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Revision as of 09:33, 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.

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

The extracellular domain of the Invariant Surface Glycoprotein 64 and 65, fused to both a StrepII-tag and 10x HIS-tag has successfully been purified using strep-tactin gravity column, see figure 3.

Figure 3: SDS gel of the protein fractions eluted from the strep-tactin column, both the flowthrough after loading the cell lysis onto the column, a few washing steps and the elution fractions.

The final 50 μl elution fraction (Elute 4) contains 283 μg/ml protein for rISG64, whereas the elution for rISG65 just contains 63 μg/ml protein. As seen from the high amounts of protein in the flowthrough, the column has reached its saturation point.

These tagged proteins, bound to the strep-tactin beads, are used for phage display selection.

Moreover, two biobricks were created of these constructs: BBa_K2387060 and BBa_K2387061. For this, the recombinant ISG gene, including the two tags, was cloned into the linearized pSB1C3 vector using biobrick assembly.

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.