Team:Heidelberg/Tour

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                 <div class="jumbotron" id="overview" style="background-image:url(https://static.igem.org/mediawiki/2013/8/8b/Heidelberg_Tour_Overview.png); background-repeat:no-repeat; background-size:110%; background-position:25% 30%">
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                 <div class="jumbotron" id="overview" style="background-image:url(https://static.igem.org/mediawiki/2013/8/8b/Heidelberg_Tour_Overview.png); background-repeat:no-repeat; background-size:115%; background-position:25% 30%">
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                       <h2>Overview</h2>
                       <h2>Overview</h2>
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                                 We wish to open up the great field of NRPS to the iGEM community. <br/> Therefore, we:  
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                                 The iGEM Team Heidelberg introduces NRPS to the iGEM community:  
<ul>
<ul>
-
  <li style="font-size:14px">Proved that <b>N</b>on-<b>R</b>ibosomal <b>P</b>eptide <b>S</b>ynthetase modules behave in a modular manner and can be shuffled</li>   
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  <li style="font-size:14px">Proved modular principle of <b>N</b>on-<b>R</b>ibosomal <b>P</b>eptide <b>S</b>ynthetase by successful shuffling domains and modules</li>   
  <li style="font-size:14px">Developed a method for easy <b>N</b>on-<b>R</b>ibosomal <b>P</b>eptide-detection</li>
  <li style="font-size:14px">Developed a method for easy <b>N</b>on-<b>R</b>ibosomal <b>P</b>eptide-detection</li>
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  <li style="font-size:14px">Employed a <b>N</b>on-<b>R</b>ibosomal <b>P</b>eptide for the recycling of gold from electronic waste</li>
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  <li style="font-size:14px">Employed and tested a concept for the recycling of gold from electronic waste using <b>N</b>on-<b>R</b>ibosomal <b>P</b>eptides </li>
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  <li style="font-size:14px">Implement a software which enables everyone to design synthetic <b>N</b>on-<b>R</b>ibosomal <b>P</b>eptides</li>
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  <li style="font-size:14px">Implemented a software which enables everyone to design synthetic <b>N</b>on-<b>R</b>ibosomal <b>P</b>eptides</li>
</ul>
</ul>
                         </p>
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                         <img src="https://static.igem.org/mediawiki/2013/3/3a/Heidelberg_GRAPHICAL_ABSTRACT_small.png" style="width:100%; margin-top:-20%">
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<h2>Recycling of Gold from Electronic Waste Using the NRP Delftibactin</h2>
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<h2>Recycling Gold from Electronic Waste Using the NRP Delftibactin</h2>
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                         <h2>Synthetic Peptides</p>
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                         <h2 >Synthetic Peptides</h2>
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Non-Ribosomal Peptide Synthetases are composed of building blocks, which are called modules. Each module shows a distinct specificity for a large variety of different monomers assembling them chain-like to a protein. Therefore, the order of modules determines the sequence of the final proteins. We used the tyrocidine synthetase from <i>Brevibacillus parabrevis</i> to:</p>
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<ul>
 +
<li style="font-size:14px">Employ this modularity to investigate the interchangeability and therefore compatibility of modules</li>
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<li style="font-size:14px">Engineer custom synthetic peptides <i>in vivo</i> by shuffling modules</li>
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<li style="font-size:14px">Demonstrate the broad variety of possible products</li>
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</ul>
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<p style="font-size:14px; text-align:justify">
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Understanding this modularity could provide new insights into combinatorial biology and approaches for creating compound libraries for screening purposes.</p>
                               <a href="/Team:Heidelberg/Project/Tyrocidine" style="margin-bottom:5%"><button typ="button" class="btn btn-default">Discover more!</button></a>
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<img src="https://static.igem.org/mediawiki/2013/3/38/Heidelberg_tour_indtag-scheme.png" style="width:30%; float:right; margin-right:5%; margin-top:15px;">
                     <h2>Indigoidine-Tag</h2>
                     <h2>Indigoidine-Tag</h2>
