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Fig. 1 | Plant Methods

Fig. 1

From: MoBiFC: development of a modular bimolecular fluorescence complementation toolkit for the analysis of chloroplast protein–protein interactions

Fig. 1

Outline of a MoClo-based approach for testing protein–protein interactions by BiFC. The MoClo syntax for the position of different modules is indicated on the top line, and is as described by Engler et al. [6]. Level 0 modules containing promoter and 5′ UTR sequences [Pro + 5U(f), Pro + 5U], N-terminal targeting sequences and tags (NT1, NT2), coding sequences for a protein of interest (CDS2ns), C-terminal tags (CT) and transcription terminators (Ter) are assembled in a one-pot restriction ligation reaction into Level 1 transcriptional units using the indicated four base-pair overhangs. The transcriptional units are then assembled in a similar fashion into single T-DNA Level 2 multigenic assemblies for BiFC assays. As an example, + and * indicate Level 0 modules selected for creating the shown Level 1 transcriptional units, that are then assembled into a Level 2 multigenic array. p35S, cauliflower mosaic virus 35S promoter; pUBI, UBQ10 promoter; CTP, chloroplast transit peptide; cYFP, C-terminal fragment of YFP for BiFC; nYFP, N-terminal fragment of YFP for BiFC; POI, protein of interest without transit peptide or stop codon; STOP, module containing a stop codon; t35S, cauliflower mosaic virus 35S terminator; tUBI, UBQ10 terminator

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Plant Methods

ISSN: 1746-4811