Difference between revisions of "Team:Harvard/Description"

 
(33 intermediate revisions by 3 users not shown)
Line 2: Line 2:
  
 
<html>
 
<html>
 +
 +
<!--Parallax scrolling-->
 +
<style>
 +
.parallax {
 +
    /* The image used */
 +
    background-image: url("https://static.igem.org/mediawiki/2017/3/3c/Harvard--High_Banner.jpeg");
 +
 +
 +
    /* Set a specific height */
 +
    min-width: 100%;
 +
    min-height: 50%; 
 +
    height: 500px;
 +
    width: 100%;
 +
   
 +
 +
 +
   
 +
 +
    /* Create the parallax scrolling effect */
 +
    background-attachment: fixed;
 +
    background-position: center;
 +
    background-repeat: no-repeat;
 +
    background-size: cover;
 +
}
 +
 +
img {
 +
    max-width: 100%;
 +
    height: auto;
 +
    width: auto\9; /* ie8 */
 +
}
 +
 +
</style>
 +
 +
<!-- make the above container -->
 +
<div class="parallax column full_size"></div>
 +
 
<div class="column full_size description" style = "margin-top: 5%">
 
<div class="column full_size description" style = "margin-top: 5%">
−
<br>
+
 
 
<h1>Project Description</h1>
 
<h1>Project Description</h1>
 +
<h3>Overview</h3>
 
<br>
 
<br>
−
 
+
Chemical synthesis is the most commonly used method of producing industrially relevant molecules, yet this practice is often accompanied by various environmental hazards. Biological synthesis, on the other hand, does not produce any toxic byproducts, nor does it require expensive starting materials. In the long run, it is a better manufacturing solution in many respects.
−
Our team will focus on developing antimicrobial biofilms and bringing production to industrial relevancy. Our project offers the potential to make the biological manufacture of antimicrobial peptides an economically feasible alternative to chemical synthesis.  
+
 
<br><br>
 
<br><br>
−
The first part of our project, functionalizing biofilms, will involve attaching functional proteins to curli fibers, the main proteinaceous component of biofilms naturally produced by Escherichia coli. In anticipation of the potential obstacles of synthesizing AMPs in a microbe, we will be using a library approach to cast a wide net on the range of documented AMPs. Through our screen of a library of AMP and curli fiber fusions, we hope to identify characteristics of AMP structure that make them more amenable to maintaining structure and function when fused to curli fibers, expressed, and secreted in E. coli. This can then act as a starting point for identifying or designing additional AMPs for functionalizing curli.
+
One strategy for biological synthesis is to adapt pre-existing systems already in nature. For instance, <i>E. coli</i> produces proteinaceous components in its biofilm called curli fibers. These fibers self-polymerize outside of the cell, forming a macroscopic agglomeration of material when isolated in sufficient bulk. The Joshi lab has already demonstrated how various functional peptide domains can be added to the self-polymerizing units of curli, so the polymers can form the basis of a variety of functional materials.  
 
<br><br>
 
<br><br>
−
The second part of our project, bringing production to industrial relevancy, can be further broken down into three aspects: pathway optimization, resource management, and bioprocess optimization. The first of those, pathway optimization, will involve finding the optimal expression ratios of the various proteins involved in the curli production pathway. This is to minimize resource and energy wasted in the production of unused chaperone or membrane proteins. The second, resource management, will focus on diverting as much of the cell’s resources and energy to the curli production pathway while still maintaining cell viability. We will be using a strain of E. coli already optimized for recombinant protein production, BL21 (DE3), but we will also test our system with other knockouts to further optimize the strain for our purposes. Lastly, the bioprocess optimization aspect will involve the development of a bioreactor that not only provides a larger fermentation environment, but also integrates the extraction and filtration of the biofilm from the cells in a smooth and continuous process.
+
Our project focuses on optimizing curli production on two fronts as a first step to developing the curli system into a robust platform for producing materials at industrially relevant yields. First, we alter ribosome binding site strengths associated with proteins involved in the curli pathway to optimize the stoichiometric ratios of these molecules in the cell. The alterations are informed by a model of curli production and export that determines the optimum ratio of pathway components to maximize the production of our desired product, extracellular curli fibers, and minimize the aggregation of unwanted byproducts within the cell. This ensures that each cell becomes a more efficient curli export machine. The second component of the project aims to maximize cell densities within culture media through the development of a bioreactor that maintains higher dissolved oxygen concentrations than standard shaker flasks. Eliminating the limiting factor of insufficient oxygenation allows bacterial cells to produce more curli per unit of feedstock. This aspect of the project aims to optimize the conditions for protein-producing cell cultures. Our work along these two lines will inform the development of the curli system as a feasible biosynthetic platform for producing scalable and programmable materials.
 +
 
