Engineered Saccharomyces cerevisiae strain BioS-OS1/2, for the detection of oxidative stress.
One of the major stress factors during space and high-altitude flight is the oxidative damage caused by the release of reactive oxygen intermediates (ROIs) in human tissues. ROIs are released in response to several stress factors including radiation in space. Since ROIs contribute to several pathological conditions, there has been a great interest in developing a biosensor that can monitor the impact of ROIs on biological systems. Toward this goal, we sought to engineer a yeast stain that can monitor oxidative stress and be easily integrated into a biosensor platform. Saccharomyces cerevisiae respond to hyperoxidative stress by activating the expression of many proteins including the transcription factor, Yap1. Activated Yap1 primarily binds to the Yap-1 response elements in the promoters of genes that combat oxidative stress. Based on these observations, we genetically altered the Yap-1 pathway in the YCR094W BY4742 strain of S. cerevisiae by fusing the YREs in the promoter region of TRX2 gene to a cDNA-insert encoding green fluorescent protein (GFP). Exposure of this engineered yeast strain BioS-OS1 to varying levels of oxidative stress, as generated by different concentrations of H(2)O(2) or diamide, elicits robust expression of GFP that can be monitored by the fluorescence of GFP by as early as 1 h. BioS-OS1 can detect a H(2)O(2) concentration from 300 microM onward. We also show that the signaling strength of the strain can be increased by engineering multiple YREs in the upstream of the cDNA-insert encoding GFP. Thus, the results presented here demonstrate that the engineered BioS-OS yeast strain can detect ROI-generating oxidative stress and validate the use of this prototypic strain for the development of a biosensor to detect and monitor oxidative stress factors during space and high altitude flights.
Jayaraman
Muralidharan
M
Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, 3307 N. Broad Street, Philadelphia, Pennsylvania 19140, USA.
Radhika
V
V
Bamne
Mikhil N
MN
Ramos
Ross
R
Briggs
Rita
R
Dhanasekaran
Danny N
DN
D015374:Biosensing Techniques / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D015202:Protein Engineering / Q000379:methods*; D017382:Reactive Oxygen Species / Q000032:analysis* / Q000494:pharmacology*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013045:Species Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D017382:Reactive Oxygen Species; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C086677:YAP1 protein, S cerevisiae; D049452:Green Fluorescent Proteins