Up to 100 μg mL−1, the growth yield was leucine limited, but the addition of higher concentrations did not lead to a further increase in the growth yield. The about 30% lower growth yield compared with the prototrophic strain remained unexplained, but the example underscores that growth in microtiter plates greatly facilitates the characterization of mutants. A further application of the growth in microtiter plates is the elucidation of biological functions of genes via the phenotypic comparison of wild type and mutants under many different conditions.

One project of our group is the characterization of the biological roles of sRNAs in H. volcanii, which is performed in collaboration with the group of Anita Marchfelder (University of Ulm, Germany). More than 100 sRNA genes have Ibrutinib been discovered by Trametinib manufacturer RNomics (Straub et al., 2009), bioinformatic predictions, followed by experimental validation (Babski et al., 2011), high-throughput sequencing (A. Marchfelder, unpublished data) and affinity isolation in a complex together with the haloarchaeal Lsm protein (Fischer et al., 2010). To elucidate the function of selected sRNAs, about 30 deletion mutants have been generated, and the construction of further mutants is under way. For their phenotypic characterization, batches of eight mutants,

the wild type and a uninoculated negative control were grown on six microtiter plates in parallel, allowing phenotypic characterization under 12 different conditions (e.g. various carbon sources, various NaCl concentrations, several stress conditions) with triplicate cultures. The power of this approach is exemplified by comparison of the growth curves of eight mutants with the wild type with xylose as the sole carbon and energy source (Fig. 6). Identification of the sRNAs, their sizes and their sequences has been published recently (Straub et al., for 2009). Five of the mutants had indistinguishable growth curves (deletion mutants of sRNA genes 63, 132, 235, 288 and 308). In this experiment, the

wild type grew slightly slower than these mutants, but the difference was not significant. Two sRNA deletion mutants clearly grew slower than the wild type and the majority of mutants (deletion mutants of sRNAs 168 and 500). Surprisingly, also, one mutant, the deletion mutant of sRNA194, was found to grow considerably faster than the wild type. While the generation and characterization of the sRNA mutants will be published separately, Fig. 6 clearly exemplifies that growth in microtiter plates enables middle- or high-throughput mutant characterization for functional genomic studies with H. volcanii, which would otherwise be impossible. In parallel to this study, an alternative phenotyping approach with H. volcanii has been developed (Blaby et al., 2010). A Bioscreen C apparatus was used that allowed parallel culturing of H. volcanii in a 100-well microtiter plate.