Clock proteins NanoLUC fusion raw data
Insertion of events that rescued mutant phenotypes were selected for performing absolute quantification using calibration curves of recombinant purified MBP-NanoLUC-3Flag-10his. Seeds were sterilised with 5% houshold bleach for 10 min and washed three time with deionised water. The seeds were then put for stratifyication at 4ºC in darkenss for 48 hours in 1.5 ml polyproplyen tubes in dionised water. After 48 hours seeds wered plated on ROBUST agar (1/2 MS salts, 1.2% Agar pH 5.8 ajudsted with KOH, no sugars). Plates were sealed with microspore tape for ensuring gas exchange. Then, placed in a Percival growth chamber at ZT0 with photperioic conditions 12L:12D at 21ºC and ~110 µmols/m^2s^2. Two weeks after growth in this conditions individual plants were transfered to F2 soil+sand compost and kept under same incubating environemnt. when the plants reached 21 days-old , rosette samples were taken at the times indicated and NanoLUC was tested in whole-cell extracts as described in Urquiza and Millar 2019.
SEEK ID: https://fairdomhub.org/assays/1980
Experimental assay
Projects: Millar group
Investigation: Absolute units for proteins in Arabidopsis clock models up to U2020.5
Study: Measuring absolute levels of clock proteins with calibrated NanoLUC assays
Assay position:
Assay type: Protein Quantification
Technology type: Enzymatic Activity Measurements
Organisms: Arabidopsis thaliana : Col-0 wild type (wild-type / wild-type) (batch)
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Created: 10th Sep 2022 at 08:54
Last updated: 19th Dec 2024 at 11:28
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Projects: Millar group, TiMet, PHYTOCAL: Phytochrome Control of Resource Allocation and Growth in Arabidopsis and in Brassicaceae crops, POP - the Parameter Optimisation Problem, Regulation of flowering time in natural conditions, PlaSMo model repository
Institutions: University of Edinburgh
https://orcid.org/0000-0003-1756-3654
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 doi 10.1038/s44320-025-00086-5.
Starting from the U2019.3 and U2020.3 models, this project rescales parameters to match protein levels that were predicted ...
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...
Transformation of RLU into absolute units using a calibration curve of recombinant MBP-NanoLUC-3FLAG-10His
Creators: Uriel Urquiza Garcia, Andrew Millar
Submitter: Uriel Urquiza Garcia
Relationship type: Not specified
Plot of linear regresion of calibration curve for inferring number of molecules from NanoLUC biolumienescence in plant extracts
"Samples of plants were collected in pre-weighed 2 ml microfuge tubes (safelock, Eppendorf) with 5 mm stainless steel grinding balls, and flash frozen in liquid nitrogen. The tissue was ground twice at 30Hz for 1 min in a Tissue Lyser (Qiagen). The samples were flash frozen between grinding steps, then placed on ice and 150 μl of BSII buffer was added to protect the samples from proteolysis, without phosphatase inhibitors (Huang et al. 2016). The tube was weighed and further BSII buffer added to ...
Creators: Andrew Millar, Uriel Urquiza Garcia
Submitter: Uriel Urquiza Garcia
Relationship type: Not specified
"Plates inoculated with Col-0 seed were grown under the same photoperiod conditions to the plants to be analysed. Plant tissue was harvested, making aliquots of 0.4 gFW. MBP-NL3F10H protein was prepared by the method described by Urquiza-Garcia U. and Millar A.J. in Plant Methods 2019. and then quantified by the linearized Bradford assay protocol using both Bovine serum albumin BSA and Ovoalbumin as standards (Ernst & Zor 2010). Then aliquots spiked with purified enzyme to generate a curve ...
Creators: Uriel Urquiza Garcia, Andrew Millar
Submitter: Uriel Urquiza Garcia
Relationship type: Not specified
