Team:Marburg SYNMIKRO/Project

From 2012.igem.org

(Difference between revisions)

Revision as of 10:34, 25 September 2012

Summary

We aim to establish a system in Escherichia coli that generates a large number of different proteins by combinatorial fusion of few protein coding sub fragments. Our project was inspired by the VDJ-recombination of human immune system using a limited number of protein coding sequences to generate a huge diversity of antibodies.We designed an A and B module containing each three different protein coding sub fragments to be recombined. Recombination of one A fragment with one B fragment is achieved by the site-specific Gin recombinase of bacteriophage Mu. Specific Gix sites arranged as direct repeats are causing deletion within the modules and create combined fragments. Other Biobricks like a Gin enhancer and the suicide gene ccdb are located in between the modules and being deleted if an A and B fragment is fused. Those are necessary to ensure proper recombination. To visualize our results we decided to fuse fluorescent proteins of different color with intracellular localization domains. As proof of principle of our recombination machine, we expect E. colis to fluoresce in different colors at different cell domains. (This might also give us the opportunity to quantify whether occurrence of curtain recombination products is related to the position of its sub fragments to the Gin enhancer)

General goals

Improvement of proteins by genetic engineering has a tremendous impact on our daily lives. We benefit from these applications for medical, industrial and environmental purposes. Hence, the generation of novel proteins is a central goal of our project. The Marburg_SYNMIKRO team 2012 created ˈThe Recombinatorˈ: an intelligent Genetically Engineered Slot Machine (iGESM). "The Recombinator" generates large numbers of novel proteins by chance recombinations of functional domains.

Combinations

The principle of recombination

In mathematical theory the number of possible combinations result from th emultiplication of elements. In our example (left) we combine three forms (triangle, square and circle) with three colors (red, blue and yellow). This results in 3 x 3 = 9 different combinations.

Recombination in the vertebrate adaptive immune system

For the creation of new fusion proteins in vivo we used the VDJ-recombination as a model. The VDJ-recombination takes place during the generation of antibodies and is essential for the recognition of diverse antigens Antibodies consist of two identical heavy (H) and light (L) chains which are covalently bound by disulfid bonds. The heavy and light chains can be subdivided into constant (C) and variable (V) regions. This/ese are for the light chain CL and VL and for the heavy chain VH and CH1, CH2 and CH3. The variable parts have the ability to bind one distinct antigen. But our immune system needs to recognize millions of different antigens from bacteria, viruses and other microorganisms. The solution to this problem is an amount of diverse variable regions of the antibodies. This is done by the VDJ-recombination. The name of this recombination is related to the gene segments which built up the variable regions. The loci (κ and λ) for the variable regions of the light chains consist of a certain number of V (Variable)- and J (Joining)-segments. The loci of the heavy variable chain exhibits an additional number of D (Diversity)-segments. The enormous rate of variable regions is caused by random fusion of segments. For the light chain one V-segment gets fused to one of the J-segments by recombination. This rearrangement is transcribed with the downstream located C (Constant)- region. The production of a variable heavy chain implies the fusion from the J-segment to the D-segment. The combined DJ-sequence is added to one V-segment and transcribed with the C-region, too. Each loci of the light chains is made up of 40 V-segments and 5 J-segments. At the loci for the heavy chain there are 50 V-, 27 D- and 6 J-segments. Taking this into consideration the number of possible combinations amounts 1,6 x 106. Furthermore mutations increase the possible rearrangements up to 1010.

Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.

Part 2

test Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.

Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.

The Experiments

Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.

Part 3

Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.

Results

Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.

@@ Line 46: / Line 46: @@
 == '''Recombination in the vertebrate adaptive immune system''' ==
 For the creation of new fusion proteins in vivo we used the VDJ-recombination as a model. The VDJ-recombination takes place during the generation of antibodies and is essential for the recognition of diverse antigens
-[[File:Syn_MR_VDJ-recombination.png]]
+[[File:Syn_MR_VDJ-recombination.png|850px]]
 Antibodies consist of two identical heavy (H) and light (L) chains which are covalently bound by disulfid bonds. The heavy and light chains can be subdivided into constant (C) and variable (V) regions. This/ese are for the light chain CL and VL and for the heavy chain VH and CH1, CH2 and CH3. The variable parts have the ability to bind one distinct antigen. But our immune system needs to recognize millions of different antigens from bacteria, viruses and other microorganisms.
 The solution to this problem is an amount of diverse variable regions of the antibodies. This is done by the VDJ-recombination.

Team:Marburg SYNMIKRO/Project

From 2012.igem.org

Revision as of 10:34, 25 September 2012

Contents

Summary

General goals

Combinations

The principle of recombination

Recombination in the vertebrate adaptive immune system

Part 2

The Experiments

Part 3

Results