 +
 
 +
<h3>Curli System</h3>
 +
 
 +
<br>
 +
Curli fibers are the main proteinaceous component of <i>E. coli</i> biofilms, which are aggregates of microorganisms embedded in a self-produced matrix of various polymeric substances. Biofilms are often associated with pathogenicity, but they also provide opportunities for development as platforms for biological synthesis of functional materials. The secretion and polymerization pathway of curli fibers is well-documented, and is depicted here in this diagram:
 +
 
 +
<br>
 +
 
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2017/2/25/Harvard_--_Curli_Pathway.jpeg" alt="Curli_Pathway.jpg" width="300" height="600"><br>
 +
<div align="middle" style="font-size:80%"> Goyal, Parveen, et al. (2014) </div>
 +
</center>
 +
 
 +
<br>
 +
 
 +
There are a number of chaperone and membrane proteins involved in this pathway:
 +
 
 +
<br>
 +
<center>
 +
<table>
 +
  <tr>
 +
    <th>Protein</th>
 +
    <th>Function</th>
 +
  </tr>
 +
  <tr>
 +
    <td>csgA</td>
 +
    <td>Major subunit that aggregates and self-polymerizes to form curli fibers</td>
 +
  </tr>
 +
  <tr>
 +
    <td>csgB</td>
 +
    <td>Interacts with csgF to initiate nucleation of csgA fibers</td>
 +
  </tr>
 +
  <tr>
 +
    <td>csgC</td>
 +
    <td>Chaperone protein that interacts with csgA and csgB and prevents them from polymerizing inside the cell</td>
 +
  </tr>
 +
  <tr>
 +
    <td>csgE</td>
 +
    <td>Periplasmic protein that interacts with csgG at the outer membrane</td>
 +
  </tr>
 +
  <tr>
 +
    <td>csgF</td>
 +
    <td>Periplasmic protein that interacts with csgG at the outer membrane</td>
 +
  </tr>
 +
  <tr>
 +
    <td>csgG</td>
 +
    <td>Outer membrane lipoprotein that is required for stability and secretion of csgA and csgB</td>
 +
  </tr>
 +
</table>
 +
</center>
 +
 
 +
<br>
 +
 
 +
We are particularly interested in curli fibers because they are extremely robust and can withstand exposure to extreme conditions like boiling in detergents or incubation in solvents. Thus, they present great potential for use in harsh environments.  
 +
 
 
<br><br>
 
<br><br>
 +
 +
Additionally, the Joshi lab has demonstrated that it is possible to fuse functional proteins to the csgA subunit and have both csgA retain its secretion and polymerization properties while having the fused protein maintain its primary function. For instance, the Joshi Lab successfully showed GFP fused to csgA retains its fluorescent properties, and enzymes can be immobilized on csgA to form catalytic biofilms. Proteins fused to csgA will not only be secreted using <i>E. coli</i> ’s natural secretion machinery, but it will also be embedded within a physical material that can be handled and applied in a variety of ways.
 +
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2017/e/e5/Harvard--Curli_fusions.png" alt="Curli_Fusions.jpg" width="600" height="300"><br>
 +
<div align="middle" style="font-size:80%"> Nguyen, Peter Q., et al. (2014) </div>
 +
</center>
 +
 +
 +
<br>
 +
<h3>References</h3>
 +
 +
<ul>
 +
  <li>Goyal, Parveen, et al. "Structural and mechanistic insights into the bacterial amyloid secretion channel CsgG." Nature 516.7530 (2014): 250-253.</li>
 +
  <li>Nguyen, Peter Q., et al. "Programmable biofilm-based materials from engineered curli nanofibres." Nature communications 5 (2014).</li>
 +
</ul>
 +
 +
<br><br><br>
 
