Difference between revisions of "Team:Wageningen UR/Results/Fluorescent"

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<h4>Choosing split sites</h4>
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<h4>Constructs</h4>
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<h4>Functional test for split proteins</h4>
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<h4>Reassembly of split proteins <i>in vitro</i></h4>
 
 
 
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Revision as of 10:21, 25 October 2017

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

Figure 1: E. coli BL21 DE3 expressing chromoproteins and fluorescent proteins under induction with 0.2% arabinose.
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
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 2: 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.