Team:Estonia TUIT/Contribution

The iGEM Registry of Standard Biological Parts collects genetic parts that iGEM teams and researchers can freely use to engineer biological systems. It makes research feasible, facilitating the growth of synthetic biology. Estonia_TUIT iGEM team has worked each year to broaden the iGEM parts collection, contributing to the global development of synthetic biology, and this year was no exception.

Fluorescent proteins

This year, our team decided to focus on fluorescent proteins, which have made a breakthrough in molecular biology. They have opened up new experimental approaches to monitor gene expression, screen transfected cells, and observe proteins in cells. After examining the iGEM fluorescent protein database, we chose EYFP, ECFP, mRFP1, mOrange, and GFPmut3 for further research.

We list the key features of the selected fluorescent proteins in Table 1:

Table 1. Fluorescent proteins found in the iGEM Registry page.

Protein	Full name	Fluorescence color	Registry part number
ECFP	Enhanced cyan fluorescent protein	Cyan	BBa_E0020
GFPmut3	Green fluorescent protein	Green	BBa_E0040
EYFP	Enhanced yellow fluorescent protein	Yellow	BBa_E0030
mOrange	Monomeric orange fluorescent protein	Orange	BBa_E2050
mRFP1	Monomeric red fluorescent protein	Red	BBa_E1010

Enhanced Cyan Fluorescent Protein (ECFP):

ECFP was obtained by mutation of tyrosine into tryptophan (Y66W) in the part of the GFP molecule that determines the color (Golub et al., 2019). Even though ECFP possesses a low quantum yield, indicating poor efficiency of photon absorption and emission, it is still a widely used fluorescent protein. The reasoning for the use of ECFP is its photostability (85 s) (ECFP :: Fluorescent Protein Database).

ECFP exhibits an excitation peak at 434 nm wavelength and an emission peak at 477 nm.

The emission and excitation spectra of ECFP are shown in Figure 1.

Figure 1. The emission and excitation spectra of ECFP (ECFP :: Fluorescent Protein Database). The violet line corresponds to the excitation spectrum, the blue line corresponds to the emission spectrum. The excitation maximum of 434 nm, the emission maximum of 477 nm, the extinction coefficient of 32500M-1*cm-1 and the quantum yield of 0.4 were recorded.

The settings for the confocal Nikon microscope A1 HD25/A1R HD25 for imaging of ECFP can be seen in Table 2 (Imaging Fluorescent Proteins | Nikon’s MicroscopyU).

Table 2. Confocal Nikon microscope A1 HD25/A1R HD25 settings for ECFP.

Excitation Laser (nm)	Excitation Filter CWL / BW (nm)	Dichromatic Mirror Cut-On (nm)	Barrier Filter CWL / BW (nm)	Relative Brightness (% of EGFP)
Diode (440)	435/40	460LP	495/50	39

Green Fluorescent Protein (GFPmut3):

GFPmut3 is a simple (constitutively fluorescent) green fluorescent protein derived from Aequorea victoria that was first published in 1996 (Tsien, 2003). It is a weak dimer that matures very quickly. The green fluorescent protein (GFP) is a protein that emits bright green fluorescence when exposed to light in the blue to ultraviolet range (Figure 2). It was first obtained from the jellyfish Aequorea victoria, and it is also known as avGFP. Other organisms with GFPs have been found, including corals, sea anemones, zoanthids, copepods, and lancelets (Prendergast & Mann, 2002).

It was discovered that the absorbance and fluorescence of GFP mutants are strongly pH-dependent in aqueous solutions and the intracellular compartments. pH titrations of purified recombinant GFP mutants indicated >10-fold reversible changes in absorbance and fluorescence with pKa values of 6.0 (GFP-F64L/S65T) with an apparent Hill coefficient of approximately 1 (M et al., 1998).

Figure 2. The emission and excitation spectra of GFPmut3b (GFPmut3b:: Fluorescent Protein Database).
The light green graph corresponds to GFPmut3 excitation spectra, and the neon green graph corresponds to GFPmut3 emission spectra. It can be seen that the excitation maximum for the GFPmut3 is at 500 nm, and the emission maximum is at 513 nm. The extinction coefficient was 89,400 M-1cm-1. The quantum yield was 0.39.

