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| - | {{:Team:BCCS-Bristol/Header}}
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| - | ==Abstract==
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| - | 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.
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| - | 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:
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| - | 1. OsmE (''Osm''otically inducible lipoprotein ''E'')
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| - | 2. fiu (siderophore receptor)
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| - | 3. FhuA (Ferrichrome-iron receptor)
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| - | 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.
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| - | ==Bioscaffold==
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| - | ====Protein Fusion Problems====
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| - | Each gene that encodes a protein naturally ends with two stop codons (5'-TAATAA-3'). Moreover when two genes are fused together an additional third stop codon is inserted in the DNA scar (DNA region that connects the two genes). Hence this would prevent the expression of the second partner in the fusion protein.
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| - | ====Existing Solutions====
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| - | The current iGEM assembly standards do not ''fully'' support protein fusions. Only assembly standard 15, a concept known as Bioscaffold, tries to address this problem in some depth. The Bioscaffold is a DNA sequence that is ligated between the two protein encoding genes and contains restriction sites of atypical Tye II restriction enzymes. The enzymes will cleave outside their recognition sequence hence removing the stop codons of the first protein in the fusion.
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| - | ====BCCS Bioscaffold====
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| - | =====Bioscaffold Features=====
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| - | [http://partsregistry.org/Part:BBa_K259002 Bioscaffold], that can be placed between the two DNA sequences (see figure below, i.e. Part A and Part B). Upon treatment with specific restriction enzymes the bioscaffold part is removed along with the stop codons. In addition a Gly-Ser linker is left behind which provides some flexibility for the correct 3D-folding of the constituent proteins in the fusion protein.
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| - | [[Image:BCCS_Bioscaffold.png|500px|thumb|center|]]
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| - | ==[https://2009.igem.org/Team:BCCS-Bristol/Notebook/Lab_Book '''Lab Book''']==
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| - | ==[https://2009.igem.org/Team:BCCS-Bristol/Notebook/Biobricks '''Biobricks''']==
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| - | ==[https://2009.igem.org/Team:BCCS-Bristol/Notebook/Lab_photos '''Lab Photos''']==
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