Team:Wageningen UR/Results/Fluorescent

Choosing the best reporter

The aim of this project is to analyze different reporter proteins to choose the best. As our project signal is based on bimolecular complementation, the reporter proteins will be split and analyzed under reassembly directed through synthetic leucine zippers. The best reporter will have to fulfill a set of characteristics:

  • First, the reporter protein has to show a bright signal, so the device can detect easily if the system has been activated.
  • Second, the reassembly and maturation of the split reporter must be a fast as possible, to be able to detect a signal in a short timeframe.
  • Last, our device will be used in tropical areas, where temperatures can be really high. Therefore, the reporter must also be able to produce a strong signal and mature fast at high temperatures.

Choosing split sites

Constructs

Functional test for split proteins

Reassembly of split proteins in vitro

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).

Table 1: Values of Absorbance at the picks maximum values.
Protein Wavelength Max. Absorbance Max. Absorbance (Split)
anm2CP
mCerulean 440 nm 0.1002±0.0008 0.0001±0.0001
mRFP 560 nm 0.053±0.001 0.0017±0.0003

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 from the evolution of fluorescence over time, showed in Figure 2. The maturation rates can be found in Table 2.
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.
Table 3: Quantum Yields (QY) calculated for split proteins.
Protein QY Full Protein Reference QY Split Protein
mRFP 0.25 0.1±0.06
eYFP 0.61 0.004±0.027
Venus 0.57 0.61±0.06
sfGFP 0.65 1.3±0.2
Cerulean 0.62 0.5±7
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.

References

  1. Biéler, Sylvain, et al. "Evaluation of Antigens for Development of a Serological Test for Human African Trypanosomiasis." PloS one 11.12 (2016): e0168074.
  2. Sullivan, Lauren, et al. "Proteomic selection of immunodiagnostic antigens for human African trypanosomiasis and generation of a prototype lateral flow immunodiagnostic device." PLoS neglected tropical diseases 7.2 (2013): e2087.
  3. Overath, P., et al. "Invariant surface proteins in bloodstream forms of Trypanosoma brucei." Parasitology Today 10.2 (1994): 53-58.