Difference between revisions of "Team:Wageningen UR/Results/Cpx BiFC"

Line 18: Line 18:
 
<li><a href="https://2017.igem.org/Team:Wageningen_UR">Home</a></li>
 
<li><a href="https://2017.igem.org/Team:Wageningen_UR">Home</a></li>
 
<li><a href="https://2017.igem.org/Team:Wageningen_UR/Results">Results</a></li>
 
<li><a href="https://2017.igem.org/Team:Wageningen_UR/Results">Results</a></li>
−
<li>Affinity molecule library</li>
+
<li>Cpx BiFC</li>
 
</ul>
 
</ul>
 
</div>
 
</div>

Revision as of 11:26, 24 October 2017

Antigen visualization using BiFC

In order to visualize antigen binding, we use E. coli's Cpx two-component signal transduction pathway. We combine its protein-protein interactions with Bimolecular Fluorescence Complementation and show how we obtain rapid and specific visual response upon activation!

We now know the native function of E. coli's Cpx two-component signal transduction pathway, and how we can use this system to detect and bind antigens (Link to ./Results/Cpx_System.html). However, we need to create to create an output signal to visually show the presence of antigen. To do so we can use the native protein-protein interactions of the Cpx pathway to our advantage.

We use visualization method called Bimolecular Fluorescence Complementation (BiFC). BiFC is based on the association of fragments of a fluorescent reporter protein fused to interacting target proteins [1] (Figure 1). A fluorophore can be split into two non-fluorescent fragments which reassemble into a fluorescent complex upon interaction between the aforementioned target proteins. BiFC can directly be used in living cells, and does not need addition of substrates. Another advantage is that a simple photo spectrometer is enough to measure the signal. We use fluorescent protein eYFP, a commonly used reporter in BiFC (Kerppola et al).

Visualization strategy

We use several protein interactions of the Cpx pathway to visualize antigen binding. Upon activation of the Cpx pathway, CpxP gets titrated away from CpxA which is activated and autophosphorylates. This phosphogroup is then transferred to CpxR, which can then homodimerize [REF]. By fusing split reporter proteins to these Cpx-proteins, Cpx pathway activation can directly be visualized! We decided to link CpxR dimerization (figure 2A) and CpxA-CpxR interaction (Figure 2B) to eYFP-termini, which are often used in BiFC.

A third possible method uses specific cleavage via TEV protease. TEV is fused to CpxR whereas eYFPn and eYFPc are fused to CpxA. The eYFP-termini are fused to antiparallel leucine zippers. Upon Cpx activation, CpxA and CpxR interact and the eYFP-Zipper fusions are cleaved off of CpxA and they can freely move through the cytoplasm. The leucine zippers have natural affinity for each other and will act as target proteins in BiFC and facilitate eYFP recomplementation (Figure 2C)

Figure 2: A) eYFPn is fused to CpxA, and eYFPc is fused to CpxR. This way, BiFC is used to visualized the phosphorylation step of the Cpx pathway. B) eYFPn and eYFPc are fused (seperately) to CpxR. This way BiFC is used to visualize the CpxR dimerization step. C) TEV protease is fused to CpxR, and eYFPn and eYFPc are fused to CpxA (seperately). Upon Cpx pathway activation, the eYFP-termini are cleaved off of CpxA. Leucine zippers are fused to the eYFP-termini to facilitate reassembly.

Cloning methods

To visualize Cpx activation via the aforementioned protein-interactions, constructs have to be made to fuse eYFP-termini to Cpx components. CpxA and CpxR were amplified from E. coli K12 genome using Q5 Polymerase. Leucine zippers were ordered and synthesized. eYFP was split between amino acids 154 and 155 (Kerppola et al). CpxA fusions were made at its cytoplasmic C-terminus; CpxR fusions were made at its C-terminus which was found to be optimal for fluorophore reassembly (2014 EFPL link)
All constructs were put under control of the inducible pBAD/araC promoter to enable controlled protein expression, and strong ribosome binding site (RBS) BBa_B0034 was placed upstream of the created fusions. All CpxR-containing fusions were cloned into high copy number standard backbone pSB1C3, whereas all CpxA-containing fusions were cloned into medium copy number backbone pSB3T5. This was done to prevent overexpression and subsequent formation of inclusion bodies containing membrane protein CpxA. All parts were brought together via Golden Gate Assembly using restriction enzyme Bsa1 and T4 Ligase, and controlled by Restriction Digest Analysis and subsequent sequencing.

