Es gibt im Moment in diese Mannschaft, oh, einige Spieler vergessen ihnen Profi was sie sind. Ich lese nicht sehr viele Zeitungen, aber ich habe gehört viele Situationen. Erstens: wir haben nicht offensiv gespielt. Es gibt keine deutsche Mannschaft spielt offensiv und die Name offensiv wie Bayern. Letzte Spiel hatten wir in Platz drei Spitzen: Elber, Jancka und dann Zickler. Wir müssen nicht vergessen Zickler. Zickler ist eine Spitzen mehr, Mehmet eh mehr Basler. Ist klar diese Wörter, ist möglich verstehen, was ich hab gesagt? Danke. Offensiv, offensiv ist wie machen wir in Platz. Zweitens: ich habe erklärt mit diese zwei Spieler: nach Dortmund brauchen vielleicht Halbzeit Pause. Ich habe auch andere Mannschaften gesehen in Europa nach diese Mittwoch. Ich habe gesehen auch zwei Tage die Training. Ein Trainer ist nicht ein Idiot! Ein Trainer sei sehen was passieren in Platz. In diese Spiel es waren zwei, drei diese Spieler waren schwach wie eine Flasche leer! Haben Sie gesehen Mittwoch, welche Mannschaft hat gespielt Mittwoch? Hat gespielt Mehmet oder gespielt Basler oder hat gespielt Trapattoni? Diese Spieler beklagen mehr als sie spielen! Wissen Sie, warum die Italienmannschaften kaufen nicht diese Spieler? Weil wir haben gesehen viele Male solche Spiel! Haben
| ID | Name | Link | Status |
|---|---|---|---|
| 1 | Material Design Color Palette | GitHub | Completed |
| 2 | Material Design Color Palette | GitHub | Completed |
| 3 | Material Design Color Palette | GitHub | Completed |
Es gibt im Moment in diese Mannschaft, oh, einige Spieler vergessen ihnen Profi was sie sind. Ich lese nicht sehr viele Zeitungen, aber ich habe gehört viele Situationen. Erstens: wir haben nicht offensiv gespielt. Es gibt keine deutsche Mannschaft spielt offensiv und die Name offensiv wie Bayern. Letzte Spiel hatten wir in Platz drei Spitzen: Elber, Jancka und dann Zickler. Wir müssen nicht vergessen Zickler. Zickler ist eine Spitzen mehr, Mehmet eh mehr Basler. Ist klar diese Wörter, ist möglich verstehen, was ich hab gesagt? Danke. Offensiv, offensiv ist wie machen wir in Platz. Zweitens: ich habe erklärt mit diese zwei Spieler: nach Dortmund brauchen vielleicht Halbzeit Pause. Ich habe auch andere Mannschaften gesehen in Europa nach diese Mittwoch. Ich habe gesehen auch zwei Tage die Training. Ein Trainer ist nicht ein Idiot! Ein Trainer sei sehen was passieren in Platz. In diese Spiel es waren zwei, drei diese Spieler waren schwach wie eine Flasche leer! Haben Sie gesehen Mittwoch, welche Mannschaft hat gespielt Mittwoch? Hat gespielt Mehmet oder gespielt Basler oder hat gespielt Trapattoni? Diese Spieler beklagen mehr als sie spielen! Wissen Sie, warum die Italienmannschaften kaufen nicht diese Spieler? Weil wir haben gesehen viele Male solche Spiel!
