Z5

Low cost cloning

Dr Sylvestre Marillonnet, Leibniz Institute of Plant Biochemistry (IPB)

TechnologieAllianz


Challenge

DNA assembly methods such as Gibson assembly (Gibson et al. 2009, Nature Methods 6, 343-345) and Golden Gate cloning (Engler et al. 2008, Plos ONE, 10.1371) are basic tools for synthetic biology. Both methods allow assembly of constructs from multiple DNA fragments in a one-pot one-step assembly reaction. Parts may include a promoter, a coding sequence or a terminator. Standardization of parts means that every part has structural features that fit a defined standard. This characteristic allows all equivalent parts of a standard (for example promoters) to be used interchangeably for assembly, allowing combinatorial assembly of parts to make a variety of constructs from a small number of parts. A limitation of current cloning protocols is the need to store DNA parts frozen at low temperature, with a cost that will be rising with the number of parts that need to be stored.


Technology

A new protocol has been developed to assemble constructs from DNA parts kept in a dry form. DNA fragments are stored in a dry form on a solid carrier in a ready-to-use format. Each aliquot of dry DNA sample contains a defined amount of DNA sufficient for a single cloning reaction. The assembly procedure takes place in a reaction vessel which contains the aliquots to be assembled, each on a separate carrier, and the buffer needed to perform the necessary reaction. The fragments are assembled directly and do not need to be put in solution separately. At the end of the reaction, the supernatant containing the assembled constructs can be transformed into competent cells and plated on selection medium.


Commercial Opportunity

The invented technology provides the opportunity for standardization of processes in synthetic biology. Standardized products can be provided for cloning reactions. Storage and shipment would be less cost intensive and complex.


Development Status

Proof of concept


Patent Situation

Patent application pending, DE102016101948A1, WO2017133738A1


Further Reading

Gibson et al. 2009, Nature Methods 6, 343-345
Engler et al. 2008, Plos ONE, 10.1371


 

Low cost cloning
Low cost cloning

Scheme for cloning using DNA fragments stored on solid carriers