User:Pakpoom Subsoontorn/Notebook/Genetically Encoded Memory/2008/10/14: Difference between revisions

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* Natural/synthetic topologies of the target DNA
* Natural/synthetic topologies of the target DNA
* Required Axillary factors:  
* Required Axillary factors:  
* Recombination Efficiency in standard host
* Recombination Efficiency in standard host, natural host, etc.
* Recombination speed
* Recombination speed
* Studies in mutated enzyme VS target sequences
* Studies in mutated enzyme VS target sequences

Revision as of 19:09, 20 October 2008

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Quantitative functional profiles of site-specific recombinase

  • Compiling together known information about recombinase
  • Focusing on information that will be useful for in vivo DNA manipulation. For now, let's focus on simple model organism like E.coli and S.cerevisiae
  • Two major sources of information:
    • mining from literature
    • large scale, parallel experiment

Database/method features

  • Key feature of the database:
    • Quantitative description,
    • Capabilities to compare and contrast
      • Standardized description
  • Expandable size of database without messing up with core structure
  • literature cross reference, updating
  • scalable experiment/measurement

Quantitative functional information

The list of information fields (need to decide: mining VS experimenting, set priority, black-box):

  • Enzyme name
  • Natural source of enzyme, host
  • Coding sequence for enzyme itself
  • Enzyme structure: amino acid sequence, functional domain,
  • Target DNA sequence: Natural target,
  • Natural/synthetic topologies of the target DNA
  • Required Axillary factors:
  • Recombination Efficiency in standard host, natural host, etc.
  • Recombination speed
  • Studies in mutated enzyme VS target sequences
  • Mechanism:


Standard System,

  • What should be our standard:
    • Host cells
    • Plasmid
    • System for expressing enzyme

Parameter we want to tune,

  • Enzyme concentration
  • Expression time
    • How long do we have to have enzyme around?

Tools

Informatics tools

  • automated system of updating the list of information in public domain.
    • The list of enzymes and the reference scientific literature (i.e. from pubmed)
    • Roughly split-up information according to fields:
    • Standard information such as sequence, structure, ontology, etc from GenBank or PDB...keep them update


Molecular Biology tools

  • Standard cassettes on plasmids so that we can try wide varieties of recombination sites and recombination enzymes
  • Tunable promoter and some reporter tags that report the levels of recombinase
  • Method for timing the recombination process. At what time point the recombination of DNA is complete? This could time scale could be much shorter than the time until the reporter of the new DNA configuration become observable. Can we pause/terminate recombination at different time point?
  • Some mechanism to accommodate the fact that the substrate (sites on genomic DNA) has very low copies.


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