IGEM:Stanford/2009/Mini Cells

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Contents

Project Summary

Engineer a system to physically insulate reactions/functions through a minicell-docking mechanism. Sort of like satellite external organelles.

Original Presentation: Physical Insulation of Pathways or Functions

Framing questions

  1. How are we going to make the mini cells?
    • Secretion from producing cells
    • artificial cell fabrication
      • Expression of a cascading genetic network within liposomes [1]
  2. How are the mini cells going to attach? ex: kinds of receptors
  3. How are the mini cells going to communicate/types of junctions? ex: gap, tight, etc,,,

Note- we can search the registry for some options- we thought we could possibly use the gene for bacteria pilus???

Experiment Ideas

Important/Interesting Papers

  • A new phylum of Archaea represented by a nanosized hyperthermophilic symbiont - a natural example of what we are trying to build.
  • The Dynamic Control of Kiss-And-Run and Vesicular Reuse Probed with Single Nanoparticles - an interesting and potentially useful mechanism to dock mini-cells with main cell, based on neurotransmitter vesicles.
  • Nature: Release of secretory products during transient vesicle fusion by G. Alvarez de Toledo
    • This was one of the sources from the kiss and run article, but it has some interesting ideas about the size of the pores through which it releases the neurotransmitters... Suzie
  • Synaptic vesicle endocytosis: fast and slow modes of membrane retrieval
    • another good article to skim... example exceprt:
    • "SNAREs and kiss-and-run exocytosis vesicle fusion is mediated partly by SNARE (soluble N-ethylmaleimide sensitive factor [NSF] attachment protein receptor) protein interactions [31]. In brief, vesicular (v)-SNAREs on the vesicle membrane bind to target (t)-SNAREs on the plasma membrane and form tight–SNARE complexes that bring the two bilayers into close apposition (Figure 1). The tight–SNARE complex is an extremely stable, low energy state and, thus, needs the action of the ATPase NSF to be dissociated so that v- and t-SNARE proteins can be recycled after fusion. At nerve terminals, Ca2+ influx followed by Ca2+ binding to synaptotagmin triggers the fusion of synaptic vesicles. The formation of a fusion pore is, thus, usually followed by full collapse of the vesicle membrane onto the plasma membrane. However, the fusion pore can sometimes transiently close, so the process of pore opening is reversible, as clearly evidenced by capacitance measurements in endocrine cells [30]. We speculate that a loose–SNARE complex, which can also lead to fusion [31] and [32], is more likely to be reversible than the tight–SNARE complex and, thus, provides a possible substrate for kiss-and-run exocytosis Figure 1). However, the conformational changes in SNARE protein (and SNARE-interacting proteins) that might favor transient fusion pore openings are still poorly understood [31] and [33] and the number of SNARE complexes that form the collar around the fusion pore neck has not been determined."
    • This could add complications to the project..?? Suzie
    • Not that complicated in fact. SNARE is one of the primary trafficking components of eukaryotes. However, implementing SNARE without a strong understanding on bacterial localization of proteins would make this process difficult. Can we produce SNARE in E. Coli? - Chris (quick google search says yes)

Questions/Discussion

  • Anusuya Ramasubramanian 12:21, 1 April 2009 (PST): For the third question in the overview (i.e. regarding how the minicells may attach), we may want to look into the docking mechanisms of actual cells, specifically clathirin and COP coatings, and see if any of these natural mechanisms are applicable.
  • This is random but there is a TED talk on quorum sensing. Enjoy - Chris
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