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mRFP controlled by inducible araC/pBAD promoter

mRFP is a red monomeric fluorescent protein. In this composite, its expression is regulated by the araC/pBad promoter and by a strong RBS. mRFP presents a maximum pick of excitation at 584 nm and a maximum pick of emission at 607 nm. mRFP was engineered from DsRed to grant it a monomeric conformation.

Sequence and Features

Functional Parameters

In order to test the functionality of this protein under the araC/pBad promoter, the absorbance and the fluorescence spectra were measured after induction with 0.02% arabinose (Figure 1).

Figure 1: Absorbance and fluorescence spectra of mRFP, measured after induction with 0.02% arabinose.

This protein is part of a collection of split proteins developed for BiFC analysis and tested with the CpxR system (BBa_K2387032). These proteins were characterized through multiple experiments using as interacting proteins antiparallel synthetic leucine zippers. The proteins of this collection are the ones found in Table 1.

For comparing our split fluorescent proteins, the first factor we determined was a comparison between the performance of split fluorescent proteins versus the full length versions. For this test, we grew E. coli expressing split and full length fluorescent proteins in M9 minimal medium while measuring at the same time the evolution of fluorescence. The relative fluorescence of the split fluorescent proteins in relation to the full length proteins was calculated comparing the maximum values of fluorescence (Figure 2).

Figure 2: Graph showing the relative fluorescence of the split fluorescent protein in comparison to their full length versions.

As can be observed, split mVenus and split sfGFP excel in comparison to the other proteins, showing that the reassembly of the split portions of these proteins is more efficient than for other ones.

Another parameter we measured to choose the best reporter was the Quantum Yields (QY) of the split fluorescent proteins, which relates the light absorbed and the emitted fluorescence. Therefore, the QY can be used as an estimation for the brightness of the fluorescent proteins. The QY must be calculated in comparison to a reference using the same wavelength for excitation. The full proteins were used as standards for the split proteins, as their QY has already been calculated previously. The calculated QY can be found in Table 2.

As observed in Table 2, mRFP and eYFP show much lower QY when compared to the full proteins. Both mVenus and mCerulean show QY values similar to those of the full proteins, which indicates that these two proteins are good candidates in terms of brightness. However, the results show that the split sfGFP is even better than the full sfGFP. This may be due to background noise in the absorbance of the full sfGFP, which would alter the results. Nevertheless, although it is likely that splitting sfGFP does not increase the QY, the high value of QY for the split sfGFP show that this split protein can be considered bright.

The next factor we took into account was the maturation rate of the fluorescent proteins. This rate can be calculated as the half-time, which is the time at which half of the maximum fluorescence is reached. The maturation rates can be found in Table 3.

According to the results, eYFP and mCerulean are the fastest ones although the difference with sfGFP is not too big. However, we have already observed that eYFP shows a weak fluorescence intensity, so the time it takes it to reach a detectable fluorescence will be longer than the one for sfGFP and mCerulean.

The last characteristic we wanted our report to has was thermostability. We tested the evolution of fluorescence for 3 hours at different temperatures: 4°C, 10°C, 20°C (Room Temperature), 30°C, 45°C (possible temperature in tropical regions) and 60°C. The results are shown in Figure 3.

Figure 3: Differences of fluorescence after incubation for three hours at different temperatures. A t-test was used to analyze differences between the values at 20°C (room temperatures) and the values at the other temperatures.

The graph shows that two of the proteins generate the most fluorescence when incubated at 45°C: mRFP and sfGFP, both in their full-length and split versions However, for mCerulean the full protein and its split version showed different behaviours. While full-length mCerulean matures best at 45°C, its split version shows more fluorescence at 20°C. This difference may be caused by a lower structural stability of the split fragments of mCerulean at higher temperatures.

Protocols

Cell lysis

1. Inoculate 10mL of LB (+appropriate antibiotics) with the bacterial strain of choice and incubate overnight at 37ºC, 200rpm. 2. The next day, measure the OD600and spin the culture down at 4,700x g for 12 minutes. 3. Add 8μL of Lysozyme mix (50mg/mL) and 8μL of DNase mix to the pellet. 4. Resuspend the pellet in 400μL B-PER™ Bacterial Protein Extraction Reagent and transfer the solution to a microcentrifuge tube. 5. Incubate the tube for 15 minutes at room temperature. 6. Centrifuge at 15,000x g for 5 minutes.

