Difference between revisions of "Team:Heidelberg/max"

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        Introduction
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            <h1>Introduction</h1>
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<h2>Protein Interactions in Directed Evolution Context</h2>
 
The proteome reflects the entirety of proteins that is expressed by a cell or an organism. Most of these proteins do not function alone. Instead, they are dependent on the interactions with a multitude of other proteins to fulfill their functions within the cell. Protein-protein interactions are responsible for almost any kind of cellular processes, such as signal transduction, cell-cell contact, transport, metabolism, cell motion or even antigen-antibody recognition (Larance, Nature Reviews Molecular Cell Biology, 2015). These interactions are enabled by electrostatic forces due to the different chemical characteristics of the amino acids. Nowadays, many biochemical or microscopy-based methods are available to investigate protein-protein interactions, for instance protein complex immunoprecipitation (Co-IP), Förster resonance energy transfer (FRET) or yeast two-hybrid screenings. As easy it is to detect a protein-protein interaction, as difficult it is to alter the strength of these interactions, although the magnitude of interaction can influence the cellular outcome, for example a stronger signal transduction or a higher expression rate. Phage-assisted continuous evolution (PACE) allows a directed evolution of different kinds of proteins within a few days as it was for example shown for RNA polymerases, proteases or aminoacyl-tRNA synthetases.  (Esvelt et al, Nature, 2011; Dickinson et al, Nat Commui., 2014; Bryson et al, Nat Chem Biol. 2017). Technically, a directed evolution towards a tighter protein-protein interaction is possible as well, as it was shown for the toxin Cry1Ac from Bacillus thuringiensis (Bt toxin) that binds a cadherin-like receptor (Badran et al, Nature, 2016). A special type of a protein-protein interaction are split enzymes that allow an auto-reassembly. They usually consist of two enzyme fragments (an N‑terminal and C‑terminal fragment) that are expressed separately, and fuse post translation. This protein fragment complementation is advantageous for any kind of application where the size of an expression cassette is a limiting factor, for instance for the packaging of DNA into virus capsids.  
 
The proteome reflects the entirety of proteins that is expressed by a cell or an organism. Most of these proteins do not function alone. Instead, they are dependent on the interactions with a multitude of other proteins to fulfill their functions within the cell. Protein-protein interactions are responsible for almost any kind of cellular processes, such as signal transduction, cell-cell contact, transport, metabolism, cell motion or even antigen-antibody recognition (Larance, Nature Reviews Molecular Cell Biology, 2015). These interactions are enabled by electrostatic forces due to the different chemical characteristics of the amino acids. Nowadays, many biochemical or microscopy-based methods are available to investigate protein-protein interactions, for instance protein complex immunoprecipitation (Co-IP), Förster resonance energy transfer (FRET) or yeast two-hybrid screenings. As easy it is to detect a protein-protein interaction, as difficult it is to alter the strength of these interactions, although the magnitude of interaction can influence the cellular outcome, for example a stronger signal transduction or a higher expression rate. Phage-assisted continuous evolution (PACE) allows a directed evolution of different kinds of proteins within a few days as it was for example shown for RNA polymerases, proteases or aminoacyl-tRNA synthetases.  (Esvelt et al, Nature, 2011; Dickinson et al, Nat Commui., 2014; Bryson et al, Nat Chem Biol. 2017). Technically, a directed evolution towards a tighter protein-protein interaction is possible as well, as it was shown for the toxin Cry1Ac from Bacillus thuringiensis (Bt toxin) that binds a cadherin-like receptor (Badran et al, Nature, 2016). A special type of a protein-protein interaction are split enzymes that allow an auto-reassembly. They usually consist of two enzyme fragments (an N‑terminal and C‑terminal fragment) that are expressed separately, and fuse post translation. This protein fragment complementation is advantageous for any kind of application where the size of an expression cassette is a limiting factor, for instance for the packaging of DNA into virus capsids.  
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                https://static.igem.org/mediawiki/2017/c/c2/T--Heidelberg--Team_Heidelberg_2017_EL222crystalstructure.png|
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                Crystal Structure of EL222 in the dark|
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                The light-inducible transcription factor EL222 is classified into three parts: the LOV domain, the conector helix and the HTH domain. (Takakado<i>et. al.</i>, 2017)|
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<h1>Motivation</h1>
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For many enzymes auto-reassembly split sites are known, however the efficiency of the joined fragments usually does not reach wildtype efficiency, as it was shown for Cas9 enzymes (Zetsche et al, Nature Biotechnology, 2015; Kaya et al, Plant Cell Physiol, 2017) Consequently, a directed evolution of split enzymes that enhance auto-reassembly is of great interest for the synthetic biology community. To demonstrate that an improved protein-protein interaction is not only possible for the Bt toxin, we used a split T7-polymerase as another example (Tiun Han et al, ACS Synthetic Biolog, 2017). We aimed at enhancing the auto-reassembly efficiency of different split sites using two in our lab established methods, PACE and PREDCEL (phage-related discontinuous evolution), which allow a fast and relatively easy directed evolution.   
 
