|
In this paper, fibre optic spectroscopy (FOSpectr) was developed for detection and quantification of recombinant green fluorescent protein (EGFP) in transgenic tobacco plants. In vitro detection was first carried out to optimize the sensitivity of the optical system. The bacterial expression vectors, pEGFP and pDsRED, were transformed into Escherichia coli host cells and fluorescent proteins were produced following induction with IPTG. Soluble EGFP and DsRED proteins were isolated from lysed bacterial cells and successfully purified by size separation under nondenaturing electrophoretic conditions and quantified. The purified proteins were serially diluted for quantitative analysis by fibre optic spectroscopy using different light sources, namely, blue LED (475 nm), tungsten halogen (350-1000 nm) and double frequency Nd:YAG green laser (532 nm). Tungsten halogen was found to be unsuitable for excitation of both EGFP and DsRED. Blue LED and green laser were the most suitable for excitation of EGFP and DsRED, respectively. The minimum concentration of EGFP detectable with blue LED excitation was 7.5 tg/ml whereas that for DsRED under excitation by green laser was 3.75 ig/ml. To determine the capability of spectroscopy detection in planta, transgenic tobacco plants expressing EGFP were first imaged under a fluorescence microscope. This was to select a panel of transformed plants expressing varying levels of the fluorescent protein. These plants were then screened via FOSpectr. The results showed that the amplitude of the fluorescence emission signal obtained from FOSpectr correlated well with the level of EGFP expressed as indicated by fluorescence microscopy. Thus, proof-of-concept for the use of FOSpectr as a potentially powerful tool for screening transgenic plants was provided in this paper.
|
