A set of monomeric chromoproteins (anm2CP, Ultramarine, Dathail) and monomeric fluorescent proteins (mRFP, eYFP, mVenus, mCerulean, sfGFP) was successfully transformed and expressed in E. coli BL21 DE3. At the start of our project, we were considering the use of chromoproteins as a possible reporter that would allow to detect the signal with the naked eye. Inducing the expression of the reporters using arabinose allowed us to observe potential candidates (Figure 1).

Figure 1: E. coli BL21 DE3 expressing chromoproteins and fluorescent proteins under induction with 0.2% arabinose.

Among the chromoproteins only anm2CP produced colored cells. Among the fluorescent proteins, mRFP produced bright pink cells and mCerulean produced bright yellow cells. The absorbance spectra of these proteins and their split versions were analyzed and in an attempt to compare quantitatively the color produced, the absorbance at the maximum pick was calculated and recorded (Table 1).

It is important to consider that this values only help us to compare between the same protein in its full and split version, because the color of the protein is caused by the full range of absorbance. For example, mRFP presents two picks and therefore it is expected that the intensity of the larger pick will still be lower that the intensity of the pick from mCerulean, which only shows one pick. Thus, with this data we cannot claim that mCerulean shows a more intense color than mRFP. The data on the table shows that the absorbance of the split proteins is lower than the absorbance of the full proteins. This correlates with the fact that colonies that expressed the split proteins showed no color. Between this fact and the development of our measurement device, we decided to continue analyzing only fluorescent proteins.

The first factor we took into account was the maturation rate of the fluorescent proteins. This rate can be calculated as the half-time obtained from the evolution of fluorescence over time, showed in Figure 2. The maturation rates can be found in Table 2.

Table 2: Maturation Rates (t1/2)
mRFP

Figure 2: Evolution of fluorescence after cells stop producing protein due to addition of chloramphenicol/geneticin in high concentrations. The increase in fluorescence is only be due to the maturation of the full and split fluorescent proteins.

According to the results, mVenus is the fastest one although the difference with mCerulean and sfGFP is not too big. Therefore, one of these three proteins would be a preliminary good candidate for our device.

As an additional paramter to choose the best reporter we calculated 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 estimate of the brightness of the fluorescent protein. 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 3.

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. However, 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 as brigth. Among the other proteins, mRFP and eYFP show lower QY than the one of the full proteins. Both mVenus and mCerulean show a QY similar to that of the full proteins, which indicates that these two proteins are good candidates in terms of brightness. As we had good results in both experiments with mVenus, we implemented mVenus to the CpxR-CpxR BiFC system in order to try to observe a decrease in the time necessary to observe the signal. Unfortunately, the system did not work properly with Venus as a reporter due to high noise.

The last characteristics we wanted our report to have was thermostability. Our split reporter should be able to mature and produce fluorescence in high temperatures, typical of tropical areas. 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 three hours at different temperatures. A statistical analysis using t-test was used to identify significant differences between the values at 20°C (room temperatures) and the values at other temperatures. ns: No significance(P>0.05); *: P≤0.05; **: P≤0.01; ***: P≤0.001.

The graphic show that only two split proteins generate most fluorescence at 45°C: mRFP and sfGFP. mCerulean is a curious case, as the full protein matures best at 45°C but the split protein as a maximum temperature of maturation at 20°C. This difference may be caused by the lower structural stability of the split fragments of mCerulean at higher temperatures. This lower structural stability does not take place in split mRFP and sfGFP due to the different sizes and sequence of the fragments between the fluorescent proteins. As split sfGFP has been proven to work at 45°C, as well as being bright and fast, it is our perfect candidate. We could not implement it in the CpxR-CpxR BiFC system due to time constrains, but we are positive that using this reporter will improve our device