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                                 Indigoidine is a blue pigment produced by the NRPS module indC. We fused indC with other NRPS modules to yield an assembly line producing peptides labelled with the <a href="https://2013.igem.org/Team:Heidelberg/Project/Indigoidine-Tag">Indigoidine-Tag</a>. This procedure opens up the possibility for the high-throughput generation of combinatorial NRPS libraries due to its simplicity in assembly, detection, validation and purification. We propose such high-throughput protocols in our <a href="https://2013.igem.org/Team:Heidelberg/RFCs#rfc99>RFC99</a>, which is integrated into our main standard, the <a href="https://2013.igem.org/Team:Heidelberg/RFCs>RFC100</a>. We optimized the indigoidine synthetase by replacing single domains with both domains from NRPS modules and fully synthetic domains and coexpression with different enzymes required for NRPS activation (see <a href="https://2013.igem.org/Team:Heidelberg/Project/Tag-Optimization">Tag-Optimization</a>).
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                                 <ul>
 +
<li style="font-size:14px">
 +
Creation of fusion NRPS producing peptides labelled with the <a href="https://2013.igem.org/Team:Heidelberg/Project/Indigoidine-Tag">Indigoidine-Tag</a>
 +
</li>
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<li style="font-size:14px">
 +
High-throughput protocols for design, construction and evaluation of combinatorial NRPS libraries (<a href="https://2013.igem.org/Team:Heidelberg/RFCs">RFC99 and RFC100</a>)
 +
</li>
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<li style="font-size:14px">
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<a href="https://2013.igem.org/Team:Heidelberg/Project/Tag-Optimization">Optimization</a> of the indigoidine synthetase by domain exchanges and coexpression with different PPTases
 +
</li>
 +
<li style="font-size:14px">
 +
Creation of functional synthetic NRPS domains based on multiple sequence alignments
 +
</li>
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                             <a href="/Team:Heidelberg/Project/Indigoidine-Tag" style="margin-bottom:5%"><button typ="button" class="btn btn-default">Read more!</button></a>
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Features:</p>
Features:</p>
<ul>
<ul>
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  <li style="font-size:14px; margin-left:45px">User input: A desired peptide consisting of more than 50 amino acids with different chirality, which are currently present in our database</li>
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  <li style="font-size:14px; margin-left:45px">Computer aided design of fully synthetic NRPS determined to produce a user-defined short peptide</li>
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  <li style="font-size:14px; margin-left:45px">The software calculates a NRPS domain sequence, composed of domains from different species, which is capable of producing the desired non-ribosomal peptide</li>
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  <li style="font-size:14px; margin-left:45px">Optimal domain assembly based on evolutionary distance</li>
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  <li style="font-size:14px; margin-left:45px">Via the integrated Software "Gibthon", a former iGEM project, a Gibson cloning strategy is designed</li>
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  <li style="font-size:14px; margin-left:45px">The curated database stores information of 658 Domains encoded on 99 DNA sequences</li>
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  <li style="font-size:14px; margin-left:45px">to make sure our tool can be used beyond this years iGEM competition we furthermore implemented the possibility of adding new domains to our database by entering a DNA sequence</li>
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  <li style="font-size:14px; margin-left:45px">Automated domain recognition for newly entered NRPS sequences</li>
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<li style="font-size:14px; margin-left:45px">On the basis of this DNA sequence a domain prediction is carried out, which is based on antismash2</li>
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<li style="font-size:14px; margin-left:45px">Integration of Gibthon to facilitate implementation of cloning strategy</li>
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<li style="font-size:14px; margin-left:45px">This de facto enables everyone to design his or her non-ribosomal peptide of interest!</li>
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                         <h2>Modeling the Feasibility of Gold Recycling with Delftibactin</h2>
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                         <p style="font-size:14px; text-align:justify">In oder to find out whether the use of delftibactin would be feasible for industrial-scale recycling of gold from electronic waste we designed a model for the cost development of such a aproach.</p>
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                         <h2 style="padding-left:50px; text-align:left">Modeling the Feasibility of Gold Recycling with Delftibactin</h2>
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                         <h2>Modeling the Indigoidine Production</h2>