</div>
 
</div>
  

Latest revision as of 03:23, 2 November 2017

Harvard OPTI POLY

Project Description

Overview


Chemical synthesis is the most commonly used method of producing industrially relevant molecules, yet this practice is often accompanied by various environmental hazards. Biological synthesis, on the other hand, does not produce any toxic byproducts, nor does it require expensive starting materials. In the long run, it is a better manufacturing solution in many respects.

One strategy for biological synthesis is to adapt pre-existing systems already in nature. For instance, E. coli produces proteinaceous components in its biofilm called curli fibers. These fibers self-polymerize outside of the cell, forming a macroscopic agglomeration of material when isolated in sufficient bulk. The Joshi lab has already demonstrated how various functional peptide domains can be added to the self-polymerizing units of curli, so the polymers can form the basis of a variety of functional materials.

Our project focuses on optimizing curli production on two fronts as a first step to developing the curli system into a robust platform for producing materials at industrially relevant yields. First, we alter ribosome binding site strengths associated with proteins involved in the curli pathway to optimize the stoichiometric ratios of these molecules in the cell. The alterations are informed by a model of curli production and export that determines the optimum ratio of pathway components to maximize the production of our desired product, extracellular curli fibers, and minimize the aggregation of unwanted byproducts within the cell. This ensures that each cell becomes a more efficient curli export machine. The second component of the project aims to maximize cell densities within culture media through the development of a bioreactor that maintains higher dissolved oxygen concentrations than standard shaker flasks. Eliminating the limiting factor of insufficient oxygenation allows bacterial cells to produce more curli per unit of feedstock. This aspect of the project aims to optimize the conditions for protein-producing cell cultures. Our work along these two lines will inform the development of the curli system as a feasible biosynthetic platform for producing scalable and programmable materials.

Curli System


Curli fibers are the main proteinaceous component of E. coli biofilms, which are aggregates of microorganisms embedded in a self-produced matrix of various polymeric substances. Biofilms are often associated with pathogenicity, but they also provide opportunities for development as platforms for biological synthesis of functional materials. The secretion and polymerization pathway of curli fibers is well-documented, and is depicted here in this diagram:
Curli_Pathway.jpg
Goyal, Parveen, et al. (2014)

There are a number of chaperone and membrane proteins involved in this pathway:
Protein Function
csgA Major subunit that aggregates and self-polymerizes to form curli fibers
csgB Interacts with csgF to initiate nucleation of csgA fibers
csgC Chaperone protein that interacts with csgA and csgB and prevents them from polymerizing inside the cell
csgE Periplasmic protein that interacts with csgG at the outer membrane
csgF Periplasmic protein that interacts with csgG at the outer membrane
csgG Outer membrane lipoprotein that is required for stability and secretion of csgA and csgB

We are particularly interested in curli fibers because they are extremely robust and can withstand exposure to extreme conditions like boiling in detergents or incubation in solvents. Thus, they present great potential for use in harsh environments.

Additionally, the Joshi lab has demonstrated that it is possible to fuse functional proteins to the csgA subunit and have both csgA retain its secretion and polymerization properties while having the fused protein maintain its primary function. For instance, the Joshi Lab successfully showed GFP fused to csgA retains its fluorescent properties, and enzymes can be immobilized on csgA to form catalytic biofilms. Proteins fused to csgA will not only be secreted using E. coli ’s natural secretion machinery, but it will also be embedded within a physical material that can be handled and applied in a variety of ways.
Curli_Fusions.jpg
Nguyen, Peter Q., et al. (2014)

References

  • Goyal, Parveen, et al. "Structural and mechanistic insights into the bacterial amyloid secretion channel CsgG." Nature 516.7530 (2014): 250-253.
  • Nguyen, Peter Q., et al. "Programmable biofilm-based materials from engineered curli nanofibres." Nature communications 5 (2014).