Difference between revisions of "Part:BBa K3610007"

Revision as of 23:55, 22 October 2020

EFR Ectodomain without signal sequence

This part is the ectodomain of the plant PRR EFR, a cell surface receptor determining the preception of the bacterial Elongation-Factor Tu.

Usage and Biology

Elongation factor-thermo unstable receptor (EFR) from A. thaliana is a plant pattern-recognition receptor (PRR). It is a cell surface receptor and part of the plants firts defence mechanism against potential pathogens. The EFR receptor is also a leucin-rich-repeats (LRR) receptor-like serine/threonine-protein kinase. The protein consists of an extracellular domain with leucin-rich repeats, a ligand binding domain found in many receptors, a single-pass transmembrane domain and finally an intracellular kinase domain. The ligand binding domain from EFR has high specificity to a bacterial pathogen-associated moleculat pattern (PAMP), namely the epitope elf18 of the abundant protein Elongation Factor Tu (EF-Tu), which is catalyzes the binding of aminoacyl-tRNA (aa-tRNA) to the ribosome in most prokaryotes and therefore is evolutionarily highly conserved. This makes the EFR a receptor that can be activated by the presence of a huge variety of bacteria. Upon binding of the ligand to the extracellular domain, the receptor dimerizes with its coreceptor BRI1-associated receptor kinase (BAK1). This interaction triggers the activation of the intracellular kinase domain of EFR and BAK1, initiating a signal cascade leading to an upregulation of immune response mechanisms. When the ectodomains of EFR and another PRR are swapped, the new chimeric receptors are sometimes still functional (depending on which second receptor is chosen). It has further been shown, that for binding of the ligand and dimerization with BAK1 the extracellular domain and the transmembrane domain are needed. The kinase domain is not necessary for initiating this interaction.

For visualizing the interaction of EFR with BAK1, the cytoplasmic domain can be replaced with a split fluorescent protein or another protein that generates a visual output. In our iGEM project, we fused this sequence to a split-mCherry protein and a split-luciferase in another experiment and then coexpressed the part with BAK1 carrying the counterpart to the split protein instead of the intracellular kinase domain in S. cerevisiae.

Characterization

Usage with YFP

We fused the ectodomain of EFR to the yellow fluorescent protein and expressed it in S. cerevisiae. For a more elaborate characterization of this part see Part:K3610045.

Fluorescent Microscopy

After successful transformation of yeast cells we checked for enhanced fluorescence with confocal fluorescence microscopy.

Imaging of the S. cerevisiae cells, which had been previously transfected with plasmids containing this construct, revealed increased fluorescence levels than the untransfected control. These results imply increased expression of YFP, indicating expression of the EFR ectodomain. Additionally, the imaging results suggest that the proteins are in part localized at the cellular membrane, which is in alignment with our expectations as there is a secretion signal peptide and a transmembrane domain in the construct.

Spectrometry

In addition to analyzing the cells with a microscope, we conducted a fluorescence assay with a plate reader. We conducted this experiment for multiple receptors at the same time. This way we were able to compare the expression levels of different receptors.

The results of these measurements are displayed in the table below.

If we set the values for the Control to 1 (Control = 1), then we get the fluorescence levels relative to the control, which is again diplayed in the table below.

Results of the plate reader suggested increased fluorescence through expression of YFP. The same was observed when cells were examined with the microscope. Therefore, S. cerevisiae cells express the plasmid containing the EFR ectodomain. Additionally, microscopy revealed localization at the cell membrane. This localization at the cell periphery is a big success as it shows, that the signal peptide from the alpha-Mating factor did, in fact induce translation into the membrane. Additionally, it was shown that the intracellular kinase domain is not necessary for membrane localization.

This expression of the EFR ectodomain in S. cerevisiae, which is accompanied by localization at the cell membrane is a big achievement that opens the doors for many future applications.

Usage with split-NanoLuc

We fused this part to the SmallBit part of the NanoBit system, a split luciferase (eEFR:SBit). We managed to express the CORE:SBit together with the ectodomain of the plant PRR BAK1 which was fused to the LargeBit part of the split NanoLuc protein (eBAK1:LBit). We were able to express both constructs in S. cerevisiae. For a more detailed description see the parts: Part:BBa K3610038 and Part:BBa K3610043.

After transfection of the cells with the two plasmids, we tested whether the split-NanoLuc proteins would be able to interact, which would reconstitute its functionality. We further were interested in the interaction between the EFR and BAK1 ectoomain. EFr and BAK1 naturally interact upon binding of elf18. Should both receptors be expressed properly in the cell membrane of S. cerevisiae, the ligand will be able to bind to the EFR ectodomain. If this is sufficient to drive dimerization of the two receptor in S. cerevisiae, then the NanoLuc parts will be more likely to interact as well, which would lead to a higher amount of functional luciferase proteins in the cell.

To test for dimerization of the luciferase parts and also the two receptors, an assay with a luminometer was performed.

Sequence and Features