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Team:Heidelberg/Tour
From 2013.igem.org
Project
Notebook
Human Practice
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 usingNon-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.
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
