The authors have declared no competing interest. We demonstrate the functionality of these vectors through biotinylation assays in tobacco ( Nicotiana benthamiana) plants. The parts of transcriptional units (modules) of 2X35S-Pro, GFP gene, and 35S-Ter were selected to form a cassette for GFP expression in plants. The new plant expression plasmid pC1300GFP was assembled by five genetic modules/fragments (Fig. So only a round of PCR with 25 to 30 cycles is required for each DNA fragment/modules, which enhances the accuracy of this method. To allow for the use of the constructs in a range of experiments we have designed assembly modules that encode the biotin ligases fused to different linkers as well as different commonly used subcellular localization sequences. Furthermore, the construction of the plasmid is completed by a single reaction. To facilitate the use of proximity-labelling in plants, we have generated a collection of constructs that can be used for the rapid cloning of TurboID and MiniTurbo fusion proteins using the Golden Gate cloning method. These biotinylated proteins can then be isolated by affinity purification using streptavidin-coated beads and identified by mass spectrometry. MacVector Home To achieve efficient assembly of PCR fragments into a vector, we suggest using a 1525 nt overlap with a Tm equal to or greater than 48C (assuming A-T pair 2C and G-C pair 4C). Expression of BioID (or of its derivates TurboID and MiniTurbo) fused to a bait protein results in the biotinylation of proximal proteins. One proximity labelling approach makes use of a promiscuous bacterial biotin ligase, termed BioID. Proximity-labelling has emerged as a powerful tool for the detection of weak and transient interactions between proteins as well as the characterization of subcellular proteomes.
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