Biology subjects

Urquiza-Garcia, U.

Publications and source records attributed to Urquiza-Garcia, U..

4 recordsLinked to original sources

bioRxiv2025

Gearing up Golden Braid assembly for plant synthetic genomics with RepTiles

We have developed RepTiles, a system for generating traces of random DNA for synthetic genomics applications. RepTiles reduced the burden associated with the manual design of long DNA from standard biological parts. We are applying RepTiles to the construction of random DNA segments that will form part of a neochromosome in Physcomitrium patens. RepTiles has a base DNA collection of 52 1.7 kb chemically synthesised Phytobricks mini-chunks. We provide a user-friendly web application that facilitates the design of DNA assemblies based on the base collection. The system generates assembly plans for generating chunks using Golden Braid. The resulting chunks can then be assembled into megachunks using Transformation Associated Recombination cloning. It thus supports the generation of ultra-long synthetic DNA from phytobricks, contributing to the vision of synthetic plant genomics from modular parts to complete genomes.

synthetic biology↗

bioRxiv2024

Abundant clock proteins point to missing molecular regulation in the plant circadian clock

Understanding the biochemistry behind whole-organism traits such as flowering time is a longstanding challenge, where mathematical models are critical. Very few models of plant gene circuits use the absolute units required for comparison to biochemical data. We refactor two detailed models of the plant circadian clock from relative to absolute units. Using absolute RNA quantification, a simple model predicted abundant clock protein levels in Arabidopsis thaliana, up to 100,000 proteins per cell. NanoLUC reporter protein fusions validated the predicted levels of clock proteins in vivo. Recalibrating the detailed models to these protein levels estimated their DNA-binding dissociation constants (Kd). We estimate the same Kd from multiple results in vitro, extending the method to any promoter sequence. The detailed models simulated the Kd range estimated from LUX DNA-binding in vitro but departed from the data for CCA1 binding, pointing to further circadian mechanisms. Our analytical and experimental methods should transfer to understand other plant gene regulatory networks, potentially including the natural sequence variation that contributes to evolutionary adaptation.

systems biology↗

bioRxiv2021

Testing the inferred transcription rates of a dynamic, gene network model in absolute units

The circadian clock coordinates plant physiology and development. Mathematical clock models have provided a rigorous framework to understand how the observed rhythms emerge from disparate, molecular processes. However, models of the plant clock have largely been built and tested against RNA timeseries data in arbitrary, relative units. This limits model transferability, refinement from biochemical data and applications in synthetic biology. Here, we incorporate absolute mass units into a detailed, gene circuit model of the clock in Arabidopsis thaliana. We re-interpret the established P2011 model, highlighting a transcriptional activator that overlaps the function of REVEILLE 8/LHY-CCA1-LIKE 5, and refactor dynamic equations for the Evening Complex. The U2020 model incorporates the repressive regulation of PRR genes, a key feature of the most detailed clock model F2014, without greatly increasing model complexity. We tested the experimental error distributions of qRT-PCR data calibrated for units of RNA transcripts/cell and of circadian period estimates, in order to link the models to data more appropriately. U2019 and U2020 models were constrained using these data types, recreating previously-described circadian behaviours with RNA metabolic processes in absolute units. To test their inferred rates, we estimated a distribution of observed, transcriptome-wide transcription rates (Plant Empirical Transcription Rates, PETR) in units of transcripts/cell/hour. The PETR distribution and the equivalent degradation rates indicated that the models predicted rates are biologically plausible, with individual exceptions. In addition to updated, explanatory models of the plant clock, this validation process represents an advance in biochemical realism for models of plant gene regulation.

systems biology↗

bioRxiv2018

Expanding the bioluminescence reporter toolkit for plant chronobiology with NanoLUC

Bioluminescence has been an important tool for gathering circadian data with the main reporter gene exploited firefly luciferase (LUC). In some circumstances the rapid inactivation of LUC could be disadvantageous, e.g. reporting total protein levels through reporter translational fusions. In the latter scenario the commercially available Nano luciferase (NanoLUC) might offer and advantage, however no data in plant has been provided so far. We tested NanoLUC under different research scenarios were LUC has been used, for example enzyme purification, expression in transient plant systems and in stable transgenic lines. We show that NanoLUC is active in these experimental scenarios. We also created a set of NanoLUC variants for example MBP-NanoLUC-3xFlag-10xHis version of NanoLUC can be easily purified and stable for several days (half-life 37.2 at 4{degrees}C) and can be used for generating calibration curves for quantifying protein as the signal is linear over a large dynamic range. In addition, we show that NanoLUC can report in-planta protein levels on circadian time scale thanks to the stability of furimazine. Therefore, opening the possibility of using NanoLUC for reporting protein dynamics in seedlings. With this new technology, we explored the dynamics of protein BROTHER OF LUX ARRHYTHMO (BOA), which has been suggested in mathematical models to be a rhythmic protein from RNA data. Using an automatic plate-reader, we were able to track BOAp:BOA-NL for an extended period of time by just adding a mix of furimazine with Triton X-100, as it is done with LUC-reporter lines. In our experimental context BOA protein does not present strong oscillatory dynamics similar to what has been reported for Phytocrhome B for which rhythmic accumulation of transcript can be observed while total protein levels remain constant under diurnal conditions. Our results support the use NanoLUC for studying the dynamics of plant proteins for extended period of time under different circumstances.

plant biology↗