Team:Heidelberg/Project

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 Project overview

Proteins are the functional basis of all biological processes and being able to control and improve their functions through design and engineering is one of the fundamental goals of synthetic biology. While conventional proteins subsist as chains of amino acids with defined beginning and end, nature has found a curious way of enhancing a protein capabilities: circularization. In head to tail circularized peptides the terminal amino acids are joined together just like in the rest of the chain, forming a circular structure. Such peptides have been discovered in all kingdoms of life during the past years and they are unified by an extreme stability towards high temperatures, proteases and changes in pH.

Given these attractive features of circular proteins, methods to circularize otherwise linear proteins have been devised, one of which is based on autocatalytic protein domains called inteins. We have applied the principle of circular peptides to synthetic biology by introducing a BioBrick-based, standardised method for circularizing any protein using inteins.

Synthetically connecting a protein's termini without disrupting its 3D structure and function is, however, a delicate task which has so far been accomplished only for relatively small proteins whose ends lie close to each other. We therefore saw the need for a comprehensive software that predicts the process of circularization. With CRAUT we have brought into existence a powerful open-source software to predict an optimal linker to support circularization of a protein preserving its 3D structure and function.
Due to our lack of calculating power we deployed this software on the distributed computing platform BOINC in an initiative we call iGEM@home.

Based on our software predictions, we constructed linkers to circularize the 871 a.a. long methyltransferase Dnmt1 and provide data suggesting that circular DNMT1 is more functional than its linear counterpart at high temperatures. Our results have strong implications for developing an innovative PCR-based technique that could revolutionize epigenetic studies and cancer research by maintaining the methylation pattern of the DNA template during amplification.

Eventually, inteins can be used to post-translationally modify any protein in a multitude of ways going far beyond circularization. We therefore created a BioBrick-based intein toolbox to allow for easy and standardised protein manipulation. We think that our toolbox will be invaluable to many systems biology projects aimed at dissecting or re-engineering the function of cellular networks.

CIRCULARIZATION

Create a linker with our crowd computing software and make your protein heat stable

OLIGOMERIZATION

Fuse multiple Proteins or Domains using Inteins

FUSION

Fuse two Proteins or Domains together using Inteins

ON/OFF

Activate or deactivate Proteins using Inteins

PURIFICATION

Placeholder
the intein
toolbox
 inducible via
light induction
Heat-stable
circular
DNA-
Methyltransferase

PCR 2.0
modify your protein
using the toolbox guide

Achievements

Establishing protein circularization as a NEW BIOENGINEERING TOOL in synthetic biology.

Contributing to iGEM with a new foundational advance!

Providing a NEW COMPREHENSIVE TOOLBOX based on inteins for modifying proteins post-translationally.

Sending 67 Biobricks to Registry of Biological parts!

Development of a NEW STANDARD to make the use of inteins easy and modular.

Establishment of a new RFC!

Showing that the toolbox WORKS: proteins are circularized and split fluorescent proteins are reconstituted.

Making Gels, Western Blots, Fluorescence-based Assays and Mass spectrometry to prove it!

Creating circular DNMT1 and showing that it is ACTIVE.

For the first time achieving the circularization of a large protein!

Developing a NEW SOFTWARE to calculate customized linkers to circularize proteins.

Making CRAUT open-source for the scientific community.

Establish a distributed computing platform called iGEM@home.

Using this platform as an entirely new way to reach out to the world with synthetic biology concepts!

Creating a NEW SOFTWARE to display the notebook on the wiki.

Distributing MidNightDOC to the iGEM community to help future teams organize their protocols!