Team:Linkoping Sweden/Project

From 2013.igem.org

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==The basics==
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The basics of our project idea is to first grow E.Coli that has been genetically modified to produce an antibody with the enzyme luciferase attached to its constant part. At first the antibody will be constructed to adhere to a peanut antigen, but our long time goal is to be able to switch to whichever antigen desired. Luciferase is an enzyme that cleaves luciferin in the presence of ATP, giving rise to a long lasting green light by the principle of luminescence. Luminescence is superior to fluorescence due to, e.g. lack of fading.
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==Antibody fusion protein==
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[[File:Ide_Bild1.png|thumb |alt=Example alt text |Fusion protein consisting of a luciferase linked with a single domain antibody]]
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The first step in our project will be to create a fusion protein consisting of a luciferase linked together with a single domain antibody from a camelid, VHH. The idea behind using these antibodies is the fact that they consist only of a single domain. This makes it possible for them to be expressed lesser organisms, such as E. coli, something that would be very hard for conventional antibodies found in other mammals.<br /><br />
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Our ulterior motive behind cloning an antibody in E. coli is that it allows us to engineer the protein after our preferences. What we want to do is connect the VHH antibody to a luciferase protein via a linker. If the translation of the fusion protein is successful, the luciferase will be active when luciferin and ATP is added to the solution.<br />
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Another factor to consider is the binding affinity that the antibody part of the fusion protein has for its antigen. This is probably the most critical part of our experiment. The challenge lies in trying to make the antibody regain most of its affinty, while being connected to another protein via a linker.
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==Antigen fusion protein==
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[[File:Ide_Bild2.png|thumb |alt=Example alt text |Fusion protein consisting of a quencher (RFP) linked with a antigen]]

Revision as of 12:03, 14 June 2013

The basics

The basics of our project idea is to first grow E.Coli that has been genetically modified to produce an antibody with the enzyme luciferase attached to its constant part. At first the antibody will be constructed to adhere to a peanut antigen, but our long time goal is to be able to switch to whichever antigen desired. Luciferase is an enzyme that cleaves luciferin in the presence of ATP, giving rise to a long lasting green light by the principle of luminescence. Luminescence is superior to fluorescence due to, e.g. lack of fading.

Antibody fusion protein

Example alt text
Fusion protein consisting of a luciferase linked with a single domain antibody

The first step in our project will be to create a fusion protein consisting of a luciferase linked together with a single domain antibody from a camelid, VHH. The idea behind using these antibodies is the fact that they consist only of a single domain. This makes it possible for them to be expressed lesser organisms, such as E. coli, something that would be very hard for conventional antibodies found in other mammals.

Our ulterior motive behind cloning an antibody in E. coli is that it allows us to engineer the protein after our preferences. What we want to do is connect the VHH antibody to a luciferase protein via a linker. If the translation of the fusion protein is successful, the luciferase will be active when luciferin and ATP is added to the solution.
Another factor to consider is the binding affinity that the antibody part of the fusion protein has for its antigen. This is probably the most critical part of our experiment. The challenge lies in trying to make the antibody regain most of its affinty, while being connected to another protein via a linker.

Antigen fusion protein

Example alt text
Fusion protein consisting of a quencher (RFP) linked with a antigen