Team:AshesiGhana/fret
What is FRET?
With the evolution of synthetic biology, the use of biosensors in designing major projects in this field has gained popularity over the years. Biosensors are devices which detect the presence of certain chemicals or compounds by use of living organisms or biological molecules. As part of the several mechanisms used in bio-sensing is the method termed Fluorescence Resonance Energy Transfer, popularly known as “FRET”. This method originated in the late 1940s by German Physical Chemist Theodor Forster (Lavdas, n.d.). This technique over the years has been widely used as a reporter method in biomedical research. This project seeks to detect the presence of gold as well as aid in its quantification, especially in refractory ores, hence the need to employ this technique as an efficient and effective way of achieving this objective.
The FRET mechanism is a physical process that works on the principle of energy transfer from a donor molecule to an acceptor molecule. In addition to this, FRET is distance-dependent process hence its use in living cells. (Held, 2005). A variety of FRET pairs have been discovered and used in different applications, however, it is the cyan-yellow fluorescent protein pair that has been the most widely used pair.
Fluorescence Resonance Energy Transfer (FRET)
Rather than the traditional Cyan-Yellow FRET pairs, this project relies on the newly identified and most effective Green-Red pair, which according to research has gained popularity due to its excitation at a longer wavelength reducing cellular auto-fluorescence and photo-toxicity while monitoring FRET (George Abraham et al., 2015). The Green-Red pair utilized in this project are, NowGFP serving as the fluorescent donor, and mRuby2 serving as the fluorescent acceptor. NowGFP, a highly efficient green fluorescent protein is an improved version of the WasCFP with tryptophan-based chromophore in anionic state (George Abraham et al., 2015). This fluorescent protein has a fluorescence lifetime of ~5ns which makes this the longest lifetime reported for any green fluorescent protein thus far (George Abraham et al., 2015). With a high emission yield of 0.76, NowGFP has several other qualities which makes it the best donor partner in the pair for our FRET mechanism in this project. To complete the green-red FRET mechanism td-tomato fluorescent protein would have been the best pair for NowGFP, however due to the td-tomato part sequence being cut by some restriction enzymes, the part could not serve the purpose for this project. In replacement for td-tomato, mRuby2 which has equally proven to be an effective FRET acceptor probe for NowGFP donor, is used. mRuby2 is an enhanced version of the mRuby red fluorescent protein (George Abraham et al., 2015).
For the design of this project, each part of the FRET (the donor and acceptor) is attached to gol B, the gold binding protein. GolB is part of the gol operon that confers resistance to A. ferroxidans. In the presence of gold, under the control of golS, golB is expressed and binds to the gold being bound to the two components of the FRET part. This therefore places each component of the FRET part in close proximity and thus decreases the distance between the donor and acceptor. In theory, the amount of gold bound to golB on each side is a determining factor in the distance between the donor and the acceptor and this therefore affects the intensity of the fluorescence from the FRET part. The more gold bound, the higher the fluorescence from the FRET part and vice versa.
This design could have made use of mCherry and mRFP which are also red fluorescent proteins. However, these fluorescent proteins are said to have very low emissions, making it difficult for these emissions to be detected above the donor emission tail which brings about problems in ratiometric imaging (Lam et al., 2012). Another option was to use a single reporter protein in the biosensor bit of our entire device which is common among bio-sensing systems, as it is an easy and sure way of detecting the presence of gold in an ore. The reporter proteins could also easily be used to detect the presence of gold when gol T, under the influence of gol S, starts to transport gold to be bound by gol B. However, the aim of the project is not just to sense the presence of gold but also, to quantify it. Using a single reporter protein defeats the purpose of this project. As mentioned earlier, the more gold bound, the more the intensity of the fluorescence as a result of the decrease in distance between the donor and acceptor. This therefore provides a rough estimate of how much gold is in an ore. Whenever gold molecules are bound to either side of a donor acceptor complex, the distance between the donor and acceptor is reduced. This reduction in distance is what gives rise to the increase in FRET intensity as there is a direct correlation between the distance between a donor acceptor complex and overall FRET efficiency (George Abraham et al., 2015).
Sugio, T., Taha, T., & Takeuchi, F. (2009). Ferrous Iron Production Mediated by Tetrathionate Hydrolase in Tetrathionate-, Sulfur-, and Iron-GrownAcidithiobacillus ferrooxidansATCC 23270 Cells. Bioscience, Biotechnology, And Biochemistry, 73(6), 1381-1386. http://dx.doi.org/10.1271/bbb.90036
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George Abraham, B., Sarkisyan, K., Mishin, A., Santala, V., Tkachenko, N., & Karp, M. (2015). Fluorescent Protein Based FRET Pairs with Improved Dynamic Range for Fluorescence Lifetime Measurements. PLOS ONE, 10(8). http://dx.doi.org/10.1371/journal.pone.0134436
Held, P. (2005). White Paper: An Introduction to Fluorescence Resonance Energy Transfer (FRET) Technology and its Application in Bioscience. Biotek.com. Retrieved 10 July 2017, from https://www.biotek.com/resources/white-papers/an-introduction-to-fluorescence-resonance-energy-transfer-fret-technology-and-its-application-in-bioscience/
Lam, A., St-Pierre, F., Gong, Y., Marshall, J., Cranfill, P., & Baird, M. et al. (2012). Improving FRET dynamic range with bright green and red fluorescent proteins. Nature Methods, 9(10), 1005-1012. http://dx.doi.org/10.1038/nmeth.2171
Lavdas, A. You May Not Know Theodor Förster but You Know His Work: FRET - Bitesize Bio. Bitesize Bio. Retrieved 16 July 2017, from http://bitesizebio.com/23012/you-may-not-know-theodor-forster-but-you-know-his-work-fret/
