Optimization and application development of marine cold-adapted DNA polymerases - OptiZyme

Isothermal amplification (IA) methods are now increasingly being used for molecular detection of pathogens, and are considered an important contribution to improve both disease control and food safety analysis. In these methods, nucleic acid amplification takes place at constant (moderate) temperatures and has no need for temperature cycling or high temperature stable enzymes as needed in the widely used polymerase chain reaction. In many of the IA methods only one enzyme is needed, the DNA polymerase, an d the effectiveness and success of the method is therefore very dependent on the enzymatic properties of the polymerase employed. The ambition of this proof of principle project is to verify and optimize enzymatic properties of selected recombinant marin e cold-adapted polymerases necessary to further develop isothermal amplification technologies. In-detail characterization of the different polymerases will provide detailed insight of the enzymatic properties and may lead to a possible usage in other mole cular applications within fields as forensic medicine and synthetic biology. Most critical challenges in order to achieve the main goal in this project lie within the protein engineering of the cold-adapted polymerases. Rational design relies on details that can be obtained only through atomic resolution structures, of which X-ray crystallography is essential. Small crystals have already been obtained, and initial diffraction experiments indicate a resolution of 3 Å. However, to obtain a high resolution structure necessary for the rational design studies in this project, further optimization of crystallization conditions is needed. In contrast molecular evolution is a random approach to create mutants with modified enzymatic properties. In order to ident ify advantageous variants for IA applications a suitable screening/selection method in high-throughput scale must be established.