The maintenance and genetic stability of the vector plasmids pBR322 and pBR325 in two genetically different Escherichia coli hosts were studied during chemostat cultivation with glucose and ammonium chloride limitation and at two different dilution rates. The plasmid pBR322 was stably maintained under all growth conditions tested. However pBR325 segregated from both hosts preferentially during glucose limitation and at low dilution rate. In addition to this general segregation process a separate loss of tetracycline resistance was observed. The remaining plasmid conferred resistance to ampicillin and chloramphenicol only, without any remarkable alteration of its molecular weight. Cultivation conditions in the chemostat were found that allowed the stable genetic inheritance of both plasmids in the hosts studied.
Liquid handling in higher density microplates (e.g., 1536-well microplates) for more efficient drug screening necessitates carefully selected and optimized parameters. The quality of a liquid handling procedure is dependent on the carryover rate of residual liquids during the pipetting process, the mixing behavior in the wells, foam and bubble formation, and evaporation. We compared and optimized these parameters in 96-, 384-, and 1536-well microplates, and herein we critically evaluate the performance of the CyBi-Well 96/384/1536 automated micropipetting device, which formed the basis of our evaluation studies.
Liquid handling in higher density microplates (e.g., 1536-well microplates) for more efficient drug screening necessitates carefully selected and optimized parameters. The quality of a liquid handling procedure is dependent on the carryover rate of residual liquids during the pipetting process, the mixing behavior in the wells, foam and bubble formation, and evaporation. We compared and optimized these parameters in 96-, 384-, and 1536-well microplates, and herein we critically evaluate the performance of the CyBi-Well 96/384/1536 automated micropipetting device, which formed the basis of our evaluation studies.
M iniaturization is one way to realize today's demands in the drug discovery process by moving from the standard 96-well plate to higher density microplate formats. In this article we describe the adaptation of a fluorescence-based enzyme assay to the challenges of the 384-and 1536-well plate format. The liquid-handling was realized by the automated micropipettor CyBi-Well™ 96/384/1536* (CyBio AG-formerly JENOPTIK Bioinstruments Gmbh-Jena, Germany). On the basis of optimized liquid-handling parameters pipetting routines were established to perform an enzyme assay (b-galactosidase) in the microplate formats of higher density. Finally, the experimental results were compared to those obtained in the well-established 96-well format. In the enzyme assay, the bioconversion of the substrate Fluorescein-di-(b-D-galactopyranoside) (FDG), occurred as a linear function of the b-galactosidase concentration comparably in all three assay formats. We conclude that miniaturization using the higher density 384-and 1536-well plate formats is advantageous as the next evolutionary step in HTS, especially using enzyme assays. A careful individual adaptation procedure for each microplate format and assay at the basis of the optimized liquid-handling parameters is essential. CyBi-Well ™ 96/384/1536 proves to be a powerful tool for a careful adaptation of the liquid-handling procedures of biological assays especially also in the 384-and 1536-well formats. * Distributed in the USA and Japan under the name of PlateMate™ until 21-12-1999.
Miniaturization of high throughput screening assays to high-density microplate formats (384 or 1536 wells) is currently the focus of research activity in modern drug discovery facilities. In this article, we describe the adaptation of a fluorescence-based functional transcription assay in yeast for assessing modulators of human progesterone receptor to the 384- and 1536-well microplate format, comparing the experimental results to those obtained in the well-established 96-well format. The experiences gained from the optimization of the liquid-handling procedures and the miniaturization of an enzyme assay (β-galactosidase) were implemented. Thus optimized pipetting protocols were developed to perform a reporter gene assay in yeast in microplate formats of higher density. In the functional transcription assay in yeast, the reporter gene expression showed the expected dependence on the ligand's dose and affinity in principle in all three microplate formats. For the first time, this assay system has been established in the 1536-well microplate format using CyBi™-Well 96/384/1536 as the liquid-handling unit. The comparison of the signal:background ratios showed a lower sensitivity of the assay in the microplate formats of higher density. This study is an example of a successful miniaturization of a yeast cell-based assay to high-density plate formats on the basis of a careful adaptation procedure and optimized liquid-handling conditions.
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