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Team:BCCS-Bristol/Notebook
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For this reason we designed and created an artificial DNA sequence, called [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. | For this reason we designed and created an artificial DNA sequence, called [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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==[https://2009.igem.org/Team:BCCS-Bristol/Notebook/Lab_Book '''Lab Book''']== | ==[https://2009.igem.org/Team:BCCS-Bristol/Notebook/Lab_Book '''Lab Book''']== | ||
Revision as of 16:56, 18 October 2009
iGEM 2009
Contents |
Abstract
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)
Bioscaffold
The current iGEM assembly standards do not fully support protein fusions because 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 fusion protein. Also sometimes the fusion of genes may change the reading frame.
For this reason we designed and created an artificial DNA sequence, called [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.