Figure 3: A) Created translational units used to visualize Cpx activation. All units are under control of the inducible araC/pBAD promoter and strong RBS BBa_B0034. A) eYFPn is fused to the C-terminus of CpxA via a flexible linker; eYFPc is fused to the C-terminus of CpxR. B) eYFPn and eYFPc are fused to the C-terminus of CpxR via flexible linkers. C) eYFPn-nZIPPER and eYFPc-cZIPPER are fused to the C-terminus of CpxA via TEV-cleavable linkers; TEV protease is fused to the C-terminus of CpxR via a flexible linker.

Promoter test and eYFP-termini affinity analysis

To test the efficiency of the araC/pBAD promoter combined with RBS BBa_B0034, we fused them directly to eYFP and measured fluorescence using the following (Link to protocol page). Figure 4a shows that the expression of eYFP is strongly linked to the concentration of added L-arabinose. On top of this, we tested the fluorescence of the individual N- and C-termini of eYFP, and if these termini have natural affinity for one another, as this could lead to high levels of background fluorescence. The results in figure 4 show that this is not the case!

Figure 4: The functionality of the araC/pBAD is used to control eYFP protein expression. This way protein expression can be quantified using eYFPs fluorescence. Several L-arabinose induction concentrations were used. On top of this, natural fluorescence of eYFP-termini (eYFPn, eYFPc) and eYFP-termini natural affinity (eYFPn-eYFPc) are tested.

Visualization results

After confirming that the araC/pBAD promoter works and creating the necessary constructs, we can start visualizing Cpx pathway activation with BiFC. In order to simplify the experiments we activate the Cpx pathway with known stress factor KCl Fleisher et al. We perform all experiments in E. coli K12. We grow the cells in saltless LB and induce protein expression with 0.2% L-arabinose. CpxA-CpxR protein interaction or CpxR dimerization and subsequent fluorescence are measured over time, the system being activated at t=20 min with 75 mM KCl efpl12. Check out the full protocol here!

The results clearly show a rapid increase in fluorescence after activation of the Cpx pathway when we visualize CpxR-CpxR dimerization, and we see that the signal gets stronger once more stress is introduced (figure 5a). It is clear that within two hours a significant fluorescent signal is detected! Unfortunately, the CpxA-CpxR protein interaction does not show any increase in fluorescence after activation (figure 5b). We already predicted a similar result in our computer models. However, we also correctly predicted a positive CpxR dimerization result[link model (integration?)]! *Place results of friday (TEV-ZIP) here!(figure 5c)

Figure 5A: The functionality of the araC/pBAD is used to control eYFP protein expression. This way protein expression can be quantified using eYFPs fluorescence. Several L-arabinose induction concentrations were used. On top of this, natural fluorescence of eYFP-termini (eYFPn, eYFPc) and eYFP-termini natural affinity (eYFPn-eYFPc) are tested.

CpxA-CpxR interaction

Figure 5B: A) Created translational units used to visualize Cpx activation. All units are under control of the inducible araC/pBAD promoter and strong RBS BBa_B0034. A) eYFPn is fused to the C-terminus of CpxA via a flexible linker; eYFPc is fused to the C-terminus of CpxR. B) eYFPn and eYFPc are fused to the C-terminus of CpxR via flexible linkers. C) eYFPn-nZIPPER and eYFPc-cZIPPER are fused to the C-terminus of CpxA via TEV-cleavable linkers; TEV protease is fused to the C-terminus of CpxR via a flexible linker.

TEV protease and CpxA-CpxR interaction

To test the efficiency of the araC/pBAD promoter combined with RBS BBa_B0034, we fused them directly to eYFP and measured fluorescence using the following (Link to protocol page). Figure 4a shows that the expression of eYFP is strongly linked to the concentration of added L-arabinose. On top of this, we tested the fluorescence of the individual N- and C-termini of eYFP, and if these termini have natural affinity for one another, as this could lead to high levels of background fluorescence. The results in figure 4 show that this is not the case!

Figure 5C: placeholder

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.