Team:UNSW Australia/Description

Our Project

The Vision

The 2016 UNSW iGEM team hopes to introduce customisable outer membrane vesicles (OMVs) as a platform technology for the application of future iGEM projects. To achieve this, we investigated the effect of various genetic alterations on the ability of E. coli to overproduce OMVs. In this hypervesiculating strain we then directed fluorescent proteins to the periplasm for uptake within OMVs, or anchored them to the outer membrane for decoration of OMVs. We hope this will serve as proof of concept for the potential for OMVs to be tailored for various functions in future projects.

The Context

Currently, synthetic biologists engineer live bacterial cells to perform new or useful functions. However, these bacteria are limited to use in the laboratory as their release poses severe biosafety risks since they may persist in the environment long after their function is required.

In comparison, OMVs are lipid bubbles, produced by bacteria when a bulge of their outer membrane pinches off and detaches from the cell surface. As the vesicle forms, it can encapsulate some of the proteins directly underlying the outer membrane in the periplasm. Thus, OMVs can potentially be easily customised for various functions by targeting cellular proteins to the periplasm or outer membrane [1]. OMVs are non-living and non-replicating, and therefore following potential release they will eventually degrade with no long-term effect on the natural ecosystem.

Research has been slow to explore the potential uses of OMVs, while their capability to be customised makes them an excellent prospective platform technology for synthetic biology. They could potentially be the medium through which synthetic biology projects can be applied in the real world, overcoming the biosafety problems posed by the classical use of live bacteria.

The Project

For our project, we first implemented various genetic alterations in E. coli that we believed would result in overproduction of OMVs [1-4]. Our final genotypes included overexpression of TolR or g3p, and knockouts of TolA or DegP. These genotypes have previously been identified to induce hypervesiculation in various bacterial species, but their relative effect on E. coli had not yet been established prior to our project. Thus, each of our E. coli genetic variants were assessed on ability to hypervesiculate, taking into account both size and amount of OMVs produced, through the use of laser scanning microscopy.

As proof of concept for OMV customisation, we then directed fluorescent proteins to the periplasm or anchored them to the outer membrane. This allowed us to test the ability of OMVs to be customised through adornment with outer membrane proteins, or packaging of periplasm-localised proteins.

We hope the results of this project will serve future teams well, enabling them to utilise OMVs as a platform technology for the application of synthetic biology projects.

Our Work

Demonstrate
Experiments
Proof
Notebook

References

  1. Deatherage B. L., Cano Lara, J., Bergsbaken, T., Rassoulian Barrett, S. L., Lara, S., Cookson, B. T. 2009. Biogenesis of bacterial membrane vesicles. Molecular Microbiology, 72(6), 1395-1407
  2. McBroom, A. J., Kuehn, M. J. 2007. Release of outer membrane vesicles by gram-negative bacteria is a novel envelope stress response. Molecular Microbiology, 63(2), 545-558
  3. Baker, J. L., Chen, L., Rosenthal, J. A., Putnam, D., DeLisa, M. P. 2014. Microbial biosynthesis of designer outer membrane vesicles. Current Opinion in Biotechnology, 29, 76-84
  4. Henry, T., Pommier, S., Journet, L., Bernadac, A., Gorvel, J., Lloubes, R. 2004. Improved methods for producing outer membrane vesicles in gram-negative bacteria. Research in Microbiology, 115, 437-446

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