Fluorescent protein reporter for monitoring transgenic plant cell cultures
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2003-05
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University of Hawaii at Manoa
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Green fluorescent protein (GFP) is a useful tool for studying online monitoring of recombinant protein in plant cell bioprocesses. Three forms of GFP were constructed for the study. They are secreted GFP, SEAP-GFP fusion and inducible GFP. The secretory GFPs were accumulated over the time course of the culture in the medium. The fluorescent spectrum of the spent medium mimics that of the pure GFP, indicating GFP as the predominant fluorescing compound in the medium. The fluorescence signal of the spent medium linearly correlated with the secreted GFP concentration. It was also demonstrated in bioreactor cultures where GFP production was positively correlated with online culture fluorescence and offline spent medium fluorescence. For SEAP-GFP fusion, both of decreased function from SEAP and GFP were found in their fusion form. The fluorescence exhibited a similar trend as cell growth in the bioreactor or flask. However, the linear correlations between the fluorescence with fusion protein and SEAP activity are only tenable before the cell reaches the growth peak because of fusion protein degradation. By using ale promoter system, GFP expression can be controlled with ethanol. The optimal dosage is between 0.1% and 1 % (v/v) for the tobacco suspension cells, with maximal fluorescent intensity occurring after 4 days. In conclusion, all above different styles of GFPs provide an efficient and versatile method to study plant bioprocesses.
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ix, 119 pages
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Theses for the degree of Master of Science (University of Hawaii at Manoa). Molecular Biosciences and Bioengineering; no. 3774
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