Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Janelle San Juan
Date created: 2015-09-17 11:00:00
Date modified: 2015-09-18 01:22:38
YFP fused to CIB1 N-terminal domain
| Types | DnaRegion |
| Roles | Reporter engineered_region |
| Sequences | BBa_K1750001_sequence (Version 1) |
Description
Yellow fluorescent protein, which produces yellow light (excitation 400nm, emission 530nm), fused to the carboxy-terminus of cryptochrome-interacting basic-helix-loop-helix1 (CIB1), a transcription factor derived from A. thaliana in which it initiates flowering upon binding the photoreceptor cryptochrome 2 (CRY2). We used the N-terminal domain of CIB1 (a.a. 1-170), that lacks the conserved basic helix-loop-helix domain which mediates dimerisation and DNA binding. This device can be used as a reporter for binding to CRY2 fused to a second fluorescent protein, e.g. cyan fluorescent protein as detected by FRET assay. This part is expressed strongly in the pETMCSII vector. Expression in E. coli showed strong YFP fluorescence but SDS-PAGE analysis suggested that the CIB1 segment may be proteolysed in the cell, which would hinder its ability to bind CRY2.Notes
Initially designed CRY2-CFP-CIB1-YFP, CRY2-YFP-CIB1-YFP, CIB1-CFP and CIB1-YFP constructs with ribosomal binding sites at the start of each fusion protein (if contained both CRY2 and CIB1 fusion proteins), HIS/MYC tags and a flexible linker between the fused proteins, and a Terminator at the end. We redesigned the construct in the order RBS-CIB1-Linker-YFP-H6-Terminator to allow purification and fluorescence measurements of individual reporter proteins. The N-terminal HIS tag also facilitated purification of the protein.Source
CIB1 - genomic sequence of A. thaliana, YFP - genomic sequence of A. thaliana A. victoria| Sequence Annotation | Location | Component / Role(s) |
| linker CIB1 (N-terminal domain) YFP linker HIS tag (H6) STOP codon | 511,516 1,510 517,1230 1231,1233 1234,1251 1252,1254 | sequence_feature feature/misc feature/protein CDS CDS feature/protein feature/misc sequence_feature sequence_feature feature/misc feature/stop stop_codon |