Assays

The list of python packages used was obtained by typing inside the Docker image

pip list -- format==columns > python_packages_pip_installed.txt

This list the version of packages installed as we have observed issues related to the use of the most current version of some python packaged for example scipy

Maps of cloned genes for rescuing selected clock mutants

The promoter regions for clock genes that present a ChIP-seq signal were extracted from TAIR10 using costume python scripts using the gene list for Kamioka et al CCA1 or Daphne Ezer et al for LUX. The promoter was considered from the TSS of the gene until the annotated end of the upstream gene. Then, this region was scanned using the Energy Matrix derived using EMA working as a classifier for bound or unbound. After classification the calibrated PBM data calibrated using in vitro data was used ...

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 ...

This section contains the links to the tools used for reproducing the computational results presented in Urquiza-Garcia et al. 2022. This is required in particular because the SloppyCell model optimisation software is at some risk. Using Docker we can assure persistence for the computational environment that allows you to run SloppyCell.

The associated git repository can be found in https://hub.docker.com/r/uurquiza/urquiza2019a_tellurium_sloppycell/tags which can be cloned.

The docker image can ...

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

This script take the scaling paramters using synthetic protein data updates model paramaters

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

Jupyter lab notebook that contains the models and data that for predicting protein levels based on mRNA data from TiMet projecto

RNA timeseries data for Arabidopsis Col wild-type plants and clock mutants, as separate mean and SD files. The raw data is available on BioDare.ed.ac.uk, and is linked as 'Attribution' from elsewhere on FAIRDOMHub.

Protein time series for clock proteins colected from the literature. Protein expression profiles were derived from images of western blot or or from plots that the orignal authors derived from quantitative western blots

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 ...