Applications
- Agriculture
- Fungi
Advantages/Benefits
- Maximizes transformation efficiency or quality event outcomes, which existing “wild-type” origins are unable to achieve
Background
Agrobacterium-mediated transformation (AMT) of plants and other eukaryotes relies on the presence of a plasmid within the bacterium that possesses a transfer DNA (T-DNA) element. This plasmid, known as a binary vector, allows DNA to be mobilized from Agrobacterium to the host. The binary vector has undergone extensive engineering to enhance its transformation properties. However, a neglected area of improvement is the modulation of the copy number of different origins of replication to achieve desired transformation outcomes. Currently, all binary vectors used in transformation depend on the natural copy number of the origin within Agrobacterium. A solution is needed that can both increase and decrease the copy number of different origins of replication within Agrobacterium in order to tailor transformation outcomes precisely and efficiently.
Technology Overview
Using a high-throughput selection, scientists at the Joint BioEnergy Institute have created a technology that isolates mutants in plasmid origins of replication in Agrobacterium tumefaciens that improve plant transformation by modulating copy number. These plasmid origins allow for either more or less T-DNA to be introduced to eukaryotic hosts, depending on the desired transformation outcome. This approach uniquely targets the modulation of plasmid copy number by identifying and isolating non-native copy number mutants. It utilizes a gentamicin resistance gene and the NahR salicylic acid inducible promoter to identify high-copy mutants, and a sacB gene in the presence of sucrose to select for low-copy mutants. This method allows precise tailoring of transformation outcomes, improving the overall transformation process.
Laboratory results demonstrate that manipulation of the binary vector origin of replication (ORI) greatly expands the dynamic range of T-DNA delivery to target cells. For a given ORI within tobacco, the scientists achieved variation of transient expression levels by 28-fold, demonstrating a large gradient of T-DNA delivery to target cells and notably improving AMT efficiency. These vectors will likely also prove useful in the transformation of non-plant eukaryotes, particularly filamentous fungi.
Development Stage: Proof of concept
Principal Investigator(s)
- Mitchell Thompson
- Matthew Szarzanowicz
- Liam Kirkpatrick
- Patrick Shih
Status: Patent Pending
Opportunities: Available for licensing or collaborative research