology, which is hosted by Massachussetts Institute of Technolgy (MIT). iGEM is considered the most prestigious competition for students in the field of biotechnology, which also makes it the world’s largest event within synthetic biology(1).

The competition started out as a month-long course at MIT, where the students had to design a biological system. This course grew to a summer competition in 2004 with just 5 teams, and since then the competition has expanded dramatically with more than 160 teams from universities all over the world in 2011(1).

The iGEM competition provide a library of standardized biological parts known as BioBricks that the teams can use for building the genetic machines. The teams can also submit their own BioBricks. Information about the BioBricks and a toolkit to make and manipulate them is provided by the Registry of Standard Biological Parts . In the beginning of the summer, the teams receive a kit of BioBricks from the Registry of Standard Biological Parts. Over the summer, the teams work at their own universities, where they use these parts and parts of their own design to build biological systems that can operate in living cells(1).

iGEM is about manipulation of genetic material, where the only limit is one’s own imagination. The iGEM competition have demonstrated a new way to arouse student interest in modern biology and to develop their independent learning skills.The project ideas are many and diverse, but overall they show great potential.

Synthetic Biology?

Synthetic biology is relatively new area of biological research that combines science and engineering. Synthetic Biology is the engineering of Biology. The goal of synthetic biology is to extend or modify the behaviour of organisms and engineer them to perform new and innovative tasks. An analogy conceptualizing both the goal and methods of synthetic biology is the computer engineering. Within computer engineering every constituent part is embedded in a more complex system that provides its context. Synthetic biology is about using standardized parts as DNA, RNA, proteins and metabolites to construct biochemical reactions that regulate physical processes. The parts can be combined in devices and modules in order to assemble complex pathways that can function as integrated circuits. Connection of pathways to each other and their integration into host cells allows the synthetic biologist to extend or modify the behaviour of cells in a programmatic fashion (2).

The world calls for a better Assembly System

The Registry of Standard Biological Parts and iGEM make use of the Standard Assembly of BioBricks formulated by Tom Knight. The Standard Assembly make use of restrictionsites for the four restriction enzymes EcoRI, XbaI, SpeI , and PstI. The BioBricks have to hold the restrictionsites for the two enzymes EcoRI and XbaI prefix and suffix the restrictionsites for the two enzymes SpeI and PstI. In order to have assembly correct the BioBrik can't contain any of these four restrictionsites within the BioBrick. However, if any of this four restriction sites are present, they will have to be eliminated by alterations like site-directed mutagenesis, which can be both time consuming and cause unwanted alterations. In developing new BioBricks from natural sources and higher organism than eukaryotes the illegal restricktion sites can be of a problem. Furthermore, when assembling BioBricks with the Standard Assembly System scars occur, which makes it impossible to create fusion proteins. Additionally, the Standard Assembly of BioBricks is limited in that it is only possible to put two BioBricks together at a time (3).

All in all, the iGEM competition and the fast growing field of synthetic biology calls for a simpler, faster and more efficient assembly system that are easily applied to both bacteria, fungi, and mammalian cells.

USER cloning

In early 1990s, the uracil excision-based (USER) cloning was invented as a ligation-independent cloning technique that could substitute the conventional assembly systems that made use of restriction enzymes and ligase. In 2003 New England Biolabs (NEB) introduced the USER Friendly Cloning Kit. Although NEBs USER kit was simple and efficient, it was not compatible with proofreading polymerases that stalled when encountering a uracil base in the DNA template (4). This made the USER Friendly Kit unattractive, although the concept was brilliant. In recent years, proofreading polymerases have been developed that are compatible with the concept of USER cloning, since they can read through uracil (5).

The USER method applies long complementary overhangs on the PCR product(s) as well as on the destination vector. The overhangs on the PCR product are custom made, between 7-15 nucleotides long and deoxy uridine nucleotides substitute selected deoxy thymidine nucleotides. The PCR products containing the customized overhangs are treated with the USER enzyme, which is a mix of DNA glycosidase and DNA glycosylase-lyase endo VIII. This treatment results in release of the DNA sequence upstream the deoxy uridine nucleotide and the resulting exposed overhangs can anneal to each other to form a stable hybridization product. This product can now be transformed directly into E.coli without prior ligation (4,5,6). In order to avoid template carry-over after PCR, the PCR product is usually treated with the restriction enzyme DpnI. DpnI cleaves only when its recognition site is methylated. Unmethylated PCR-derived DNA will be left intact (5).

Outline of the Plug 'n' Play with DNA idea

We introduce a standardized assembly system based on the principle of USER cloning and the USER fusion Assembly standard introduced by the 2009 iGEM of DTU. The new assembly standrad called The Plug 'n' Play allowing easier cloning and is a combined standard and system. The Plug 'n' Play assembly standard can be found here (BBF RFC 80).

Successful assembly of up to six biological parts in one reaction have in the conducted project proof to be possible. The upper limit of fragments that efficiently can be assembled has not been delineated. The time for construction of devices and plasmids to create new synthetic biology system have hereby significantly been reduce. This is essential for move beyond imagination of what we can create with synthetic biology and solve fundamental problems in our communities.

The mission of Plug 'n' Play with DNA

DTU-Denmark-2 introduces the standardized and versatile system called "Plug ‘n’ Play with DNA", where certain categories of biological parts can be gathered. We imagine that the parts is in the form of pre-produced PCR-products, which directly can be mixed with a backbone vector to make assembly of expression vectors possible in less than one day. All our parts in the form of PCR-products should be distributed like the original iGEM BioBricks in microtiter plates, but directly ready for cloning. Furthermore, the "Plug ’n’ Play" kit will contain a back-up plasmid of all parts to ensure amplification from a mutation free template if needed. The simple and easy use of the system have been demonstrated by developing a reporter targeting system for the filamentous fungi of Aspergilli as well as for mammalian cells. In collaboration with the Copenhagen iGEM team the system have been demonstrate the easy use for the bacteria E. coli.

Application area

The Plug ‘n’ Play assembly standard can be applied in a lot of different contexts. We have in this project succeeded in creating a reporter system for both mammalian and fungi in less than 2 months. Our assembly system therefore is of great value in creating DNA libraries fast and simple.

For the institute of System Biology as well as The Center of Microbial Biology at DTU our system represents the possibility for creating high trough-put cloning. The system is therefore in the process of being implemented.

For cell factories and heterologous protein expression our assembly standard provides huge potential for easy cloning and making gen expressions analyses.

The construction of expression vectors represents a bottleneck in the development for the productions of biopharmaceuticals, given that mammalian cells possess sophisticated molecular biology. The genetic tools used for mammalian cells vectors are based on outworn methods. Commonly the mammalian expression vectors have a multiple cloning site (MCS). The gene of interest is therefore required to hold restriction sites compatible with the expression vector and the insertion of the gene is achieve by ligase.

References

[1] https://igem.org/About (Website, accessed 19/09/2011)

[2] Andrianantoandro, E. et. al. Synthetic biology: new engineering rules for an emerging discipline. Molecular Systems Biology: 10: 1-14 (2006).

[3] http://partsregistry.org/Assembly:Standard_assembly (Website, accessed 19/09/2011) [4] New England Biolabs, 2004

[5] Hussam H. Nour-Eldin, Fernando Geu-Flores, and Barbara A. Halkier. USER Cloning and USER Fusion: The Ideal Cloning Techniques for Small and Big Laboratories. Methods in Molecular Biology 643.