Difference between revisions of "Http://2017.igem.org/2017.igem.org/Team:Potsdam/Abstract"

Line 16: Line 16:
  
 
<div align="justify">
 
<div align="justify">
−
Bringing enzymes closer together for increased reaction efficiency by using DNA scaffolds In fields like biotechnology, synthetic biology and in medical applications it becomes increasingly important to produce more and more complex substrates efficiently. For this reason, a high product yield is always sought after.  
+
Bringing enzymes closer together for increased reaction efficiency by using DNA scaffolds. In fields like biotechnology, synthetic biology and in medical applications it becomes increasingly important to produce more and more complex substrates efficiently. For this reason, a high product yield is always sought after.  
 
<br>A relatively straight forward method to achieve this is a close proximity between cooperating enzymes, this so called metabolic channeling (MC) has already been widely observed in natural processes <sup>[1]</sup> like the proteasome for molecular degradation.  
 
<br>A relatively straight forward method to achieve this is a close proximity between cooperating enzymes, this so called metabolic channeling (MC) has already been widely observed in natural processes <sup>[1]</sup> like the proteasome for molecular degradation.  
 
</div>
 
</div>
Line 38: Line 38:
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
−
     <td align="justify"width="50%";>The first method centers on the usage of a low copy plasmid and a high copy plasmid. <br>The low copy plasmid contains coding regions for dCas9, sgRNA-aptamer-fusions and enzyme-RNA-binding-protein-fusions (RBPs corresponding to the aptamers). There will also be a slightly altered version with a direct dCas9-enzyme-fusion and no aptamers or RBPs.
+
     <td align="justify"width="50%";>The first method centers on the usage of a low copy plasmid and a high copy plasmid. <br>The low copy plasmid contains coding regions for dCas9, sgRNA-aptamer-fusions and pathway enzymes fused to RNA-binding-proteins (RBPs corresponding to the aptamers).
 +
 
 
<br>The high copy plasmid contains many repeated target regions for the sgRNAs.
 
<br>The high copy plasmid contains many repeated target regions for the sgRNAs.
 
<br>After successful transformation of the plasmids the sgRNA-aptamer-fusions will bind to the scaffold DNA directed by dCas9. The enzymes will then be attached to the aptamers with the connected RBPs. Through this arrangement the enzymes will be immobilized on the scaffold close together which should lead to increased efficiency.
 
<br>After successful transformation of the plasmids the sgRNA-aptamer-fusions will bind to the scaffold DNA directed by dCas9. The enzymes will then be attached to the aptamers with the connected RBPs. Through this arrangement the enzymes will be immobilized on the scaffold close together which should lead to increased efficiency.
 
</td>
 
</td>
 
     <td align="justify"><p style="margin-left:30px;">
 
     <td align="justify"><p style="margin-left:30px;">
−
The second method revolves around Liquid-liquid-phase separation, a process naturally occurring in cells<sup>[2]</sup>.Through this process, proteins aggregate together to form droplets or membrane-less bodies in the cytoplasm or nucleoplasm. Examples are stress granules or cajal bodies. The formation of droplets has already been studied by attaching YFP fluorescent protein to DDX48<sup>[3]</sup>, a protein known for aggregation. There are also indicators for this to happen in bacteria.
+
The second method revolves around Liquid-liquid-phase separation, a process naturally occurring in cells<sup>[2]</sup>. Through this process, proteins aggregate to form droplets or membraneless bodies in the cytoplasm or nucleoplasm. Examples are stress granules or cajal bodies. The formation of droplets has already been studied by attaching YFP fluorescent protein to DDX4<sup>[3]</sup>, a protein known for aggregation. There are also indicators for this to happen in bacteria.
 
<br>To use this mechanism for improvement of enzyme efficiency, the YFP-coding region will be replaced by the coding region of the desired enzymes and expressed in yeast (due to the size of the droplets). </p>
 
<br>To use this mechanism for improvement of enzyme efficiency, the YFP-coding region will be replaced by the coding region of the desired enzymes and expressed in yeast (due to the size of the droplets). </p>
 
</td>
 
</td>

Revision as of 10:04, 1 November 2017

No Sidebar - Escape Velocity by HTML5 UP

Abstract
Bringing enzymes closer together for increased reaction efficiency by using DNA scaffolds. In fields like biotechnology, synthetic biology and in medical applications it becomes increasingly important to produce more and more complex substrates efficiently. For this reason, a high product yield is always sought after.
A relatively straight forward method to achieve this is a close proximity between cooperating enzymes, this so called metabolic channeling (MC) has already been widely observed in natural processes [1] like the proteasome for molecular degradation.

To achieve this artificially, we propose two approaches:
Bild Bild
MC using dCas9

 MC by Liquid-liquid-phase separation

The first method centers on the usage of a low copy plasmid and a high copy plasmid.
The low copy plasmid contains coding regions for dCas9, sgRNA-aptamer-fusions and pathway enzymes fused to RNA-binding-proteins (RBPs corresponding to the aptamers).
The high copy plasmid contains many repeated target regions for the sgRNAs.
After successful transformation of the plasmids the sgRNA-aptamer-fusions will bind to the scaffold DNA directed by dCas9. The enzymes will then be attached to the aptamers with the connected RBPs. Through this arrangement the enzymes will be immobilized on the scaffold close together which should lead to increased efficiency.

The second method revolves around Liquid-liquid-phase separation, a process naturally occurring in cells[2]. Through this process, proteins aggregate to form droplets or membraneless bodies in the cytoplasm or nucleoplasm. Examples are stress granules or cajal bodies. The formation of droplets has already been studied by attaching YFP fluorescent protein to DDX4[3], a protein known for aggregation. There are also indicators for this to happen in bacteria.
To use this mechanism for improvement of enzyme efficiency, the YFP-coding region will be replaced by the coding region of the desired enzymes and expressed in yeast (due to the size of the droplets).


In both approaches we will introduce the auxin pathway and compare auxin output with and without metabolic channeling.

[1] Lester J. Reed (1973), Multienzyme Complexes, Clayton Foundation Biochemical Institute and Department of Chemistry, University of Texas at Austin, 40-46
[2] Anthony A. Hyman et al. (2014), Liquid-Liquid Phase Separation in Biology, Max Planck Institute of Molecular Cell Biology and Genetics Dresden and Max Planck Institute for the Physics of Complex Systems, Dresden, 39-58
[3] Nott et al. (2015), Phase Transition of a Disordered Nuage Protein Generates Environmentally Responsive Membraneless Organelles, Mol. Cell, 57, 936-947 4 Yuan A. H., Hochschild A. (2017).A bacterial global regulator forms a prion. Science355, 198–201