Strategies and Solutions to Maintain and Retain Data from High Content Imaging, Analysis, and Screening Assays.

Data analysis and management in high content screening (HCS) has progressed significantly in the past 10 years. The analysis of the large volume of data generated in HCS experiments represents a significant challenge and is currently a bottleneck in many screening projects. In most screening laboratories, HCS has become a standard technology applied routinely to various applications from target identification to hit identification to lead optimization. An HCS data management and analysis infrastructure shared by several research groups can allow efficient use of existing IT resources and ensures company-wide standards for data quality and result generation. This chapter outlines typical HCS workflows and presents IT infrastructure requirements for multi-well plate-based HCS.

Strengthening the dimerisation interface of Lac repressor increases its thermostability by 40 deg. C.

We increased drastically the heat stability of Lac repressor (LacR) of Escherichia coli. Wild-type tetrameric LacR denatures irreversibly at 53 degrees C. Improving hydrophobic packing at the dimerisation interface by a single substitution increases LacR heat-resistance by 40 deg. C without abolishing inducer binding at high and low temperatures. Tetrameric LacR mutants carrying substitutions of the positively charged amino acid Lys84 by each of the hydrophobic amino acids Leu, Ile and Met resist heating to temperatures up to 93 degrees C. We performed IPTG binding assays at 80 degrees C and found the mutant Lac repressors active and, thus, the core intact. Furthermore, the activity of LacR following heating is shown at room temperature by a gel retardation assay, which demonstrates normal oligomerisation state and function of the headpiece. The same mutations (K84L/I/M) in the dimer LacR331stop, carrying a stop codon in amino acid 331, increase thermostability of the dimer from 47 degrees C to 87 degrees C. LacRK84M represses beta-galactosidase activity in vivo as well as the wild-type and is sufficiently induced to allow growth on lactose. The results with both tetramer and dimer variants of LacR indicate mutual stabilisation of the tetramerisation region and the stable core.