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When devising a reporter for our measurement system we figured that optical feedback is both, a very simple and low-tech mechanism and a mechanism that can be measured qualitatively and quantitatively. In the end we came up with the standard reporter luciferase (LUC) but with a twist.

Generally, the luciferase-luciferin reaction in yeast is rather problematic due to a specific peroxisomal targeting signal at the C-terminus of the luciferase (Gould et al., 1989), heterologously expressed insect luciferase (from Photinus pyralis in our case) is transported into peroxisomes (Gould et al., 1990). “When the luciferase is transported into the peroxisome the concentration of externally added D-luciferin may be a limiting factor, as it has to penetrate across both the cytoplasmic and peroxisomal membranes to find its target” (Leskinen et al., 2003).

Since our system is based on a yeast chassis we had to face this very fundamental problem: luciferase might be a simple reporter and light-emission is easy measurable but the problem lies in the chassis itself due to this luciferin bottleneck that might increase measurement times. Normally, one would have to break down the yeast cells in order to measure luciferase activity in the cell lysates. However, the extraction of cell lysates is either time-consuming (e.g. autolysis takes up to 48h), needs additional enzymes (e.g. enzymatic lysis), elaborate machines (e.g. homogenization or freezing and grinding), or results in variable breakage (e.g. glass bead vortexing) (European Molecular Biology Laboratory). Even when one has obtained a perfect cell lysate the light emission is still dependent on the medium’s pH. The whole measurement protocol would be rather complicated and would oppose our low-tech idea.

In their study on heterologously expressed insect luciferase in yeast Leskinen et al. (2003) removed the peroxisomal targeting signal by modifying the enzyme’s gene accordingly. The modified luciferase did not accumulate in peroxisomes anymore which led to higher growth rates in comparison to unmodified luciferase (Leskinen et al., 2003). Also, “in all measurements the modified luciferase construct gave at least two orders higher levels [of fluorescence] than the wild-type luciferase” (Leskinen et al., 2003). Most importantly, with the modified luciferase cell lysis is not necessary anymore when making bioluminescence measurements and “it is possible to measure luciferase activity from intact living yeast cells just be adding D-luciferin solution” (Leskinen et al., 2003). According to Leskinen et al. (2003) background luminescence is reduced when using the modified luciferase together with an inducible promoter – which is exactly what we are planning. Therefore, we are confident that even quantitative luminescence measurements will be possible with our progestin measurement system when using this modified luciferase.

Leskinen et al. (2003) found that for their modified luciferase normalized luminescence does not depend on cell number as long as all measurements are conducted during the log-phase of growth and pH dependency is reduced. This is very beneficial for our system since only minor calculations have to be made when analyzing luminescence data. Also, both luminescence measurements as well as the determination of growth phase of the cells can be made with the same simple photometer.

All in all, the modified luciferase meets all our requirements for low-tech measurements. In our measurement system we will place the luciferase gene downstream of a promoter which is regulated by our inverter.

References

EUROPEAN MOLECULAR BIOLOGY LABORATORY. Protein Purification - Extraction and Clarification - Preparation of cell lysates from yeast [Online]. Available: http://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/cell_lysates_yeast/ [Accessed 2.10. 2013].

GOULD, S. J., KELLER, G. A., HOSKEN, N., WILKINSON, J. & SUBRAMANI, S. 1989. A conserved tripeptide sorts proteins to peroxisomes. The Journal of Cell Biology, 108, 1657-1664.

GOULD, S. J., KELLER, G. A., SCHNEIDER, M., HOWELL, S. H., GARRARD, L. J., GOODMAN, J. M., DISTEL, B., TABAK, H. & SUBRAMANI, S. 1990. Peroxisomal protein import is conserved between yeast, plants, insects and mammals. Embo j, 9, 85-90.

LESKINEN, P., VIRTA, M. & KARP, M. 2003. One-step measurement of firefly luciferase activity in yeast. Yeast, 20, 1109-1113.