For many enzymes auto-reassembly split sites are known, however the efficiency of the joined fragments usually does not reach wildtype efficiency, as it was shown for Cas9 enzymes (Zetsche et al, Nature Biotechnology, 2015; Kaya et al, Plant Cell Physiol, 2017) Consequently, a directed evolution of split enzymes that enhance auto-reassembly is of great interest for the synthetic biology community. To demonstrate that an improved protein-protein interaction is not only possible for the Bt toxin, we used a split T7-polymerase as another example (Tiun Han et al, ACS Synthetic Biolog, 2017). We aimed at enhancing the auto-reassembly efficiency of different split sites using two in our lab established methods, PACE and PREDCEL (phage-related discontinuous evolution), which allow a fast and relatively easy directed evolution.   
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Revision as of 06:28, 1 November 2017


Protein Interaction
Improve Protein Reassembly
Interaction of proteins can be changed by using directed evolution. In the shown case above, stronger interaction between two proteins is achieved.

Introduction

Protein Interactions in Directed Evolution Context

The proteome reflects the entirety of proteins that is expressed by a cell or an organism. Most of these proteins do not function alone. Instead, they are dependent on the interactions with a multitude of other proteins to fulfill their functions within the cell. Protein-protein interactions are responsible for almost any kind of cellular processes, such as signal transduction, cell-cell contact, transport, metabolism, cell motion or even antigen-antibody recognition (Larance, Nature Reviews Molecular Cell Biology, 2015). These interactions are enabled by electrostatic forces due to the different chemical characteristics of the amino acids. Nowadays, many biochemical or microscopy-based methods are available to investigate protein-protein interactions, for instance protein complex immunoprecipitation (Co-IP), Förster resonance energy transfer (FRET) or yeast two-hybrid screenings. As easy it is to detect a protein-protein interaction, as difficult it is to alter the strength of these interactions, although the magnitude of interaction can influence the cellular outcome, for example a stronger signal transduction or a higher expression rate. Phage-assisted continuous evolution (PACE) allows a directed evolution of different kinds of proteins within a few days as it was for example shown for RNA polymerases, proteases or aminoacyl-tRNA synthetases. (Esvelt et al, Nature, 2011; Dickinson et al, Nat Commui., 2014; Bryson et al, Nat Chem Biol. 2017). Technically, a directed evolution towards a tighter protein-protein interaction is possible as well, as it was shown for the toxin Cry1Ac from Bacillus thuringiensis (Bt toxin) that binds a cadherin-like receptor (Badran et al, Nature, 2016). A special type of a protein-protein interaction are split enzymes that allow an auto-reassembly. They usually consist of two enzyme fragments (an N‑terminal and C‑terminal fragment) that are expressed separately, and fuse post translation. This protein fragment complementation is advantageous for any kind of application where the size of an expression cassette is a limiting factor, for instance for the packaging of DNA into virus capsids.
Crystal Structure of EL222 in the dark
The light-inducible transcription factor EL222 is classified into three parts: the LOV domain, the conector helix and the HTH domain. (Takakadoet. al., 2017)

Motivation

For many enzymes auto-reassembly split sites are known, however the efficiency of the joined fragments usually does not reach wildtype efficiency, as it was shown for Cas9 enzymes (Zetsche et al, Nature Biotechnology, 2015; Kaya et al, Plant Cell Physiol, 2017) Consequently, a directed evolution of split enzymes that enhance auto-reassembly is of great interest for the synthetic biology community. To demonstrate that an improved protein-protein interaction is not only possible for the Bt toxin, we used a split T7-polymerase as another example (Tiun Han et al, ACS Synthetic Biolog, 2017). We aimed at enhancing the auto-reassembly efficiency of different split sites using two in our lab established methods, PACE and PREDCEL (phage-related discontinuous evolution), which allow a fast and relatively easy directed evolution.
Title
Subtitle
General rules: if html tags (the things starting with "<" and ending with ">") occur, the whole block in which they occur, have to be inside the following (replacing 'content') content Pages on the wiki can be edited or created by going the URL where the page shall be and choosing wiki tools -> Edit/Create Simply paste the text there and save the page. Images can be uploaded by choosing 'upload files' under 'wiki tools'. Filenames shall be T--Heidelberg--Team_Heidelberg_2017_real-name.png If a file was uploaded, a page showing the file occurs, click on 'original file' and take the URL you are getting to. This URL is the image URL used to embed the image. An editor that matches braces is pretty useful. Check if the braces of main are close properly and the chances are good thta the page will work. headers can be written as follows:

Large header

or in smaller:

Smaller Header

this is bold text this produces a line break
This text is italic Referencing works iGEMHD. Links to other pages work like this. Images can be embedded over the half page:
Title
Subtitle
Or over the full page:
Title
Subtitle
HTML tables can be generated from excel tables via http://tableizer.journalistopia.com/tableizer.php. Please delete the 'class="tableizer-table"' and 'class="tableizer-firstrow"' They are put in tableboxes as follows:

Title Subtitle

html table

References