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November 14, 2005

Synthetic Clock PhD plan

Writing about web page http://blogs.warwick.ac.uk/ulrichjanus/entry/todo_1/

Writing about an entry you don't have permission to view

Results of the meeting with Markus and Isabelle last Friday (11.Nov). Email to get the E.Coli strain used by Atkinson 2003 is under way..

Motivating or guiding Question

  • How accurately can synthetic systems be described by mathematical models?
  • How well can specific parameters of the in vivo system be changed?

Main Goals:

  • Review the theoretical model (kinetics and structure) to make it more realistic.
  • Identify relevant parameters to move system into the region of sustained oscillations.
  • Achieve this parameter shift by mutagenesis of transcription factors by error prone PCR.
  • Compare responses of theoretical and in vivo system.

Postulated Results:

  • Model predictions can be improved by using more realistic kinetic description of the regulation events.
  • Mutagenesis by PCR allows are more precise manipulation of model parameters than the method used previously.

Schedule of the Experimental Work:

  • Check if the strains are still rythmic, i.e. if the previous work can be reproduced (~ 1mth.)
  • Intoduce a luciferase reporter gene to measure clock function via luminescence assays (~ 1mth.)
  • Test, if cells can be grown on agar plates rather than in liquid cultures
  • mutagenise the sequence of relevant transcription factor to change chosen model parameters by erroneous PCR (~ 2mth)
  • Identify mutated colonies by changed clock behaviour caused by changes in the affinites of the transcription factor (~ 4mths)
    These are 8mths, make it 12mths.

about the advisor(s):
Postponed…


October 27, 2005

PhD proposal

Ok, where to start. Maybe with the original proposal presented by Markus and Isabelle in the summer. Let's browse through the main points. It also looks like its reading time again…

This proposal is as I have heard, obsolete now, as the the described system is already investigated by a Japanese lab. I stilss is a good starting point.

Summary:
Experimental and mathematical study of a synthetic genetic network.

Biological Background:
In cyanobacteria clock has KaiA, KaiB and KaiC as major components forming an osciallatory transcriptional/translational feedback loop. But rhythmic phosphorylation of KaiC can persist in the absence of rhythmic transcription and translation. The KaiA,B,C cycle was reconstituted in vitro suggesting that it constitutes a minimal circadian oscillator [Nakajima 2005 ].

Proposal:

  • Reconstitute this minimal clock in E. Coli under the control of the lac promoter
  • investigate its properties (sensitivity of dynamics to varying expression levels of the components)
  • Control expression levels by titrating the level of the IPTG inducer (what the hell is that?)
  • Monitor KaiA, KaiC interaction by tagging them with split GFP.

Experimental work:

  • Examine clock behaviour of System in E.coli to changes in condition (IPTG level, temperature etc.
  • directed evolution selecting for "improved" (in what sense) properties
  • mutagenise Kai genes by error-pron PCR, transfer back into E.coli, screen for desired properties
  • sequence mutation, determine nature of mutation and how they affect clock component interaction

Mathematical Analysis:

  • kinetic model of the clock based on mass action kinetics
  • use elementary flux mode
  • effects of small copy number investigated by Fokker-Planck birth-death master equations

Goals / Direction of research

  • Special interest in the predicted effect of changes in the binding constants between different Kai proteins. To be compated with the effect of mutation altering the rate of KaiC phosphorylation.
  • Predict the effevts of increased noise to artificialy small copy numbers of proteins. Derive estimates of the noise in relatio to cellular copy numbers.

Remarks:

  • Model and experiment investigate clock at the post-translational model in contrast with the typical transcriptional networks.
  • Long term goal: understand the relationship between the protein-protein and the genetic network in a "systematic" way. Setup allows combinging both levels by design.

October 24, 2005

What's up these days

I am stumbling forward into the first term of this year with several things to do at once plus a substantial workload of supervisions. Well, my choice. Let's have a look:

PhD outline


Certainly the most important. So far it is presumed to involve

  • Generalization of the elementary flux mode analysis (see groups of Heinrich and Schuster ) to oscillations, i.e. decompose a reaction network into oscillatory subnetworks.

  • Study of a synthetic clock as a model system. No references right now.

  • Some yet to be defined project connecting network approaches of different research areas of the groups involved in UniNet

As we see this needs to be boiled down and clarified a bit. Also there are several issues with the plan so far, that have to be resolved:

  1. The structural network analysis relies on the stoichiometric matrix of reaction networks. Can this be transferred to regulatory genetic networks at all? If not, then any application to transcriptional networks like circadian clocks would not be feasible.

  2. What kind of experiments with synthetic systems are feasible and how much time will it involve. After all, my main focus is supposed to be mathematical. It was also suggested that I might rather continue work on the Arabidopsis clock as this is the main focus of the Carre lab.

Well, until the end of this year I should have a clearer picture of all this.

Loose ends of the Arabidopsis clock project


Reminds me to put all my reports of the MOAC projects somewhere. I will check and have the data ready next week, no worries there. What remains is to go over the code I wrote and document it, so I can hand it back with a clear conscience.

A short term follow up project might involve taking the approximation model, rewrite the code from scratch and turn it into a usable tool for the Carre lab. But let them sort that out.


Ulrich Janus

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