Table 3. Confocal Nikon microscope A1 HD25/A1R HD25 settings for GFP (wt) (ECFP :: Fluorescent Protein Database).

Excitation Laser (nm)	Excitation Filter CWL / BW (nm)	Dichromatic Mirror Cut-On (nm)	Barrier Filter CWL / BW (nm)	Relative Brightness (% of EGFP)
Argon (488)	450/50	480LP	510/50	48

Enhanced Yellow Fluorescent Protein (EYFP):

EYFP is a rapidly-maturing yellow/green fluorescent protein derived from Aequorea victoria (Nagai et al., 2002a) that can be used for both mammalian and bacterial gene expression studies (Enhanced Yellow Fluorescent Protein (EYFP) Tubulin Localization | Nikon’s MicroscopyU). It is a weak dimer with high acid sensitivity that can be quenched by chloride ion (Cl-) (Nagai et al., 2002). Besides fast maturation of only 9 minutes, EYFP has a quite high quantum field of 0.67 units (EYFP :: Fluorescent Protein Database). EYFP is one of the brightest fluorescent proteins (brighter than EGFP, ECFP, and mRFP1) (Jusuk et al., 2015) and therefore is specifically useful for super-resolution microscopy. The photostability of EYFP is 60 seconds which is lower than that of GFP and ECFP.

EYFP has an excitation maximum at 513 nm wavelength and emission peak at 527 nm wavelength that can be seen on Figure 3.

Figure 3. The emission and excitation spectra of EYFP (EYFP :: Fluorescent Protein Database, n.d.).

The confocal microscopy setup for vizualisation of EYFP protein can be found in Table 4

Table 4. Fluorescent Nikon microscope A1 HD25/A1R HD25 settings for EYFP.

Excitation Laser (nm)	Excitation Filter CWL / BW (nm)	Dichromatic Mirror Cut-On (nm)	Barrier Filter CWL / BW (nm)	Relative Brightness (% of EGFP)
Argon (514)	490/40	515LP	540/30	151

Monomeric orange fluorescent protein (mOrange):

mOrange is an extremely bright orange fluorescent protein that can be expressed both in bacterial and mammalian cells (Kremers et al., 2009). It has a high extinction coefficient and quantum yield, which allows it to be a perfect FRET (fluorescence resonance energy transfer) acceptor. mOrange has a half-life time of 2.5 hours for maturation at 37°C (mOrange :: Fluorescent Protein Database).

Due to a low photostability (6.4 s), which is approximately 5 % that of EGFP, its usage is limited. However, by applying a directed evolution approach to select for higher photostability, the drawback was overcome. This improvement resulted in the mOrange2 derivative, which has a photostability 30% higher than EGFP (Formation Stages of the mOrange Fluorescent Protein Chromophore).

Figure 4. The emission and excitation spectra of mOrange (mOrange:: Fluorescent Protein Database). It can be seen on Figure 4 that the excitation maximum for the mOrange is at 548 nm, and the emission maximum is at 562 nm. The extinction coefficient was 71000 M^-1cm^-1. Quantum yield is 0.69.

In the Nikon microscope, settings for mOrange imaging are as in the Table 5.

Table 5. Confocal Nikon microscope A1 HD25/A1R HD25 settings for mOrange (Imaging Fluorescent Proteins | Nikon’s MicroscopyU).

Excitation Laser (nm)	Excitation Filter CWL / BW (nm)	Dichromatic Mirror Cut-On (nm)	Barrier Filter CWL / BW (nm)	Relative Brightness (% of EGFP)
He-Ne (543)	525/40	550LP	585/50	146

Monomeric red fluorescent protein 1 (mRFP1):

mRFP1 is a substantially mutated monomeric form of DsRed, cloned from Discosoma coral (Jach et al., 2006). mRFP1 was obtained from the poorly fluorescent dimer T1-I125R of DsRed by applying directed evolution with a combination of targeted and random mutagenesis. mRFP1 is ideal for multicolor imaging in combination with GFP as they have minimal spectral overlap. Thus, mRFP1 has almost no emission when excited at wavelengths optimal for GFP and vice versa. mRFP1 is relatively rapidly maturing, with a maturation time of 60 minutes at 37 °C. mRFP1 has low acid sensitivity with its pKa equaling 4.5 (Campbell et al., 2002). Its photostability half-life is 6.2 s, and it can be used in both mammalian (HeLa) and bacterial (E. coli) cells (Campbell et al., 2002).

