SEEK ID: https://fairdomhub.org/assays/188
Assay type: Experimental Assay Type
Technology type: Technology Type
Organisms: No organisms
Created: 2nd May 2012 at 17:31
Last updated: 8th Nov 2017 at 15:21
I am a Postdoc at Keith Matthews lab in the Institute of Immunology and Infection Research, Edinburgh University. As part of the SilicoTryp project we are in charge of performing Targeted disruption and Overexpression of critical enzymes of Trypanosoma brucei redox metabolism enzymes and developmental perturbations to provide part of the necessary data for the construction of the model. Also generate consistent samples, so that data can be integrated and quantification results are guarateed to
The SilicoTryp project aims at the creation of a “Silicon Trypanosome”, a comprehensive, experiment-based, multi-scale mathematical model of trypanosome physiology.
Trypanosomes are blood-stream parasites transmitted by tsetse flies; they cause African sleeping sickness in humans and livestock. Currently available drugs have severe side effects, and the parasites are rapidly developing resistance.
In this project, we collect a wide range of new experimental data on the parasite in its various
Multiply perturbations of trypanosome redox metabolism, closing the feedback loop between experimentation and in silioc modelling, allowing model refinement or, where there are unexpected outcomes, re-evaluation.
Providing a dynamic picture of cell physiology by examining programmed metabolic changes during the developmental life-cycle of these parasites as they adapt to very different external milieus, including distinct levels of oxidative stress and unique adaptations of their redox balance
Snapshots: No snapshots
Key enzymes of critical points in the pathways will be targeted for disruption by the generation of RNAi cell lines and lines which drive tetracycline-regulatable ectopic over expression of either wild type enzyme or, if appropriate, dominant-negative or mis-targeted mutants of these. In all cases perturbed lines will be analysed with respect to the mRNA, protein or enzymatic activities of other components of the subsystem, this being directed iteratively by the predictions from systems modelling.
Person responsible: Federico Rojas
Snapshots: No snapshots
We routinely select specific RNAi gene targets (400–600 bp) and primers using the RNAit software http://trypanofan.path.cam.ac.uk/software/RNAit.html. A single pair of PCR primers are designed that incorporate four selected restriction sites (not present in the RNAi target fragment) such that a single PCR product can be differentially digested and sequentially cloned. For example, using MCS1/2, the following primers could be used to clone antisense followed by sense fragments: Primer 1, XbaI–BamHI-5′