Team:DTU-Denmark-2

The emerging and fast growing field of molecular and synthetic biology calls for simpler, faster and more efficient cloning techniques. Conventional cloning techniques include restriction digestion and ligation, which can be both time consuming and complex – especially when working with mammalian cells and fungi. So in order to optimize the cloning process to become faster and more efficient, it would be convenient to avoid the use of restriction enzymes. In early 1990s, the uracil excision-based cloning was invented as a ligation-independent cloning technique that could substitute the conventional cloning with restriction enzymes and ligase. This technique was further developed, and a few years later, New England Biolabs (NEB) introduced the USERTM Friendly Cloning Kit, which was to ensure cloning without the use of restriction enzymes. However, the kit was not compatible with proofreading polymerases, since they stalled when encountering a uracil base in the DNA template, as it is a promutanegic event (Nour-Eldin et al., 2010; New England Biolabs, 2004). However, newer proofreading polymerases have been developed that can read through the uracils and thus be compatible with the concept of USER. This has opened the door to a more simple and flexible approach to molecular cloning techniques. The method applies long complementary overhangs on the PCR product as well as the destination vector. These overhangs can anneal each other to form a stable hybridization product. The overhangs on the PCR product are custom made and independent of restriction sites, which is very convenient when working with fungi and mammalian cells. We will develop a standardized cloning system, called “Plug’n’Play”, where certain categories of biological parts can be gathered. This means that the parts in the form of pre-produced PCR-products are directly mixed with a vector, which makes assembly of an expression vector possible within a few hours. All the parts in the form of PCR-products will be distributed in microtiter plates directly ready for cloning. Furthermore, the “Plug’n’Play” kit will contain a back-up where all parts are contained on a plasmid to ensure amplification of a mutation free template if needed. The simple and easy use of the system will be demonstrated by developing a reporter targeting system for the fungus Aspergillus niger. Furthermore, will we demonstrate its application in mammalian cells."