Why Yeast?
Most microbial biosensors and bioassays employ bacteria as the central biological component, whereas GreenScreen GC employs brewer's yeast. Yeast cells are more like human cells than bacteria. They share the same basic cellular architecture as mammalian cells and possess some endogenous p450 proteins for metabolising compounds. Yeast are thus a more appropriate model for the detection of mutagens and carcinogens than bacteria. Unlike bacteria, the cellular mechanisms yeast use to deal with this gene damage are strikingly similar to those in human cells. So much so that elements of the cell division and DNA repair mechanisms from yeast and human cells are functionally interchangeable. Yeast cells are also more physically robust than bacteria and can therefore function in a wider range of environmental conditions, making them a more versatile tool.
A key advantage of using yeast is that aliquots of the culture can stored in a laboratory freezer and are easily resuscitated ready for use after a 72 hour (i.e. over weekend) incubation. The stock culture can be used immediately for assays and remaining stocks are disposed of after 1 week. There is no requirement for on-going passage and maintenance of cell lines, and thus the assay can be handled in a basic laboratory setting.
The Principles Underlying the GreenScreen GC Assay System:
The central component of the GreenScreen GC bioassay technology is a genetically engineered yeast strain (Saccharomyces cerevisiae). In the genetically engineered cells, the yeast promoter for a DNA repair gene (RAD54) has been copied and placed in front of a green fluorescent protein (GFP) gene (patented technology). As a result, cells that sustain chromosome damage produce increasing quantities of fluorescent protein, which can be detected using a standard laboratory fluorescence microplate reader.
The repair of genetic and cellular damage requires expenditure of energy in manufacturing and assembling new replacement components. This required energy must be diverted from normal cellular growth and proliferation, and hence causes measurable effects on cell yield in the carefully formulated GreenScreen growth medium. It is this principle that underlies GreenScreen GC's ability to also detect cytotoxicity. The assay thus uses the whole genome to sense genotoxicity and the whole cell to sense cytotoxicity.
Assay Protocol:
Nine, 2-fold dilutions of each compound together with positive controls are set out in the microplate and growing cells are added to each well. After overnight incubation and measurement in a microplate reader, simple software gives automated decisions and a clear graphical output.