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Team:BCCS-Bristol/Project/Wet lab
From 2009.igem.org
iGEM 2009
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| Check out our selection of submitted BioBricks! | In-frame fusion of arbitrary bio-bricks with no scarring? no problem! Read about our BioScaffold solution here. | View our team's lab book, charting our progress throughout the course of the project. | Take a look at our photos from the wet lab. |
Introduction
We wanted to exploit the fact that outer membrane vesicles (OMVs) were naturally produced in gram negative bacteria such as E. coli. The aim was to use them in our advantage as a directed delivery system to cells of proteins of our interest.This can potentially be used for the safe delivery of drugs into cells or for cell-cell communication purposes.We believe that the inclusion of proteins in vesicles will not only protect them from degradation in the extracellular environment but will also protect other cells if the cargo proteins packaged into vesicles are toxic or harmful.
Because the exact mechanism by which OMVs are produced in gram negative cells is not yet elucidated (although 3 models have been proposed, Lauren M. Mashburn-Warren et al, 2006) we decided to make protein fusions of proteins that were normally included in OMVs with proteins of our interest. Experiments with protein fusions to the toxin ClyA were already made with success (Jae-Young Kim et al, 2008) and the desired proteins were delivered to OMVs. But for the purposes that we would be using OMVs we thought that it would be safer to use harmless proteins that would act as carriers instead of ClyA. Hence we selected 3 possible candidate protein carriers (Eun-Young Lee et al, 2007) to be used in our project.These proteins are:
1. OsmE (Osmotically inducible lipoprotein E)
2. fiu (siderophore receptor)
3. FhuA (Ferrichrome-iron receptor)
In order to assess the production of our fusion proteins and at the same time monitor their introduction into the OMVs we used GFP (Green Fluorescent Protein) as our cargo protein fused to one of the three carriers stated above.
If you want to find out more about our project, as far as the laboratory work in concerned, click on the available links!!
