Difference between revisions of "Part:BBa K3610040"

Latest revision as of 00:18, 28 October 2020

EFR ectodomain / mCherry C-terminal for S. cerevisiae

This part entails the ectodomain of the plant pattern recognition receptor EFR from A. thaliana which is fused to the C-terminal domain of the mCherry protein via a 15 amino acid linker. The C-terminal sequence of the receptor protein, encoding the signal peptide, was removed from the sequence. To ensure localization, the secretion signal of the alpha Factor from yeast is added instead.

Usage and Biology

EFR

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 leucine-rich-repeats (LRR) receptor-like serine/threonine-protein kinase. The protein consists of an extracellular domain with leucine-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.

Usage with mCherry

In this case, the C-terminal domain of EFR, entailing the intracellular kinase domain, was removed from the sequence. Instead, the C-terminal domain of the split mCherry was fused to the C-terminal domain via a 15 amino acid linker.

The ligand-dependent interaction of EFR with its coreceptor BAK1 is driven by the extracellular ligand-binding domain. Further necessary is the transmembrane domain, including the juxtamembrane domain. Therefore, dimerization can be achieved without the intracellular kinase domain of neither EFR nor BAK1. Coexpressed with Part:BBa_K3610034, which is the ectodomain of BAK1 fused to the N-terminal part of mCherry, elf18-induced interaction between BAK1 and EFR drives the reassembly of the C-terminal and N-terminal domain of the split-mCherry, reconstituting its functionality as a fluorescent protein. This part, therefore, allows visualization of the ligand-dependent interaction of the plant PRRs EFR and BAK1. In our project, we used this part, in coordination with Part:BBa_K3610034, as we designed a system to visually capture the presence of the elf18 epitope in water samples as the elf18 pattern will induce interaciton between the receptors, causing the split-mCherry parts to rejoin and generate a funcitonal fluorescent protein.

Characterization

For our iGEM project, we were interested in seeing wether we would achieve ligand-dependent dimerization of the EFR receptor with its coreceptor after expression in S. cerevisiaey and whether we would be able to visualize the interaction of the two receptors. We, therefore, fused split-reporter proteins to the cytoplasmic domain (intracellular kinase domain had been removed) and expressed both constructs in yeast.

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 the N-terminal domain of mCherry and the receptor protein (only ectodomain), 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 a a kanamycin acetyltransferase gene. This protein makes yeast resistant to the aminoglycoside G418 Geneticin. This enabled us to use the same plasmid for expression in S. cerevisiae.

For coexpression with Part:BBa_K3610034, we had to use two different plasmids. The part with the coreceptor was assembled in the same manner as this part, the vector, however, contained the TRP1 gene, which encodes phosphoribosylanthranilate isomerase, an enzyme that catalyzes the third step in tryptophan biosynthesis. Transformation with both plasmids was done simultaneously and then the cells were plated on a medium which selected for both plasmids. As the error propensity is higher when cotransforming the cells with two plasmids at the same time, we also set up sequential transformation, in case the double-transformation should fail.

After successful transformation of S. cerevisiae cells, we examined the cells with a fluorometer. The goal of this experiment was to see whether fluorescence in cells transfected with the plasmids would be increased when compared with wild-type yeast cells. Should the constructs be expressed at sufficiently high levels and allow dimerization of the two split-mCherry parts, we would expect transformed cells to show increased fluorescence intensity. Additionally, if the dimerization of the mCherry proteins was driven by the ligand-dependent receptor interaction, presence of the bacterial epitope should further increase fluorescence intensity.

A fluorescence assay was performed (λEX = 587nm and λEM = 610nm) with a luminometer of the type Synergy H1. For this assay, the samples were incubated in liquid medium for several hours at 30°C. After this time, the samples were resuspended to obtain samples of the same optical density (OD600 = 0.5). We had two different types of samples, samples which were transformed with plasmids containing this sequence and plasmids containing Part:BBa_K3610042 and the second sample was an untreated control. With each sample two types of emasurements were performed. Once fluorescence levels were measured directly from the dilutet samples and for the other type of measurement, the bacterial elicitor elf18, which initiates the interaciton between the BAK1 and EFR receptors in plants, was added right before the measurement started.

Figure 1: Comparison of average luminescence measurements over time for different samples.

Surprisingly, fluorescence levels did increase when the bacterial elicitor was present, however, this was the case for both types of samples. We must be, however, aware of the fact that the variance was rather large and this one time assay does not provide enough data to confirm a significant effect of the bacterial elicitor. What was an even bigger surprise is that the measured fluorescence intensities were higher for the samples containing the untransformed yeast cells, both when the bacterial elicitor was present and when it had not been added. The reasons for these results are unclear. It could be due to an error when adjusting the samples to the same OD600. A difference in the OD could lead to increased fluorescence levels. This would still not explain why the bacterial elicitor seemed to increase autofluorescence for the untransformed yeast cells.

It is clear that this deserves further examination. We propose to repeat the measurements to increase the data coverage and rule out errors during pipetting or labelling the samples. It should further be attempted to lower autofluorescence for yeast cells that resembles the fluorescence of mCherry. It further needs to examined, whether the same type of results are obtained when a different split-reporter is fused to the receptor domain (something which we were able to carry out during our project).

Sequence and Features