Figure 5. The emission and excitation spectra of mRFP1. (mRFP1 :: Fluorescent Protein Database, 2021). The light orange line corresponds to the absorption spectrum, the dark orange line corresponds to the excitation spectrum, the red line corresponds to the emission spectrum. The excitation maximum of 584 nm, the emission maximum of 607 nm, the extinction coefficient of 50000M-1*cm-1 and the quantum yield of 0.25 were recorded.

Confocal microscopy settings for mRFP1 can be found in Table 6.

Table 6. Confocal Nikon microscope A1 HD25/A1R HD25 settings for mRFP1 detection (Imaging Fluorescent Proteins | Nikon’s MicroscopyU).

Excitation Laser (nm)	Excitation Filter CWL / BW (nm)	Dichromatic Mirror Cut-On (nm)	Barrier Filter CWL / BW (nm)	Relative Brightness (% of EGFP)
Diode (594)	560/55	590LP	630/60	37

Characterization of yeast inducible promoters

The second part of our contribution this year was yeast promoter characterization. We examined iGEM registry parts and chose GAL1 and CUP1 inducible promoters (Table 7), and we screened scientific articles to find available information on these promoters. For promoter characterization, we cloned the gene encoding for Venus fluorescent protein under GAL1 or CUP1 promoters in different copy number plasmids. Plasmids were transformed into yeast cells, followed by fluorescent microscopy to analyze the Venus expression level.

Table 7. The main features of the promoters.

Promoter	Type	Gene regulated by the promoter and its function	Registry part number
GAL1	Galactose-inducible,bidirectional (Peng et al., 2015)	The promoter controls a gene encoding GAL1, a galactokinase that drives phosphorylation of alpha-D-galactose to alpha-D-galactose-1-phosphate as the primary step of galactose catabolism (GAL1 | SGD).	BBa_J63006
CUP1	Copper ions inducible promoter (Leblanc et al., 2000a)	CUP1 promoter regulates the expression of the CUP1 gene encoding for metallothioneins, molecules responsible for metal binding and controlling metal toxicity (Wang et al., 2016a).	BBa_K586000

CUP1 promoter

CUP1 promoter regulates the expression of the CUP1 gene encoding for metallothioneins, molecules responsible for metal binding and controlling metal toxicity (Wang et al., 2016b). S. cerevisiae CUP1 promoter is a copper-inducible promoter. Copper ions bind to the N-terminal domain of the transcriptional activator Ace1 to cause a conformational change that triggers recognition of the activating sequences in the CUP1 promoter. Transcriptional activation occurs through the C-terminus of Ace1. CUP1 can also be induced with heat shock instead of copper ions (copper-independent pathway, (Leblanc et al., 2000b)).

GAL1 promoter

GAL1 promoter regulates the expression of the galactokinase gene. It is induced by galactose and repressed by glucose. The presence of galactose causes a 1000-fold increase in GAL1 gene transcription. Regulatory proteins Gal4 and Gal80 control the transcription. Without galactose, Gal80 inhibits GAL gene transcription. Galactose induction removes the Gal80 inhibition complex, allowing transcription activation by GAL4, which binds the upstream region of the GAL gene (Flick & Johnston, 1990).

Plasmid construction

To characterize CUP1 and GAL1 promoters, we constructed plasmids using Golden Gate assembly and the MoClo yeast toolkit parts (Lee et al., 2015). Four plasmids were designed: low-copy number (CEN6/ARS origin of replication), and integrative plasmids (URA 3'-homology) with either GAL1 or CUP1 promoter. We cloned the gene encoding for the Venus fluorescent reporter protein to these vectors under the control of our target promoters. The idea behind that is to detect, quantify and compare Venus fluorescence signals from both promoters and from plasmids with different copy numbers. The main features of the constructed plasmids are shown in Table 8.

