volcanii in microtiter
plates has been developed (Blaby et al., 2010). The advantages and disadvantages of the two approaches are compared. Three H. volcanii strains used in this study were obtained from Thorsten Allers (University of Nottingham, UK). H26 is a pyrE deletion strain and is thus auxotrophic for uracil, but has wild-type characteristics for all the features analyzed in this study. The other two strains were derived from H26. H53 is a trpA deletion strain and is auxotrophic for tryptophan; H66 is a leuB deletion strain and is auxotrophic for leucine (Allers et al., 2004). In addition, deletion mutants of the following eight sRNA genes were used: sRNA63, sRNA132, sRNA168, sRNA194, sRNA235, sRNA288, sRNA308 and sRNA500. The identification of the sRNA genes and the generation of two deletion mutants including LY2606368 concentration a deletion mutant of sRNA63 have already been described (Straub et al., 2009); the remaining seven mutants were constructed using the same protocol. The sequences of the oligonucleotides used for mutant construction are available upon request. Haloferax volcanii was grown in a complex medium and a synthetic medium as described
previously (Dambeck & Soppa, 2008). Cultures were grown in Erlenmeyer flasks in a rotary shaker at 42 °C and 250 r.p.m. During BGB324 research buy the first trials to grow H. volcanii cultures in 96-well microtiter plates, pelleting of the cells was a problem. This problem was solved using an orbital shaker (1.5 mm orbit) and increasing the shaking velocity to 1100 r.p.m. A further problem was the evaporation of water during the incubation at the optimal growth temperature of 42 °C over several days, which led to an uneven loss of volume in the inner and outer wells and precipitation of NaCl in some wells. This problem was solved using the outer wells not for cell growth, but for the creation of an ‘evaporation barrier’. However, filling them with water led to a very fast evaporation
Meloxicam in the outer wells and to a volume increase in the inner, medium-containing wells. Therefore, salt solutions of different concentrations were tested, and a solution of 150 μL of 1 M NaCl turned out to be optimal. The evaporated water in the outmost wells was replaced daily; the volume of the inner wells remained constant throughout the experiments. Using the outmost wells for the ‘evaporation barrier’ 60 wells remained for cell culturing, which enables to test, for example, 20 conditions simultaneously using triplicate cultures, or to compare many mutants with the wild type under few conditions. Precultures were grown in Erlenmeyer flasks in a synthetic medium with glucose as described above to the early exponential growth phase (OD600 nm=0.3±0.1). The vitamin solution contained nine different vitamins (Sigma, Taufkirchen, Germany; order no. B6891) and was diluted 1 : 1000 into the medium.