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. Protein splicing, a process in which a catalytically active internal polypeptide termed INTEIN excises itself from a precursor protein, has been used by our team to alter the functionalities of proteins in various ways post-translationally.

We have developed a comprehensive toolbox compromising five sets of standardized parts that provide standard mechanisms for specific modifications of proteins: (1) A purification standard to faciliate purification of recombinant proteins. (2) A construct that allows for post-translational fusion of protein domains to produce synthetic proteins (3) A standard that allows for switching proteins on and off. (4) One to oligomerize protein monomers. And (5) perhaps the most intriguing standard construct of this toolbox that was built to produce heat stable proteins, which can be achieved by circularization. In order to offer a comfortable way to apply any of the above mechanisms to a protein and a biological system we designed a toolbox guide that provides step by step instructions for cloning based on the intein standard parts described in our RFC.

Besides achieving a solid and broad foundation for future intein applications in synthetic biology, we aimed on deeply exploring one of the functions provided in our toolbox: circularization of proteins. 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. 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. With CRAUT we have brought into existence a powerful open-source software to predict an optimal rigid linker to support stabilizing circularization of a protein preserving its 3D structure and function. In order to provide us - and future iGEM teams - with a standard way to conduct the needed consuming modelling calculations we deployed a distributed computing platform we call iGEM@home that we also developed into a powerful science communication platform. As an evaluation of the linker software we screened linkers generated by the software by circularizing lamda lysozyme and experimentally evaluating the heat stability of the products.

Based on the calibrated software, 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.

Besides a protein with potential medical and one with biotechnological applications we chose the hemicellulose xylanase as a third target for circularization, that has applications in large scale production in paper and food industry.

As an additional feature we decided to add another dimension to intein mediated modifications of our toolbox by developing optically induced small inteins that are photocaged by an As LOV2 domain. While sterically hampering intein dimerization in the dark, the LOV domain opens up when exposed to blue light, releasing the intein to proceed the splicing reaction.