Fig.1: Postcard Heidelberg
Es gibt im Moment in diese Mannschaft, oh, einige Spieler vergessen ihnen Profi was sie sind. Ich lese nicht sehr viele Zeitungen, aber ich habe gehört viele Situationen. Erstens: wir haben nicht offensiv gespielt. Es gibt keine deutsche Mannschaft spielt offensiv und die Name offensiv wie Bayern. Letzte Spiel hatten wir in Platz drei Spitzen: Elber, Jancka und dann Zickler. Wir müssen nicht vergessen Zickler. Zickler ist eine Spitzen mehr, Mehmet eh mehr Basler. Ist klar diese Wörter, ist möglich verstehen, was ich hab gesagt? Danke. Offensiv, offensiv ist wie machen wir in Platz. Zweitens: ich habe erklärt mit diese zwei Spieler: nach Dortmund brauchen vielleicht Halbzeit Pause. Ich habe auch andere Mannschaften gesehen in Europa nach diese Mittwoch. Ich habe gesehen auch zwei Tage die Training. Ein Trainer ist nicht ein Idiot! Ein Trainer sei sehen was passieren in Platz. In diese Spiel es waren zwei, drei diese Spieler waren schwach wie eine Flasche leer! Haben Sie gesehen Mittwoch, welche Mannschaft hat gespielt Mittwoch? Hat gespielt Mehmet oder gespielt Basler oder hat gespielt Trapattoni? Diese Spieler beklagen mehr als sie spielen! Wissen Sie, warum die Italienmannschaften kaufen nicht diese Spieler? Weil wir haben gesehen viele Male solche Spiel!
OptoPACE
Enzymes represent a major tool for many branches of the chemical industry, including food, brewing, paper, detergent or biofuel. Millions of years of evolution have allowed these proteins to performed extremely specific chemical modifications that are not only essential for living organisms but can also be of great benefit to produce useful molecules for our life, efficiently and at low cost. A major limitation of the use of enzyme for industrial application and in general out of their natural environment is their stability. They can be destroyed by other enzymes and they can unfold and take non-functional conformation when exposed to non-physiological temperature and pH. Such limitations has motivated research in species that can grow at extreme temperatures . Another major area of chemical research is the design of strategies to stabilize enzymes, and more generally proteins and peptides. Protein circularization, meaning ligation of the N- and C-terminal ends of a protein, represents a promising way to achieve this stabilization. While conserving the functionality of their linear counterpart, circular proteins can be superior in terms of thermostability, resistance against chemical denaturation and protection from exopeptidases . Moreover, a circular backbone can improve in vivo stability of therapeutical proteins and peptides. All these remarkable properties motivated us to develop new tools to circularize any protein of interest. Our Toolbox Guide provides a step-by-step strategy to clone a circularization linker and express it in E. coli. Moreover, in case of complex structures where the protein extremities are far from each other, we have developed the software tool CRAUT that will design the appropriate rigid linkers.
Please find more information about the circularization of proteins and the theory that laid the foundation for the circularization kit of the toolbox on the Circularization pages.
Circularization Constructs
The most promising approaches to circularize proteins are protein trans-splicing using split inteins and Sortase A-catalyzed cyclization. Both methods require the addition of specific proteins domains or peptides to the protein to be circularized. Consequently, on DNA level, creating circular proteins is equivalent to creating fusion proteins. However, existing protein fusion standards like RFC[23] cause scars. Those scars on protein level may affect protein function and further complicate 3D-structure modeling. Therefore, we decided to create the new RFC[i] that allows scarless cloning of inteins. Our intein circularization constructs apply to this standard, while our sortase constructs are closely related and can be used similarly. Detailed instructions on how to use our constructs are provided in our Toolbox Guide.
Split Intein Circularization
NpuDnaE intein RFC [i] circularization construct
Between the coding sequences of the Npu DnaE C-intein and the N-intein we placed. Exteins, RFC [i] standard overhangs and BsaI sites have to be added to the coding sequence of the protein to be circularized without start- and stop codons by PCR. By Golden Gate assembly, the mRFP selection marker has to be replaced with the protein insert. After addition of an inducible promotor the circular protein is ready to be expressed. For detailed step-by-step instructions please use our Toolbox Guide.
Upon expression of the fusion protein, the split intein domains reassemble to the active intein and thus ligate the termini of the protein to be circularized in trans-splicing reaction.