Table 8. The main features of constructed plasmids

Promoter	Reporter	Origin of replication	Yeast integration site	Yeast selection gene	Copy number
pCUP1	Venus	CEN6/ARS4	-	URA3	Low
pCUP1	Venus	-	URA 3'-homology	URA3	Low
pGAL1	Venus	CEN6/ARS4	-	URA3	Low
pGAL1	Venus	-	URA 3'-homology	URA3	Low

Yeast strain construction

Constructed integration vectors were restricted with NotI and used for transformation of S. cerevisiae DOM90 (MATα {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]) strain. The CEN6/ARS4 plasmids were used without linearization. Transformants were selected for URA+ phenotype on uracil-dropout CSM plates containing 2% glucose. All yeast strains generated and used for promoter characterization are listed in Table 9.

Table 8. The main features of constructed plasmids

Strain name	Genotype	Description
DOM90	MATα {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]	Background strain without Venus (control)
ET51	pYTK074-CEN-pCUP1-Venus-tENO1-URA3	Strain with Venus under CUP1 promoter, low copy number
ET52	ura3-1::pYTK074-Integration-pCUP1-Venus-tENO1-URA3	Strain with Venus under CUP1 promoter, integration into ura3-1 locus
ET53	pYTK074-CEN-pGAL1-Venus-tENO1-URA3	Strain with Venus under GAL1 promoter, low copy number
ET54	ura3-1::pYTK074-Integration-pGAL1-Venus-tENO1-URA3	Strain with Venus under GAL1 promoter, integration into ura3-1 locus

Microscopy

Before microscopy, ET51, and ET52 strains were grown in 3 ml of uracil-dropout CSM (Complete Supplement Mixture, 100 mM MES buffer (2-ethanesulfonic acid diluted in 6M NaOH till pH=5.5); 2% glucose) for 3 hours at 30˚C. After that, every strain culture was split into two: in one 300 µM CuSO4 was added and the second culture was used as a no-induction control. Cultures were grown for another 3 hours to OD600 0.2-0.8. ET53, and ET54 strain cultures were grown in 3 ml of uracil-dropout CSM (2% raffinose) for 3 hours at 30˚C. After that, every strain culture was splitted into two: in one 2% galactose was added and the second culture was used as a no-induction control (with 2% glucose for GAL1 promoter repression). Cultures were grown for another 3 hours to OD600 0.2-0.8. After that, 0.5 µl of cell culture was pipetted onto a 0.08 mm cover glass slip and covered with 1.5% agar-CSM (low melting temperature agarose was used). Zeiss Observer Z1 microscope with an automated stage, 63C/1.4NA oil immersion objective, and Axiocam 506 mono camera was used for imaging. During imaging, the focus was kept using Definite Focus and the sample was kept at 30 °C using PeCon TempControl 37-2. ImageJ was used for image processing.

Results

We characterized the expression from GAL1 and CUP1 promoters using Venus fluorescent protein as the reporter gene. The expression cassettes were either integrated into the yeast genome or on a centromeric low-copy plasmid, and the Venus expression was monitored in the presence and absence of the induction. GAL1 promoter was induced by the addition of galactose and CUP1 promoter with CuSO4. The experiments confirmed that both studied promoters are strongly regulated by the presence of an inducible agent, as the Venus fluorescence intensity increased drastically upon induction (Figure 6) of both promoters. The expression from the CUP1 promoter was slightly higher than the Venus expression from the GAL1 promoter. Further, expression from the chromosomal locus was lower than from the plasmid. These experiments confirm that GAL1 and CUP1 are tightly-regulated promoters that mediate strong expression of the gene in the presence of the inductor.

Figure 6. Expression from GAL1 and CUP1 promoters is tightly controlled.

The activity of promoters was measured by quantifying the fluorescence intensity of Venus that is expressed from the respective promoters. The bars show mean fluorescence signals, error bars show standard deviation.

References

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EYFP :: Fluorescent Protein Database. (n.d.-b). Retrieved October 19, 2021, from https://www.fpbase.org/protein/eyfp/

Flick, J. S., & Johnston, M. (1990). Two Systems of Glucose Repression of the GAL] Promoter in Saccharomyces cerevisiae. MOLECULAR AND CELLULAR BIOLOGY, 10(9), 4757–4769.

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