Together with NorStruct (Dr. Altermark) and ArcticZymes, the production and characterization of enzymes in terms of catalytic efficiency and thermal stability will carried out. Mutations suggested to be important for either catalytic activity and tuning of the enthalpy-entropy balance (WP1) or thermal stability (WP2) will be produced and assayed. Both collaborators have extensive hands-on expertise from working with the relevant enzymes. Production protocols with good yields and kinetic assays are available for both cold- and warm-active enzyme.

With our international collaborators we will start out to characterise changes in the translational machinery due to cold environments. So far, there is basically no data available for the performance of the translational apparatus in psychrophilic species but there are clear sequence characteristics found for the 16S rRNA. Thus, the project will attempt to isolate and culture psychrophilic bacteria from, e.g., Vibrio and Psychrobacter species in order to extract components of the translation system. With our in-house in vitro translation capabilities it will then be possible to examine the speed of protein synthesis both for full reporter genes and short synthetic mRNAs with fast kinetics methods. The effects of external factors such as temperature and pressure can then be studied.  Evolutionary experiments on E. Coli and Salmonella will also be attempted, where these are submitted to cold growth conditions and selected for fitness in many rounds. With subsequence genome sequencing relevant genetic changes both in the translation machinery, which is usually limiting for growth in these species, and regular enzymes can be detected and analyzed. This will provide highly interesting data for subsequent computational analysis in WP1.