Team:MSU-Michigan/Measurement

Measurement

Using Current for Protein Expression

MSU-iGEM 2017 developed a measurement tool that researchers throughout synthetic biology can utilize. Green fluorescent proteins (GFP) are used under aerobic conditions to signal protein expression (2,5,8). Unfortunately, for researchers working under anaerobic conditions, GFP does not mature and thus fluoresce under anaerobic conditions (3). Therefore, we showed that current induced via IPTG produces a reproducible signal for protein expression under anaerobic conditions. Current induction occurs 18 min after IPTG is added displaying a more rapid response than GFP expression. This measurement tool even proves to be more efficient than GFP expression under aerobic conditions due to the rapid current increased after induction. Also, current can be measured using simpler measuring tools than other anaerobic protein expression tools. Some examples of these other tools include: measuring hydrogen production and sampling for several metabolic fluxes (4,7,9). MSU-iGEM’s simplistic bioreactors utilizing Arduino boards(1,6,10) are a model example of a system that can quantitatively measure protein expression via current increase in a more versatile way than using tools for measuring GFP fluorescence.

GFP Fluorescence Measurement Study

MSU iGEM character demo

Discussion

Current was measured anaerobically and samples were taken before and after IPTG was added to allow the GFP to mature with exposure to oxygen for one hour. These measurements were then correlated to the current data along the same time scale. When comparing the graphs, current shows a response approximately in 18 min. and a peak about 13 hours post IPTG. On the contrary, 13 hours was needed to just allow for enough GFP protein to be made to then see a response once aerobically matured. This displays the advantage of current as a reporter for protein expression.

References

(1) Aristizabal, D. H.; Giraldo, D. A.; Sanchez, S.; Taborda, G.; Baeza, A. J. Phys. Conf. Ser. 2017, 365, 11001.
(2) Chiu Wan-Ling; Niwa, Y.; Zeng, W.; Hirano, T.; Kobayashi, H.; Sheen, J. Curr. Biol. 1996, 6 (3), 325–330.
(3) Drepper, T.; Eggert, T.; Circolone, F.; Heck, A.; Krauß, U.; Guterl, J.-K.; Wendorff, M.; Losi, A.; Gärtner, W.; Jaeger, K.-E. Nat. Biotechnol. 2007, 25 (4), 443–445.
(4) Fuchs, S.; Pané-Farré, J.; Kohler, C.; Hecker, M.; Engelmann, S. J. Bacteriol. 2007, 189 (11), 4275–4289.
(5) Heim, R.; Prasher, D. C.; Tsien, R. Y. Proc. Natl. Acad. Sci. 1994, 91 (26), 12501–12504.
(6) Jannelli, N.; Anna Nastro, R.; Cigolotti, V.; Minutillo, M.; Falcucci, G. Appl. Energy 2017, 192, 543–550.
(7) Mus, F.; Dubini, A.; Seibert, M.; Posewitz, M. C.; Grossman, A. R. J. Biol. Chem. 2007, 282 (35), 25475–25486.
(8) Scholz, O.; Thiel, A.; Hillen, W.; Niederweis, M. Eur. J. Biochem. 2000, 267 (6), 1565–1570.
(9) Sørensen, H. P.; Mortensen, K. K. Journal of Biotechnology. 2005, pp 113–128.
(10) Yang, Y.; Ren, H.; Ben-Tzvi, P.; Yang, X.; He, Z. Int. J. Hydrogen Energy 2017, 42 (31), 20260–20268.

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