Construction of NanoLUC-tagged plants
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Clock mutants for lhy-1/cca1-11, prr9/7, toc1, lux-4, elf3-1 were transformed with the genomic regions of the associated clock genes tagged with NanoLUC-3FLAG-10His. The tagged genomic constructs were transformed in the mutants using Agrobcterium ABI strain (kindly donated by Prof. Seth Davis University of York). T3 plants resistant to homozygous for BASTA resistance were phenotyped by luciferase imaging asessing period phenotype or plant architecture. Rescuing lines were then used for performing abosolute quantification by using recombinant MBP-NanoLUC-3FLAG-10His protein.
SEEK ID: https://fairdomhub.org/studies/1125
Absolute units for proteins in Arabidopsis clock models up to U2020.5
Projects: Millar group
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Created: 21st Jan 2023 at 12:50
Last updated: 19th Dec 2024 at 11:28
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Projects: Millar group, PlaSMo model repository, PHYTOCAL: Phytochrome Control of Resource Allocation and Growth in Arabidopsis and in Brassicaceae crops, Light and plant development, Light control of leaf development, Toggle switch, Reduce Complexity (RCO) reconstruction, Model Driven Prime Editing, PULSE 2.0, Plant optogenetics
Institutions: University of Edinburgh, Heinrich Heine University of Düsseldorf
https://orcid.org/0000-0002-7975-5013
SynthSys is the University of Edinburgh's research organisation in interdisciplinary, Synthetic and Systems Biology, founded in 2012 as the successor to the Centre for Systems Biology at Edinburgh (CSBE).
Projects: Millar group, PHYTOCAL: Phytochrome Control of Resource Allocation and Growth in Arabidopsis and in Brassicaceae crops, TiMet, POP - the Parameter Optimisation Problem, Regulation of flowering time in natural conditions, PlaSMo model repository
Web page: http://www.synthsys.ed.ac.uk
Andrew Millar's research group, University of Edinburgh
Programme: SynthSys
Public web page: http://www.amillar.org
The dataset presents mathematical models of the gene regulatory network of the circadian clock, in the plant Arabidopsis thaliana. The work will be published as Urquiza-Garcia, Molina, Halliday and Millar, title "Abundant clock proteins point to missing molecular regulation in the plant circadian clock", in Molecular Systems Biology, 2025.
Starting from the U2019.3 and U2020.3 models, this project rescales parameters to match protein levels that were predicted using a simple model from the TiMet ...
Submitter: Andrew Millar
Studies: Construction of NanoLUC-tagged plants, Estimating DNA-binding affinities for Arabidopsis proteins, Measuring absolute levels of clock proteins with calibrated NanoLUC assays, Predicting absolute levels of clock proteins with a simple model, Recalibrating the clock models for absolute protein levels, to create mo..., Reproducibility documentation
Assays: Clock protein number determination with NanoLUC calibration, Clock proteins NanoLUC fusion raw data, Gatway maps of genomic regions of clock genes, In vivo bioluminescence of clock protein-NanoLUC fusions: example experi..., Jupyter notebook Predicting Protein Numbers, Propagating scaling factors into model parameters for U2019.4->U2019.5 a..., Protein level time series, Python packages, Reproducibility tool set, Selection of complemented transgenic lines, TiMet RNA timeseries data, promoter binding affinity calculations on the genome based on PBMs and E...
Maps of cloned genes for rescuing selected clock mutants
Submitter: Uriel Urquiza Garcia
Assay type: Transformation
Technology type: Technology Type
Investigation: Absolute units for proteins in Arabidopsis cloc...
The reporter fusion constructs expressing clock proteins fused to NanoLUC or firefly FLUC were transformed into the cognate, clock-mutant host plants. Each host also contained a transcriptional FLUC fusion that was used to score the circadian period of each transgenic line in constant light. Transformants that expressed a functionally normal level of clock protein were selected by choosing lines that complemented the mutant's period defect back close to the wild type period. Note that the reporters ...
Submitter: Andrew Millar
Assay type: Transcriptional reporter gene
Technology type: Imaging
Investigation: Absolute units for proteins in Arabidopsis cloc...
Construct fof TOC1 for comparision with NanoLUC
Creator: Uriel Urquiza Garcia
Submitter: Uriel Urquiza Garcia
Binary vector used for transforming with Agro ABI the elf3-2 CCA1p:LUC for rescuing elf3-2 mutation. In this particular case the plants selected were based on hypocotyl length. Based on the data of LUX in which we observed that rhytmicity was rescued in all lines, hypocotyl elongation varied between lines. Therefore, we used hypocotyl length for assesing complementation
Creator: Uriel Urquiza Garcia
Submitter: Uriel Urquiza Garcia
Donor plasmid for Gatway cloning
Creator: Uriel Urquiza Garcia
Submitter: Uriel Urquiza Garcia
Donor plasmid for Gatway cloning
Creator: Uriel Urquiza Garcia
Submitter: Uriel Urquiza Garcia
Binary vector used for transforming with Agro ABI the cca1-1/lhy-1p:LUC for rescuing cca1-1 mutation.
Creator: Uriel Urquiza Garcia
Submitter: Uriel Urquiza Garcia
Binary vector used for transforming with Agro ABI the cca1-11/lhy-1 CCA1p:LUC for rescuing lhy-1 mutation.
Creator: Uriel Urquiza Garcia
Submitter: Uriel Urquiza Garcia
Binary vector used for transforming with Agro ABI the cca1-1/lhy-1p:LUC for rescuing lhy-11 mutation. This was an alternative to NanoLUC and also for testing the behaviour of LHY.
Creator: Uriel Urquiza Garcia
Submitter: Uriel Urquiza Garcia
Binary vector used for transforming with Agro ABI the prr9/7-9 CCR2:LUC for rescuing prr7-9 mutation.
Creator: Uriel Urquiza Garcia
Submitter: Uriel Urquiza Garcia
Binary vector for transformation with Agro. This construct was intended for comparision with NanoLUC
Creator: Uriel Urquiza Garcia
Submitter: Uriel Urquiza Garcia
Binary vector used for transforming with Agro ABI the toc1-2 CCA1p:LUC for rescuing the toc1-2 mutant.
Creator: Uriel Urquiza Garcia
Submitter: Uriel Urquiza Garcia
Donor plasmid for Gatway cloning for CCA1 genomic region
Creator: Uriel Urquiza Garcia
Submitter: Uriel Urquiza Garcia
Donor plasmid for Gatway cloning
Creator: Uriel Urquiza Garcia
Submitter: Uriel Urquiza Garcia
Donor plasmid for Gatway cloning
Creator: Uriel Urquiza Garcia
Submitter: Uriel Urquiza Garcia
CCA1 genomic fusion with LUC+ for comparision between NanoLUC and Firfly luciferase
Creators: Uriel Urquiza Garcia, Andrew Millar
Submitter: Uriel Urquiza Garcia
