IGEM:Stanford/2009/Interspecies Membrane Transfer

Project Summary

Goal: To design a system that allows for the transfer of membrane-bound proteins across species between a factory cell and a target cell. This transfer will be mediated by vesicles.

Proposed application: To use E. coli as a factory/production center for carotenoids, which will be transferred to cyanobacteria (target cell).

General Outline/Steps

1. Express carotenoids in E. coli.
2. Ensure that the carotenoids colocalize at membrane-forming loci and are integrated into the outer membrane of E. coli.
3. Show that OMVs contain carotenoids.
4. Fuse E. coli-derived OMVs to outer membrane of cyanobacteria.
5. Determine difference in absorption spectrum of cyanobacteria.

What We Don't Know (but need to know)

• How do we ensure transport of proteins to the outer and not the inner membrane? Does it matter? -Chris

• Tuesday 4/21 discussion
  • Do we know the genes that causes blebbing
  • Is cyanobacteria gram-negative
  • How do we penetrate the cell wall
  • What receptors are required for quorum sensing
  • Will the mixed cultures affect growth time. Would the bacteria "kill" the cyanobacteria?
    • Possibly separate blebs from cells by centrifuge
    • Possibly test bleb transfer to new cells by using antibiotics to kill old cells and keep the target cells alive.
  • If blebbing is very easy to induce:
    • How can we improve a simple system?
    • How can we make a generic system species specific
  • Using SNARE.
    • Robert suggests similar approach to UCSF blockage problem in lysosomes.

Experiment Ideas

• Use blebbistatin to control blebbing

Important/Interesting Papers and Notes

• Notes from Gram-negative outer membrane vesicles: beyond the cell surface. Geobiology. 2008 Jun;6(3):214-9. [1]
  • Certain proteins are enriched in membrane vesicles, which implies that these proteins are selectively sorted during MV formation.
  • Kadurugamuwa and Beveridge demonstrated the stable fusion of MVs with the outer membrane of Gram-negative bacteria. The situation is more complicated when the target cell is Gram-negative, but studies have suggested that even fusion doesn't occur between MVs and Gram-positive bacteria, the MVs can attach to Gram-positive targets and deposit antibacterial factors, possibly via salt-bridges.
  • Another group showed that treatment of a bacterial infection with soluble gentamicin (an antibiotic that increases outer membrane permeability) did not succeed, but when the antibiotic was loaded into MVs, the infection was successfully treated.
  • Biofilms rely on MVs and have been purified from biofilm matrices. Biofilms produce more MVs than cultures of planktonic bacteria, and whether the bacteria are biofilm or planktonic determines the protein content in the MVs.

• Organisation and evolution of the tol-pal gene cluster.J Mol Microbiol Biotechnol. 2001 Jan;3(1):113-22. [2]
• Res Microbiol. 2004 Jul-Aug;155(6):437-46. [3]
  • Mutations in these genes have been shown to amplify the level of vesicle formation. Can we use these genes to decrease the stability of the outer membrane in our factory E. coli to induce greater vesicular formation? The resulting E. coli would exhibit unregulated expression of carotenoids and unregulated formation of vesicles.

• Outer Membrane Vesicle Production by Escherichia coli Is Independent of Membrane Instability. Journal of Bacteriology, August 2006, p. 5385-5392, Vol. 188, No. 15 [4]
  • In this study, the E. coli genome was screened for genes that affect levels of vesicle formation. Almost all of the mutations in the tested genes resulted in high-vesiculation, ranging from a 5-fold decrease to a 200-fold increase in comparison to wild-type levels.
• Review paper on blebbing
• Article on the math and visualization of blebs.
• Another good review on outer membrane vesicles
• The classic 1974 paper on membrane vesicles.
• Blebs in quorum sensing