Difference between revisions of "Team:UNSW Australia/Description"
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<h2>The Project</h2> | <h2>The Project</h2> | ||
| − | < | + | <h4 style="font-family: Slim-Joe;">Hypervesiculating Strain Library</h4> |
| − | <p>For our project, we first implemented various genetic alterations in E. coli that we believed would result in overproduction of OMVs. The final stains we thus obtained are shown in the table below: | + | <p>For our project, we first implemented various genetic alterations in E. coli that we believed would result in overproduction of OMVs. The final stains we thus obtained are shown in the table below:<br><br> |
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<table class="table table-striped table-bordered"> | <table class="table table-striped table-bordered"> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| − | <th></th> | + | <th rowspan="2">Knock-in Mutations</th> |
<th colspan="3">Recipient Strains</th> | <th colspan="3">Recipient Strains</th> | ||
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| − | < | + | <td>NEB T7 E.coli</td> |
| − | < | + | <td>ΔTolA E. coli</td> |
| − | < | + | <td>ΔDegP E. coli</td> |
</tr> | </tr> | ||
</thead> | </thead> | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | < | + | <td>None</td> |
<td></td> | <td></td> | ||
<td>ΔTolA E. coli</td> | <td>ΔTolA E. coli</td> | ||
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| − | < | + | <td>g3p</td> |
<td>E. coli + g3p</td> | <td>E. coli + g3p</td> | ||
<td>ΔTolA E. coli + g3p</td> | <td>ΔTolA E. coli + g3p</td> | ||
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</tr> | </tr> | ||
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| − | < | + | <td>TolR</td> |
<td>E. coli + TolR</td> | <td>E. coli + TolR</td> | ||
<td>ΔTolA E. coli + TolR</td> | <td>ΔTolA E. coli + TolR</td> | ||
Revision as of 15:33, 19 October 2016
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
Hypervesiculating Strain Library
For our project, we first implemented various genetic alterations in E. coli that we believed would result in overproduction of OMVs. The final stains we thus obtained are shown in the table below:
| Knock-in Mutations | Recipient Strains | ||
|---|---|---|---|
| NEB T7 E.coli | ΔTolA E. coli | ΔDegP E. coli | |
| None | ΔTolA E. coli | ΔDegP E. coli | |
| g3p | E. coli + g3p | ΔTolA E. coli + g3p | ΔDegP E. coli + g3p |
| TolR | E. coli + TolR | ΔTolA E. coli + TolR | ΔDegP E. coli + TolR |
TolR (periplasmic domain) or g3p (N-terminal domain) were expressed in a T7 promoter-driven vector (pET or pRSF Duet, respectively). These knock-in mutations were selected for our strain library as they have both been demonstrated to destabilise the outer membrane of numerous gram negative strains and induce increased production of OMVs.
Attachment protein g3p has been shown to interact with TolA, a protein which confers outer membrane stability. Interaction between TolA and g3p destabilises the outer membrane, promoting OMV production. Interaction is mediated by both the N1 and N2 domains of g3p (Lubkowski et al., 1999). To date, however, only N1 has been assessed for its OMV-inducing capacity; thus, this is the terminus we also worked with.
TolR, meanwhile is part of the Tol-Pal complex which tethers the outer membrane to the cytoplasmic membrane (Figure 1). Overexpression of tolR has been linked to loss of outer membrane integrity, and increased OMV production (Baker et al., 2014).
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.
References
- 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
- 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
- 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
- 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
