Difference between revisions of "Part:BBa K3610034"

Latest revision as of 21:17, 27 October 2020

BAK1 ectodomain / mCherry N-terminal

This part contains the sequence for the ectodomain of the plant surface receptor BAK1 from A. thaliana fused to the N-terminal part of a split-mCherry protein. Additionally, instead of the signal peptide native to the plant receptor, there is the secretion signal of the alpha factor from yeast at the N-terminal domain of the receptor, replacing the original signal sequence.

Biology and Usage

BAK1

The BRI1-associated receptor kinase (BAK1) is a leucine-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 leucine-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.

Usage with split-mCherry

In this case, the C-terminal domain of BAK1, entailing the intracellular kinase domain, was removed from the sequence. Instead, the N-terminal domain of the split mCherry was fused to the C-terminal domain via a 15 amino acid linker.

Interaction between the BAK1, which is a coreceptor to many other PRRs, 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. Coexpressed with, for example, Part:BBa_K3610040, which is the PRR EFR that contains the C-terminal domain of the split-mCherry protein instead of the intracellular kinase domain, elf18-induced interaction between BAK1 and EFR is driving the reassembly of the C-terminal and N-terminal domain of the split-mCherry, reconstituting its functionality as a fluorescent protein. In our project, we aimed at visualization of the ligand-dependent interaction between BAK1 and the respective plant PRR. This part has the potential to, in coordination with different PRRs, test for the presence of the epitopes which are recognized by the plant receptors.

Characterization

Coexpression with EFR / mCherry C-terminal in S. cerevisiae

For our iGEM project, we were interested in seeing wether we would achieve ligand-dependent dimerization of the BAK1 receptor with the target-receptors after expression in S. cerevisiaey/i> 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 <i>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 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 (AP4) cells were transfected with the plasmid and then plated on the selective medium.

For coexpression with Part:BBa_K3610040, we had to use two different plasmids. The part with the target receptor was assembled in the same manner as this part, the vector, however, contained a a kanamycin acetyltransferase gene. This protein makes yeast resistant to the aminoglycoside G418 Geneticin. 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