Difference between revisions of "Part:BBa K3610016"

Latest revision as of 03:23, 28 October 2020

SmallBit NanoLuc - codon optimized for S. cerevisiae

This part is a modification of Part:BBa_K1761006. It has been codon optimized for expression in S. cerevisiae.

Usage and Biology

NanoLuc is a small, bright and stable luciferase from O. gracilirostris and has been proven to be an efficient reporter protein since its introduction by Promega. One way to use the NanoLuc luciferase is in a protein-fragement complementation assay (PCA) to visualize protein-protein or protein-ligand interaction. The protein-protein interaction is observed by fusing spli-reporter proteins, like the LargeBit and the SmallBit from NanoLuc, to the proteins of interest. Interaction of the two proteins will associate the reporter fragments and lead to a reconstitution of the full length and fully functional NanoLuc protein.

To enhance translation, we codon optimized this part for the expression in S. cerevisiae as codon bias has shown to influence mRNA stability and translational efficiacy.

We used this part our biosensing system which relies on the dimerization of plant pathogen recognition receptors (PRRs). The general idea is that a microbe assiciated molecular pattern (MAMP), like the epitope flg22 of flagellin, binds to a cell surface receptor, which drives the interaction of the receptor with a coreceptor. The split luciferase proteins are fused to the N-terminal intracellular domains of the receptor protein.

Usage iGEM UZurich

We used this part in our constructs Part:BBa K3610043 and Part:BBa K3610051 which encode the ectodomain of the plant PRR EFR and COREfused to this part via a 15 amino acid linker (eEFR and CORE). We managed to also assemble a construct containing the ectodomain of the plant PRR BAK1 which has also been fused to the LargeBit part (eBAK), giving us the part Part:BBa K3610038.

We then coexpressed eEFR or CORE with eBAK in S. cerevisiae and conducted a luminescence assay to test whether the SmallBit and the LargeBit would be able to interact and become a functional NanoLuc protein.

The goal behind this experiment was to test whether bacterial elicitor driven interaction between the receptors mediates the interaction between the two NanoLuc parts.

Figure 1: Dimerization assay with PRR ectodomains fused to split-NanoBits

Multiple assays suggested that interaction between the LargeBit and the SmallBit is possible when fused to plant PRRs, although this is not mediated by ligand-dependent interaction of the plant PRRs.

For more details on these experiments see: Part:BBa K3610003, Part:BBa K3610038, Part:BBa K3610043, Part:BBa K3610051.

Sequence and Features