Measuring Cell Lysates and Calculating Quantum Yields

1. Inoculate 10mL of LB (+appropriate antibiotics) with the bacterial strain of choice and incubate overnight at 37ºC, 200rpm. 2. To 5mL of LB (+appropriate antibiotics), containing 0.2% arabinose, add such a volume of overnight culture that the final OD600is 0.1. 3. Incubatethe new culture at 37ºC, 200rpm for 3 hours. 4. Afterwards, leave the cultures overnight at 4ºC, to allow for protein maturation. 5. Lyse samples according to the protocol(s) above and measure 100μLfrom three dilutions (1:1, 1:10 and 1:100, dilute with MQ) of the supernatants in the Synergy Mx™ Microplate Reader. Measure absorbance from 340nm to 750nm in intervals of 10nm. Measure the fluorescence for the fluorescent proteins were measured using the maximum excitation value. Emissions were recorded from 340nm to 750nm in intervals of 10nm. 6. Calculate the quantum yield, using the following formula:

QY=QY_std*(grad/grad_std)

QY: Quantum Yield QY_std:Standardized Quantum Yield grad:Gradient, arbitrary units grad_std: Standardized gradient, arbitrary units

“Grad” refers to the linear gradient that correlates absorbance and integrated fluorescence (area of the emission spectrum). The range of absorbance will be achieved with the different dilutions used in the fluorescence measurement (1:1, 1:10 and 1:100).

Measuring Fluorescence Generation Over Time

1. Inoculate 10mL of M9 medium (+appropriate antibiotics) with the bacteria containing the plasmids coding for full/split proteins and incubate overnight at 37ºC, 200rpm. 2. To 5mL of M9 (+appropriate antibiotics), add such a volume of overnight culture that the final OD600is 0.1. 3. In a 96-wells plate, mix 50μL of culture with 50μL of M9 media, containing the following final concentrations of arabinose: 0%, 0.004%, 0.04% and 0.4%. 4. Measure the plate in a Synergy Mx™ Microplate Reader during 18-24 hours at 30ºC, with fast shaking enabled. Make a measurement every 30 minutes, using the maximum values for excitation and emission for each protein.

Measuring the Effect of Temperature

1. Inoculate 10mL of LB medium (+appropriate antibiotics) with the bacteria expressing full/split proteins and incubate overnight in 37ºC, 200rpm. 2. To 5mL of LB (+appropriate antibiotics), containing 0.2% arabinose, add such a volume of overnight culture that the final OD600is 0.1. 3. Incubate at 37ºC, with shaking, for 2 hours. 4. Spin the culture down at 4,700x g for 12 minutes and discard the supernatant. 5. Resuspend the pellet in 5mL MQ and transfer to a 96-wells plate. 6. Incubate the 96-wells plate overnight at the following temperatures: 4ºC, 10ºC, 20ºC, 30ºC, 45ºC and 60ºC. 7. Use the Synergy Mx™ Microplate Reader to measure the fluorescence, using the maximum excitation and emission values for each protein.

Calculation of Maturation Rates

1. Inoculate 10mL of M9 medium (+appropriate antibiotics) with the bacteria expressing full/split proteins and incubate overnight in 37ºC, 200rpm. 2. To 5mL of LB (+appropriate antibiotics), containing 0.2% arabinose, add such a volume of overnight culture that the final OD600is 0.1. 3. Incubate at 37ºC, with shaking, for 2 hours. 4. Transfer cultures to a 96-wells plate and add geneticin until a final concentration of 50μM. 5. Measure in a Synergy Mx™ Microplate Reader for 3 hours, using the following settings:-Take measurement every 5 minutes of the OD600and the fluorescence, using maximum excitation and emission wavelengths for each protein as displayed in the table below. 6. Calculate the maturation rate, followingthe signal generation timeformula.

References

Guido, Jach, et al. "An improved mRFP1 adds red to bimolecular fluorescence complementation." Nature Methods 13.8 (2006): 597-600.

Nagai, Takeharu, et al. "A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications" Nature Biotechnology 20 (2002): 87-90.

Pédelacq, Jean-Denis, et al. "Engineering and characterization of a superfolder green fluorescent protein" Nature Biotechnology 24 (2004): 79-88.

Rizzo, Mark A., et al. "An improved cyan fluorescent protein variant useful for FRET" Nature Biotechnology 22 (2004): 445-449.