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                    Non-ribosomal peptide synthetases expressed in natural organisms help to develop evolutionary advantages over competitors. This ability has been recognized at the industrial level for example, by pharmaceutical companies. Of course, we were also fascinated by the idea to elevate our system to a larger scale and to test its industrial feasibility. Accompanying our experimental results confirming the ability of delftibactin to precipitate gold, we attempt to use theoretical considerations and metabolic modeling to show the realistic potential of our idea. </p>
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                         <h2 style="padding-left:50px; text-align:left">Modeling the Indigoidine Production</h2>
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                                 A challenge we had to face during the characterization and optimization of indC was to identify the production kinetics of indigoidine. In order to disentangle the underlying mechanisms of bacterial growth and peptide synthesis, we decided to set up a mathematical model based on coupled ordinary differential equations. Calibrated with our experimental time-resolved data, the mathematical model could potentially not only elucidate how indigoidine production influences growth of bacteria but also provide a more quantitative understanding of the synthesis efficiency of the different T domains and PPTases that were tested.  
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                         <p style="font-size:14px; text-align:justify">The aim of science and synthetic biology in particular is to <b>improve lives by solving problems</b>. We as researchers (-to-be) are therefore working for society. Yet, we can only offer solutions, which have to be approved and applied by the public.
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                         <p style="font-size:14px; text-align:justify">The aim of science and synthetic biology in particular is to <b>improve lives by solving problems</b>. We as researchers are therefore working for society. Yet, we can only offer solutions, which have to be approved and applied by the public.
</p>
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<p style="font-size:14px; text-align:justify">
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We as iGEM Team Heidelberg have therefore put great effort in communicating with many groups within society to open minds, broaden horizons as well as minimize prejudices and concerns:
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We as iGEM Team Heidelberg have therefore put great effort in communicating with many groups within society to open minds, broaden horizons as well as minimize prejudices and concerns by:
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                         <h2> Results</h2>
                         <h2> Results</h2>
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                               <li style="font-size:14px">We opened up the usage of NRPS to the iGEM community</li>
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                               <li style="font-size:14px">Automated Design of <b>Custom Non-Ribosomal Peptide Synthetases</b></li>
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                               <li style="font-size:14px">We proved modularity of NRPS on different hierachical layers</li>
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                               <li style="font-size:14px">Production of <b>Synthetic Peptides</b> in different engineered E. coli Hosts</li>
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                               <li style="font-size:14px">We established a <a href="https://2013.igem.org/Team:Heidelberg/Project/Indigoidine-Tag">universal and specific tag for the labeling of NRPs</a></li>
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                               <li style="font-size:14px">Invention of a Universal Tag for Non-Ribosomal Peptide labeling in vivo </li>
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                              <li style="font-size:14px">We submitted a standardized assembly line production procedure for synthetic NRPs: <a href="/Team:Heidelberg/RFCs">RFC 99 and RFC 100</a></li>
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                              <li style="font-size:14px">Using the Tag for peptide characterization and quantification of production </a></li>
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                              <li style="font-size:14px">We implemented a software which enables everyone to <a href="/Team:Heidelberg/Project_Software">create synthetic NRPS!</a></li>
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                              <li style="font-size:14px">Introducing our standardized Framework for Production of Synthetic Peptides to the Synbio Community:  <a href="/Team:Heidelberg/RFCs">RFC 99 and RFC 100</a></li>
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                              <li style="font-size:14px">We had a great summer with lots of <a href="/Team:Heidelberg/Team/Gallery">fun and new experiences!</a></li>
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                              <li style="font-size:14px">Having lots of <a href="/Team:Heidelberg/Team/Gallery">fun</a> and very much looking forward to the Jamboree in Boston</a></li>
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Latest revision as of 03:55, 29 October 2013

