Measuring absolute levels of clock proteins with calibrated NanoLUC assays

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).

Andrew Millar's research group, University of Edinburgh

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 ...

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 ...

Analysis for inferring the number of molecules of clock proteins using recombinant NanoLUC

Seedlings of the transgenic lines complemented with CCA1-NL and TOC1-NL were tested under 12L:12D cycles followed by constant light, to test how well the reporter signal in living plants reflected the expected patterns of protein expression. One example is linked below, from the BioDare2 repository record, because FAIRDOMHub's Data File is not accepting these URLs.

BioDare2 ID 11391; Plate reader experiment CCA1 TOC1 NanoLUC; permalink: https://biodare2.ed.ac.uk/experiment/11391

Transformation of RLU into absolute units using a calibration curve of recombinant MBP-NanoLUC-3FLAG-10His

Plot of linear regresion of calibration curve for inferring number of molecules from NanoLUC biolumienescence in plant extracts

4 seeds of stable NanoLUC T3 homozygous lines for LHY, PRR7, TOC1, and ELF3 were seeded in 96-well flat white plate that contained 150 µl of ROBUST media and stratified for 2 days at 4ºC. Then plates were incubated in a 2 hours pulse of white light given and transferred to 22 hours darkness at 21 ºC. Then transferred 12L:12D photoperiod for 10 days. On day 10, 50 µl of 1:50 Furimazine:0.05% Triton X-100 added to each well for tracking NanoLUC bioluminescence. A) Measurements using a Tristar plate ...

"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 ...

"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 ...

Jupyter notebook that contains the linear regression for inferring numnber of molecules from NanoLUC biolumiescent data in plant extracts using as calibration curve recombinant MBP-NanoLUC-3FLAG-10His