Difference between revisions of "Part:BBa K3610051"

Latest revision as of 01:12, 28 October 2020

CORE ectodomain / SmallBit NanoLuc for S. cerevisiae

This part includes the ectodomain of the plant pattern recognition receptor CORE fused to the SmallBit part of the split-NanoLuc system. To ensure localization at the membrane, this part further contains the sequence for the signal peptide of the alpha factor from S. cerevisiae.

Usage and Biology

CORE

The cold shock protein receptor (CORE) is a plant pattern recognition receptor (PRR) and as such activates host innate immunity through detection of pathogen-associated molecular patterns (PAMPs). CORE is a leucine-rich repeat receptor-like kinase with 22 LRRs, there additionally is a 6 amino acid insert at LRR 11. It consists of an extracellular domain that perceives an epitope, csp22, from the highly conserved nucleic acid binding motif RNP-1 of bacterial cold-shock proteins (CSPs), which are highly abundant proteins found in the cytosol of bacteria. Further domains are a single pass transmembrane domain and an intracellular kinase domain (The sequence encoding the kinase domain is not in this part). Interaction of CORE with brassinosteroid-associated kinase (BAK)1 is necessary for inducing an immune response in the plant. The dimerization of CORE and BAK1 depends on the csp22, the ligand of CORE. The function of CORE in S. lycopersicum has been confirmed by expressing the receptor in A. thaliana, which made the plant responsive to csp22, a PAMP that is otherwise not perceived by PRRs from A. thaliana.

Usage with NanoLuc

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

The ligand-dependent interaction of CORE 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 CORE nor BAK1. Coexpressed with the ectodomain of BAK1 fused to the LargeBit part of the NanoLuc luciferase, csp22-induced interaction between BAK1 and EFR can drive the reassembly of both parts from the NanoLuc luciferase, reconstituting its function to react with furimazine in the presence of oxigen, yielding furimamide and a luminescent output. This part, therefore, allows visualization of the ligand-dependent interaction of the plant PRRs CORE and BAK1. This enables us to use this part, in coordination with the BAK1 ectodomain and LargeBit NanoLuc, to visually capture the presence of the csp22 epitope in water samples, as the csp22 pattern will induce interaction between the receptors, causing the split-NanoLuc luciferase parts to rejoin and generate a functional protein, which gives a visual uotput with the substrate furimazine.

Characterization

We coexpressed this part together with Part:BBa_K3610038 (eBAK1), which is the ectodomain of the co-receptor BAK1 fused to the LargeBit part of the NanoBit system, in S. cerevisiae. The parts were assembled with Golden Gate Cloning in two different vectors. This part (eCORE) was assembled in a plasmid containing a kanamycin resistance gene, while the eBAK1 construct was assembled in a plasmid with a TRP1 gene, which encodes an enzyme necessary for tryptophan synthesis. These two genes allowed for selction on selective media.

After coexpressing the two plasmids in S. cerevisiae, a dimerization assay under a luminometer was performed with a plate reader of the type Synergy H1. Should both proteins be expressed and able to interact, then it is possible that functionality of the split-NanoLuc protein get reconstituted, which would enable it to catalyze the reaction of furimazine to furimamide, a reaction which is accompanied by luminescence.

Luminescence assay

Samples with cells which were transfected with the two mentioned plasmids (eBAK1 and eCORE) and samples containing S. cerevisiae cells that were not transfected with any plasmids (UT). Additionally, we also examined cells which expressed the eEFR construct instead of eCORE.

Optical densities (OD600) of all samples were adjusted to 0.34.
For each type of sample, three types of measurements were made:

• 1 µL of deionized water added (no elicitor)
• 1 µL of epitope elf18 added
• 1 µL of epitope csp22 added

Each measurement was done four times with sample size 50µL. To each well 50 µL NanoGlo solution was added (50:1 buffer to furimazine)

Of all 4 measurements, the average was taken an is summarized in the chart below.

Figure 1: Average Luminescence Levels over time after 30 minutes incubation

As expected the control sample (UT) did not show any luminescence in the presence of the NanoLuc substrate.

eBAK1 coexpressed with eCORE showed an increase in luminescence, although the effect was much smaller when compared with eEFR. The results again suggest, that addition of the bacterial elicitor csp22, which initiates interaciton between CORE and BAK1 does not increase the luminescence levels as samples without csp22 added showed greater luminescence than samples which were treated with this bacterial epitope.

Another observation was that the increase in luminescence levels was much more pronounced when eEFR was expressed instead of eCORE.

These results led us to the conclusion that our plasmids get expressed. It further has been shown that the NanoBit parts fused to the receptors are able to interact and reconstitute their functionality as a funcitonal NanoLuc protein which catalyzes the reaction of furimazine to furimamide, which gives a luminescent output. In our case, however, receptor-specific bacterial epitopes did not increase luminescence levels when the receptors were expressed in S. cerevisiae. It seems that there is no or very little csp22-driven dimerization of the two receptor-ectodomains CORE and BAK1.

Sequence and Features