Take the Tour! Take Your Chance and Join Our Quest!

Overview

The iGEM Team Heidelberg introduces NRPS to the iGEM community:

  • Proved modular principle of Non-Ribosomal Peptide Synthetase by successful shuffling domains and modules
  • Developed a method for easy Non-Ribosomal Peptide-detection
  • Employed and tested a concept for the recycling of gold from electronic waste using Non-Ribosomal Peptides
  • Implemented a software which enables everyone to design synthetic Non-Ribosomal Peptides

Recycling Gold from Electronic Waste Using the NRP Delftibactin

Due to the fast turn-over of today’s high-tech equipment, millions of tons of electronic waste accumulate each year. It contains tons of gold which is very valuable to the society, not only because of its use in jewelry but also for medical applications. The main approach nowadays is to recycle gold by electrolysis which is highly inefficient and expensive, preventing most of the gold from being recovered. In our project we worked on finding a more efficient and environmentally friendly method to recover this precious metal:

  • We managed to recover gold from electronic waste using delftibactin, a NRP produced by Delftia acidovorans
  • We managed to clone all genes needed for delftibactin production into E. coli
  • We managed to recombinantly express the NRPS responsible for delftibactin production in E. coli

Synthetic Peptides

Non-Ribosomal Peptide Synthetases are composed of building blocks, which are called modules. Each module shows a distinct specificity for a large variety of different monomers assembling them chain-like to a protein. Therefore, the order of modules determines the sequence of the final proteins. We used the tyrocidine synthetase from Brevibacillus parabrevis to:

  • Employ this modularity to investigate the interchangeability and therefore compatibility of modules
  • Engineer custom synthetic peptides in vivo by shuffling modules
  • Demonstrate the broad variety of possible products

Understanding this modularity could provide new insights into combinatorial biology and approaches for creating compound libraries for screening purposes.

Indigoidine-Tag

  • Creation of fusion NRPS producing peptides labelled with the Indigoidine-Tag
  • High-throughput protocols for design, construction and evaluation of combinatorial NRPS libraries (RFC99 and RFC100)
  • Optimization of the indigoidine synthetase by domain exchanges and coexpression with different PPTases
  • Creation of functional synthetic NRPS domains based on multiple sequence alignments

Software: NRPSDesigner

Features:

  • Computer aided design of fully synthetic NRPS determined to produce a user-defined short peptide
  • Optimal domain assembly based on evolutionary distance
  • The curated database stores information of 658 Domains encoded on 99 DNA sequences
  • Automated domain recognition for newly entered NRPS sequences
  • Integration of Gibthon to facilitate implementation of cloning strategy
  • Parts registry interface and SBOL output format

Modeling the Feasibility of Gold Recycling with Delftibactin


Non-ribosomal peptide synthetases expressed in natural organisms help to develop evolutionary advantages over competitors. This ability has been recognized at the industrial level for example, by pharmaceutical companies. Of course, we were also fascinated by the idea to elevate our system to a larger scale and to test its industrial feasibility. Accompanying our experimental results confirming the ability of delftibactin to precipitate gold, we attempt to use theoretical considerations and metabolic modeling to show the realistic potential of our idea.

Modeling the Indigoidine Production

A challenge we had to face during the characterization and optimization of indC was to identify the production kinetics of indigoidine. In order to disentangle the underlying mechanisms of bacterial growth and peptide synthesis, we decided to set up a mathematical model based on coupled ordinary differential equations. Calibrated with our experimental time-resolved data, the mathematical model could potentially not only elucidate how indigoidine production influences growth of bacteria but also provide a more quantitative understanding of the synthesis efficiency of the different T domains and PPTases that were tested.

Human Practice

The aim of science and synthetic biology in particular is to improve lives by solving problems. We as researchers are therefore working for society. Yet, we can only offer solutions, which have to be approved and applied by the public.

We as iGEM Team Heidelberg have therefore put great effort in communicating with many groups within society to open minds, broaden horizons as well as minimize prejudices and concerns by:

  • Involving experts
  • Engaging the broad public
  • Getting inspired by artists
  • Intruiging the next generation of scientists

And finally bringing them all together to an open talk evening addressing the question "On the Way to a Synthetic Future?"

Results

  • Automated Design of Custom Non-Ribosomal Peptide Synthetases
  • Production of Synthetic Peptides in different engineered E. coli Hosts
  • Invention of a Universal Tag for Non-Ribosomal Peptide labeling in vivo
  • Using the Tag for peptide characterization and quantification of production
  • Introducing our standardized Framework for Production of Synthetic Peptides to the Synbio Community: RFC 99 and RFC 100
  • Having lots of fun and very much looking forward to the Jamboree in Boston

Thanks to