Growth of individual transformants was studied by streaking single colonies onto solid media (2% glycerol, 2% lactate in uracil drop-out medium, pH5.5+2% glucose or+0.05% raffinose and 2% galactose) at 30°C and following their growth for increasing periods of time.
In order to have better insights about the affinity for glucose of both transporters, the growth rates of the transformants expressing MstG and MstH were studied in the presence of different glucose concentrations.
The fermentation performance of the different C. acetobutylicum transformant strains was studied and compared with those of the wild-type strains (WT) in batch cultures performed in bioreactors with a 1 L-working volume.
Resulting clones were transformed with a third plasmid encoding the protein to be studied, and triple transformants were selected on SC-URA-LEU-HIS.
Single colony transformants were isolated from minimal medium agar plates containing 2%% maltose and the ability of both genes to restore growth of the EBY.VW.4000 transformant strain in the presence of different monosaccharides was studied.
These transformants were re-grown and transformed with the third plasmid encoding the protein to be studied.
For AFM studies, transformants were cultured in leucine-free synthetic media as follows.
Only single-copy transformants were included in the study.
Fifteen transformants were randomly chosen for further studies, and plasmid DNA was isolated from a 3.0-ml broth culture using the GeneJET plasmid Miniprep Kit (Fermentas).
In the present study, yeast transformants were immediately selected on YEPD rich media plate supplemented with 200 μg/ml of G418.
The resulting transformants are functional for studying dynamic bacterial processes in living host cells.