These constructs were successfully used to circularize lambda lysozyme and xylanase and probably DNMT1.
Sortase Circularization
Sortase A circularization construct (with His6)
An mRFP selection marker BBa_J04450, which can be removed by restriction with BsaI, is flanked by TEV protease cleavage site (left) and a sorting signal (right) with a His6 tag.
BsaI sites and overhangs corresponding to the sortase A circularization construct have to be added to the coding sequence of the protein to be circularized without start- and stop codons by PCR. By Golden Gate assembly, the mRFP selection marker has to be replaced with the protein insert. After addition of an inducible promotor protein is ready to be expressed. The protein has to be purified and treated with TEV protease and sortase A. The sortase A catalyzes the transpeptidation reaction that leads to backbone circularization.
Please find more information about our the toolkit for cirularization on the Circularization Construct pages.
Oligomerization
Split inteins constitute a useful tool to produce huge polymers in vivo: Hauptmann et al. managed to fabricate synthetic spider silk with microfiber structure. The results using an easy-to-handle split intein system were stunning: The polymers had a molecular weight of 250 kDa and more [7]. Further application of oligomerization by inteins includes the posttranslational complexation of multi-domain proteins, after their domains have been expressed individually. This approach is very valuable considering great difficulities of expressing large eukaryotic proteins in E.coli.
Standardization of oligomerization
The valuable properties of spider silk, for example its exceptional strength and elasticity, result from numerous repeats of certain protein motifs. Convenitonal methods to multimerize these motifs bear a lot of difficulties: Often genetic and mRNA instability constitute a barrier for the production of multimers as fusion proteins [1]. Posttranslational assembly through split inteins is therefore the solution to overcome these problems. The successfull polimerization of spider silk potein motifs demonstrates the potential of split inteins to be a useful tool for the production of new biomaterials by performing oligomerization reactions with split inteins. The iGEM team Heidelberg standardized (lik to toolbox guide) the oligomerization procedure with split inteins to allow easy handling with different proteins.
The use of non-orthogonal split inteins can further be exploited to direct the oligomerisation of several protein domains at once.
The mechanism
Oligomerization with inteins
Oligomerization reactions require the same constructs as the ones used for protein circularization.
Circularization is achieved by bringing the N and C terminus of a protein very close together, so both intein parts can asseble, cut out off the protein and thereby circularize it. In contrast, oligomerization occurs when both termini of a protein cannot reach each other and the intein parts of two neighbouring proteins assemble.
Fusion and Tagging
Introduction
Post-translational modifications are present in nature in great numbers.Synthetic Biology, however, has not yet made use of the innumerable possibilities nature has developed. With our collection of intein assembly constructs we expand the arsenal of synthetic biology by enabling unlimited changes of a protein's amino acid sequence even after translation.
Standard Construction
To be able to fuse any two halves of a protein together can have many different uses. We therefore saw the need for a standardised construct, the intein assembly part. This BioBrick part allows the user to clone two DNA-sequences coding for two parts of a peptide into a plasmid prepared with selection markers and standardised overhangs. Those parts were all send in with additional hexahistidine-tags to enable quick analysis on a western blot, however there are highly customisable parts available as well.Visit our parts page to get an overview of our assembly constructs. In an extensive assay we proved the principle behind split protein assembly by showing that GFP can be artificially split into two halves and thereafter be reassembled so the fluorescence is restored. Visit the split Fluorescent Protein.
N-intein fusion construct
C-intein fusion construct
Posttranslational Modifications
Introduction
Posttranslational modifications are highly prevalent in nature: Almost every protein in a cell is modified after having been translated, adding numerous varieties of the protein to the mere protein backbone. Synthetic Biology, however, can expand the possibilities offered by nature and introduce synthetic posttranslational modifications or attachments, such as biophysical probes. As the two parts of a split intein assemble in a highly specific manner, the modifications are introduced controllable a certain locus. There are different publications on intein-based introduction of posttranslational modifications, including phosphorylation, lipidation, glycosylation, acetylation and ubiquitination [4]. Phosphorylation, for example has been applied with tyrosine kinase C-terminal Src kinase (Csk) in order to be able to study the structure and function of this specifically modified protein [5].
