Absolute units for proteins in Arabidopsis clock models up to U2020.5

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

Files required for reproducibility of computational results. This include Docker file and python packages

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 ...

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 ...

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

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 ...

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

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

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

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 ...

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

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.

https://doi.org/10.1105/tpc.15.00737

https://doi.org/10.1038%2Fnplants.2017.87

The file contains the matrices that come from the MCMH sampling during the inference process from PBMs

The file contains the matrices that come from the MCMH sampling during the inference process from PBMs

https://doi.org/10.1073/pnas.1316278111

https://doi.org/10.1016%2Fj.cub.2010.12.021

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 ...

4 seeds of stable NanoLUC T3 homozygous lines for LHY, 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 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 at which 50 µl of 1:50 Furimazine:0.05% Triton X-100 added to each well for tracking NanoLUC bioluminescence. Measurements using a Tristar plate reader were performed automatically ...

Small data base of clock proteins profiles obtained with western blots by several authors of A. thaliana collected from the literature. This data can be fed into simple models for making prediction of abosute number of protein when combined with RNA data in absolute units. For example the TiMet data set

"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 ...

This time series were obtained from the literature by perroming rough quantiftification from western blot images. In some cases the data was quantified by the authors and graphs were provided in the publications. In this case we used ImageJ or https://automeris.io/WebPlotDigitizer/

This files contains the predicions generated using a simple model of translation, described in the manuscript. This synthetic data was used to rescale U219.3 resulting in U2019.4 and U2020.3 into U2020.4. The .4 models are only resceled for the mass scale of protein and still present the dynamics of the .3 version

SD values of clock gene RNA data in absolute units of RNA copies per cell (calculated from copies per gFW, / 25 million cells/gFW) from TiMet WP1.1, RNA dataset ros (from rosettes). Note the Col data are from WP1.1, not substituted with Col from the LD12:12 of the WP1.2 photoperiod data set, as they were in Flis et al. 2015. Note also that cL_m in these data is taken from CCA1 only, not the average of CCA1 and LHY as in the data sets used for optimisation of P2011.2.1 in Flis et al. 2015.

The ...

Mean values of clock gene RNA data in absolute units of RNA copies per cell (calculated from copies per gFW, / 25 million cells/gFW) from TiMet WP1.1, RNA dataset ros (from rosettes). Note the Col data are from WP1.1, not substituted with Col from the LD12:12 of the WP1.2 photoperiod data set, as they were in Flis et al. 2015. Note also that cL_m in these data is taken from CCA1 only, not the average of CCA1 and LHY as in the data sets used for optimisation of P2011.2.1 in Flis et al. 2015.

The ...

Parameters rescaled and scaling factors set to 1

This is the scaled version of U2020.4 in sbml file. It already contains the scaling factors

Paramters rescaled and scaling factors set to 1

Paramteres rescaled and scaling factors set to 1

Parameters rescaled and scaling factors set to 1

Derived from U2019.3 from Testing the inferred rate of dynamic, gene regulatory network in absolute units

Sbml version of U2019.4 with reacaling factors values already incoporated in the model. This was generated autmatically using tellurium python package

This file was derived from U2020.3 by introducing the scalig factors in the required locations in the model. This files is used then for numerically rescaling the model for matching synthetic protein data.

Construct fof TOC1 for comparision with NanoLUC

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

Donor plasmid for Gatway cloning

https://benchling.com/s/02xiS4zk?m=slm-xxhynNMU6VkdMdqdfpFr

Donor plasmid for Gatway cloning

https://benchling.com/s/3OtoUun0?m=slm-pL4lmqV9bCWOW3WvoSFL

Binary vector used for transforming with Agro ABI the cca1-1/lhy-1p:LUC for rescuing cca1-1 mutation.

https://benchling.com/s/Zu0nJONs?m=slm-5zxnih8JIOujB9nYIG8E

Binary vector used for transforming with Agro ABI the cca1-11/lhy-1 CCA1p:LUC for rescuing lhy-1 mutation.

https://benchling.com/s/GP25kROo?m=slm-ocCyiEfwuTW5mJsbNQGn

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.

Binary vector used for transforming with Agro ABI the prr9/7-9 CCR2:LUC for rescuing prr7-9 mutation.

Binary vector for transformation with Agro. This construct was intended for comparision with NanoLUC

Binary vector used for transforming with Agro ABI the toc1-2 CCA1p:LUC for rescuing the toc1-2 mutant.

List of python packages for reproducing the modelling and data analysis results

Donor plasmid for Gatway cloning for CCA1 genomic region

https://benchling.com/s/HcUdo0bf?m=slm-v3TyQ8AupdMj0SCqtZ2J

Donor plasmid for Gatway cloning

https://benchling.com/s/PpY2tH3D?m=slm-pRd1S0YhNNVNUhMkT4N1

Donor plasmid for Gatway cloning

https://benchling.com/s/g08AhXBj?m=slm-bGbYOjeh3kiwQQUdokaH

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

Documents the model paramter rescaling and set the scaling factors to 1

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 ...