Difference between revisions of "Part:BBa K3610003"

Revision as of 17:19, 19 October 2020

BAK1 Ectodomain from Arabidopsis thaliana

This part is a version of the BAK1 receptor from A. thaliana (Part:BBa_K3610001 for full length) without the intracellular kinase domain and without the native signal sequence.

Usage and Biology

The BRI1-associated receptor kinase (BAK1) is a leucin-rich repeat receptor kinase (LRR-RK) which interacts with multiple other LRR-RKs with different functions in hormone signalling and defense response. BAK1 localizes at the plasma membrane and the endosome. The BAK1 protein forms a structure with an extracellular domain with leucin-rich repeats, a single pass transmembrane domain and an intracellular domain with a kinase function.

Among others, BAK1 interacts with the LRR-RKs EF-Tu receptor (EFR), Flagellin sensing 2 (FLS2) and cold-shock protein receptor (CORE), all of which are pathogen recognition receptors (PRR) in brassicaceae plants. Upon binding of a microbe-associated molecular pattern at the LRR domain of the PRR, BAK1 forms a heterodimer with the PRR which triggers a phosphorylation cascade, leading to upregulation of defense mechanisms.

This part includes only the ectodomain and the transmembrane domain. The intracellular kinase domain was cleaved off. Additionally, the natural signal peptide sequence, which is necessary for localization at the cell membrane in the plant, has been cleaved off as well. Therefore, this part needs to be used together with a signal peptide from the organism in which the protein is expressed.

Plant Immunity Based Biosensing

For visualizing the interaction of BAK1 with other plant receptors the cytoplasmic domain can be replaced with a split fluorescent protein or another protein that generates a visual output. For our iGEM project, we designed a system to use this mechanism for detection of bacterial epitopes in water. We used this part for multiple experiments.

Expression with YFP

We fused this part together with the yellow fluorescent protein venus with a 15 amino acids long linker and added the secretion signal from the alpha-Factor, a protein from S. cerevisiae. The corresponding composite part is Part:BBa_K3610032 (the results for this experiment are also on this parts page). We expressed this protein in S. cerevisiae to test two things. First of all, we wanted to see whether the protein gets expressed at all, and secondly, we were interested in seeing, whether the secretion signal from yeast and the receptor without the intracellular kinase domain would be sufficient for localization at the cell periphery.

In a first step we inserted the single fragments making up this part into a plasmid with a gentamycin-3-acetyltransferase gene and transformed E. coli (DH10alpha) with the plasmids for amplification. In the next step we assembled the fragments in a plasmid with a spectinomycin acetyltransferase and amplified the plasmids again in the same E. coli strain. For this step we applied the techniques of Golden Gate Cloning to get the fragments in the right order into the plasmid. The restriction enzyme we chose was BsaI. For expressing this part consisting of YFP and the receptor protein, we initially intended to use promoters of different strength to get more quantitative data. Finally, we got the construct in a plasmid with a truncated version of the ADH1 promoter from S. cerevisiae. For termination, this part has the terminator sequence of the enolase 2 protein from S. cerevisiae. The plasmid also contained the TRP1 gene, which encodes phosphoribosylanthranilate isomerase, an enzyme that catalyzes the third step in tryptophan biosynthesis. This enabled us to use the same plasmid for expression in S. cerevisiae. We prepared a medium containing YNB and free amino acids, without tryptophan. S. cerevisiae cells (AP4) were transfected with the plasmid and then plated on the selective medium.

After successful transformation of yeast cells we checked for expression of the protein under a confocal microscope.

Confocal microscopy confirmed increased fluorescence in the S. cerevisiae cells that had been previously transfected with plasmids containing BAK1 ectodomain fused to YFP. This increased fluorescence indicates expression of our genes. Additionally, this imaging experiment revealed that the fluorescent protein is in part localized at the cell periphery. This is in alignment with our expectations as our construct includes a secretion signal protein and a receptor coding protein with the transmembrane domain. These results suggest that the secretion peptide fused to the receptor ectodomain, including the transmembrane domain can be expressed in S. cerevisiae and that the components are sufficient for localization at the cell membrane.

Expression with split-mCherry

For another part of our project, we fused the ectodomain to the N-terminal domain of the split-mCherry protein instead of YFP. The goal was to coexpress this construct (Part:BBa_K3610032) with one of the plant PRRs that show ligand-dependent interaction with BAK1, like EFR. We managed to design a construct consisting of the ectodomain of EFR which was fused to a secretion signal and the C-terminal domain of mCherry (Part:BBa_K3610039. Coexpressed in S. cerevisiae, these two parts are able to interact upon ligand-binding. This dimerization will reconstitute the functionality of the mCherry protein, leading to a fluorescent output.

Expression with split-NanoLuc

For another part of our project, we fused the ectodomain to the LargeBit part of the split-NanoLuc protein instead of the intracellular kinase domain. The goal was to coexpress this construct (Part:BBa_K3610032) with one of the plant PRRs that show ligand-dependent interaction with BAK1, for example EFR. We managed to design a construct consisting of the ectodomain of EFR which was fused to a secretion signal and the SmallBit of NanoLuc (Part:BBa_K3610043. Coexpressed in S. cerevisiae, these two parts are able to interact upon ligand-binding. This dimerization will reconstitute the functionality of the NanoLuc protein, leading to a chemiluminescent output in the presence of furimazine.

Sequence and Features