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In our assays we scored for survival of yeast-transformants that could survive at and beyond 750 mM NaCl.

The P. pastoris transformant that could resist higher concentrations of geneticin G418 might contain multiple copies of integration of a heterologous gene into the P. pastoris genome, which could potentially lead to a higher expression level of the heterologous protein as elucidated in the manual of the Multi-Copy Pichia Expression Kit (Invitrogen, Carlsbad, CA, USA).

From the transformants obtained, those that could not grow on plates containing galactose (for induction of αSyn expression) and 5-FOA (for loss of the cDNA plasmid) were selected for an αSyn toxicity test.

This suggests a metabolic defect in transformants with a slow-growth phenotype on minimal medium that could be rescued by supplying metabolites in the medium.

However, it must be considered that the transformants still produce the mutated version of the protein that could exert aberrant functions, in this case resulting in mislocalization of fruiting bodies.

Transformants that were generated through co-transformation with plasmid pHsp70A-GLuc were also analyzed qualitatively for luciferase activity, and several of these transformants showed chemiluminescence (data not shown).

Transformants were streaked onto 5-FOA plates (US Biologicals) to select for candidates that could grow at permissible temperatures but not at non-permissible temperatures.

For those transformants that were growth retarded, we analyzed the insert of the pB42 fusion to see if the strain carried a hybrid that could account for the aberrant growth.

Transformation of DGLA-producing A. thaliana with this construct yielded transformants that successfully produced prostaglandin F.

The transformants that were able to grow on resistant plates were considered genetically stable.

Leu+ transformants were replica-plated onto YPD plates at 36°C, and the plasmid DNA was recovered from transformants that exhibited plasmid-dependent rescue.

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