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Measurment

To achieve the best measurement of our oscillating system, we chose two different kinds of reporter gene as our downstream reporter, fluorescent protein gene and luciferase gene, which are both widely used in the biotechnological labs.

Fluorescent protein

In search for the part kit provided by the iGEM headquarter, we found several available fluorescent proteins. Under the instruction of our fluorescent protein judging model, we finally chose mCherry and EYFP as our fluorescent protein reporter.

Table 1. Chosen fluorescent proteins
Protein	λ_em(nm)	λ_em(nm)	part serial number	site
mCherry	587	610	Part:BBa_E2060	3-7L
EYFP	513	527	Part:BBa_E2030	3-7J

Figure 1. Mechanism of fluorescent proteins

Luciferase

Luciferase is a popular choice as a reporter for gene expression because functional enzyme is created immediately upon translation and the assay is rapid, reliable and easy to perform.^[1,2] The fluorescent output of luciferase gene is due to the chemical reaction between luciferase and corresponding substrate, which can be easily detected by the luminoskan microplate reader.
We used firefly luciferase(Fluc) gene purchased from Promega and Nanoluc luciferase(Nluc) gene submitted by Team Tuebingen 2015 as another two reporters for our KaiABC system.

Table 2. Chosen luciferase
luciferase	λ_em(nm)	resource	Detail
Fluc	560	Bought from Promega	pGL4 Luciferase Reporter Vectors
Nluc	460	Part:BBa_K1680009	Kit site:7-3N

Figure 2. Mechanism of luciferase

Figure 3.The bioluminescent reaction catalyzed by Fluc and Nluc

How did we design our measurement plan?

When reconstructed in Escherichia coli, KaiABC oscillator showed a periodicity of 24 hours^[3]. To best verify our system can function in Saccharomyces cerevisiae , we planned to measure our system using different reporter at 3-hour intervals for 1 day, 2 days, 3 days respectively.
As for the measurement of fluorescent proteins, we not only used the common detecting instrument, fourescence spectrophotometer, but also make an attempt to record the fluorescent variation via the cutting-edge Leica DMi8 inverted microscope.
Another thing worth mentioning is that, we bought all of the luciferase assay system from company Promega, among them there was a new kind of Nanoluc luciferase system----Nano-Glo^® Live Cell Assay System, which allows experimenters to detect live cell luminescent signals without lytic process, suitable for the 3-day assay. Despite there is no reference indicating that this product, a commonly used assay system in mammalian cells, could be used in Saccharomyces cerevisiae, we decided to take a try and have successfully proved its validity. Consequently, we chose Nluc solely to complete our long time luminescent detection .

Table3.1 day’s measurement ----- Fluorescent protein detection
Experimental Group	pABaC +pCiRbS +gal1+mCherry
	pABaC +pbCiRS +gal1+mCherry
	pABaC +pCiRbS+gal1+EYFP
	pABaC +pbCiRS +gal1+EYFP
Negative Control Group	pABaC +gal1+mCherry
	pCiRbS +gal1+mCherry
	pbCiRS +gal1+mCherry
	pABaC +gal1+EYFP
	pbCiRS +gal1+EYFP
	pCiRbS +gal1+EYFP

Figure 4. Procedures for measuring mCherry/EYFP via fluorescent spectrophotometer

Figure 5. Procedures for measuring luciferase via luminaskan microplate reader

Detailed methods are available in our Experiment.

Table 4.2 days’ measurement ----- Fluc&Nluc detection in lytic cells
Experimental Group	pABaC +pCiRbS +gal1+Fluc
	pABaC +pbCiRS +gal1+Fluc
	pABaC +pCiRbS +gal2+Nanoluc
	pABaC +pbCiRS +gal2+Nanoluc
Negative Control Group	pABaC +gal1+Fluc
	pbCiRS +gal1+Fluc
	pCiRbS +gal1+Fluc
	pABaC +gal2+Nanoluc
	pbCiRS +gal2+Nanoluc
	pCiRbS +gal2+Nanoluc

Table 5.3 days’ measurement ----- Nluc detection in live cells
Experimental Group	pABaC +pCiRbS +gal1+Nanoluc
	pABaC +pbCiRS +gal1+Nanoluc
	pABaC +pCiRbS +gal2+Nanoluc
	pABaC +pbCiRS 1+gal2+Nanoluc
Control Group	pABaC +gal2+Nanoluc
	pbCiRS +gal2+Nanoluc
	pCiRbS +gal2+Nanoluc
	TDH3P+NanoLuc

EYFP measurement using Leica DMi8

Leica DMi8 is an excellent inverted microscope for life science research, which is utilized extensively in materials inspection and measurement. For live cell research, the DMi8 platform is a complete solution whether precisely follow the development of a single cell in a dish, screen through multiple assays, obtain single molecule resolution, or tease out behaviors of complex processes.
Combined with easy to use software, high resolution cameras, and brilliant LED illumination, these inverted research systems are flexible to see the expression of the fluorescent protein EYFP in Saccharomyces cerevisiae. Through high speed imaging, using the external fluorescent filter wheels, we obtained fast and accurate fluorescent images as following show. It tells that our bacteria that we constructed express EYFP successfully and continuously in oscillating system. Meanwhile, the fluorescence intensity detected was very bright, indicating that the expression of EYFP was at a high level.

Figure 6. pABaC +pCiRbS+gal1+EYFP photoed by Leica DMi8

Measuement result

Figure 7. Experimental result reported by pABaC+pbCiRS+gal1+Fluc

Figure 8. Experimental result reported by pABaC+pCiRbS+gal1+Fluc

Figure 9. Experimental result reported by pABaC+pbCiRS+gal1+Nluc
(obtained by live cell assays)

Figure 10.Experimental result reported by pABaC+pCiRbS+gal1+Nluc
(obtained by live cell assays)

Figure 11.Experimental result reported by pABaC+pbCiRS+gal2+Nluc
(obtained by live cell assays)

Figure 12.Experimental result reported by pABaC+pCiRbS+gal2+Nluc
(obtained by live cell assays)

Some basic information can be gained from the above figures:

Compared with the corresponding control groups, all the experimental groups have showed apparent oscillatory signals.
In the measured 3500 min, the oscillations in 700-2000 min are extremely unstable and nearly absent, while the oscillations for the rest of the time are relatively stable.
The cycle is about 500min (about 8.3h).

Note: final measurement results and analysis should be accessed in Result page.

Measuement instrument

Fluorescent protein:
Hitachi F-2500 Flourescence Spectrophotometer;
Leica DMi8 inverted microscope
Luciferase : Thermoscientific Luminoskan Microplate Reader

References

[1]Ow, D.W. et al. (1986) Transient and stable expression of the firefly luciferase gene in plant cells and trans-genic plants. Science 234, 856–9.

[2]De Wet, J.R. et al. (1987) Firefly luciferase gene: Structure and expression in mammalian cells. Mol. Cell. Biol. 7, 725–37.

[3]Chen, AH (Chen, Anna H.); Lubkowicz,D (Lubkowicz, David); Yeong, V (Yeong, Vivian); Chang, RL (Chang, Roger L.); Silver, PA(Silver, Pamela A.) Transplantability of a circadian clock to a noncircadian organism. SCIENCE ADVANCES(2015) DOI: 10.1126/sciadv.1500358

Team:Tianjin/Measurement

References