Standard construction
Applying the intein assembly constructs the iGEM team Heidelberg provides a tool for all kinds of natural as well as synthetic posttranslational modifications by chemoselective addition of a peptide to a recombinant protein. The principle is based on intein-mediated protein fusion using the SspDnaB split intein for N-terminal modifications and SspDnaX-S11 for C-terminal ones. The split SspDnaB intein has a very short N-terminal part, consisting of only 11 amino acids intein and 5 amino acids extein sequence and a much longer C-terminal part. By contrast, SspDnaX-S11 has a C-terminal part consisting of only 6 amino acids intein and 3 amino acids extein sequence, but a much longer N-terminal part. The short part including the desired modification is easy to obtain by chemical synthesis. This offers the possibility to introduce this modification at a specific locus.
In general, split inteins are powerful tools to easily introduce all kinds of posttranslational modifications in a highly chemoselective manner.
Use our toolbox guide to attach posttranslational modifications and attachments to your protein of interest!
Translocation
Introduction
Adding and removal of translocation tags is one application example for split intein-mediated fusion of two protein domains. Translocation tags offer the possibility to transfer proteins to a certain locus inside the cell by attaching a short tag sequence to the terminus of one's protein of interest. Expressed fused to the protein, which is the conventional way, such a tag is neither removable nor attachable to a protein at a certain time point. Usage of split inteins for tagging offers new dimensions of mobility and control: Tags can be attached (using fusion constructs) or removed (using the intein protease) posttranslationally at a specific time point.
References
[1] McGinness, KE et al.: Engineering controllable protein degradation. Molecular Cell 22, 701–707, June 9, 2006. DOI 10.1016/j.molcel.2006.04.027
[2] Volkmann, G. et al: Controllable protein cleavages through intein fragment complementation. Protein Sci, 18 (2009), pp. 2393–2402. DOI: 10.1002/pro.249
[3] Volkmann, Gerrit et al.: Site-specific protein cleavage in vivo by an intein-derived protease. FEBS Letters 586 (2012) 79–84. doi:10.1016/j.febslet.2011.11.028.
[4] Vila-Perello, Miquel et al.: Biological Applications of Protein Splicing. Cell 143. October 15, 2010. DOI 10.1016/j.cell.2010.09.031.
[5] Muir, Tom W. et al.: Expressed protein ligation: A general method for protein engineering. Proc. Natl. Acad. Sci. USA 95 (1998).
[6] Lu, Wei et al.: Split intein facilitated tag affinity purification for recombinant proteins with controllable tag removal by inducible auto-cleavage. J. Chromatogr. A 1218 (2011)
[7] Hauptmann, V. et al.: Native-sized spider silk proteins synthesized in planta via intein-based multimerization. Transgenic Res (2013) 22:369–377. DOI 10.1007/s11248-012-9655-6.
[8/D] Vila-Perello, Miquel et al.: Biological Applications of Protein Splicing. Cell 143. October 15, 2010. DOI 10.1016/j.cell.2010.09.031.
[9/E] Muir, Tom W. et al.: Expressed protein ligation: A general method for protein engineering. Proc. Natl. Acad. Sci. USA 95 (1998).
[10] McGinness, KE et al.: Engineering controllable protein degradation. Molecular Cell 22, 701–707, June 9, 2006. DOI 10.1016/j.molcel.2006.04.027
[11] Volkmann, G. et al: Controllable protein cleavages through intein fragment complementation. Protein Sci, 18 (2009), pp. 2393–2402. DOI: 10.1002/pro.249
[12] Volkmann, Gerrit et al.: Site-specific protein cleavage in vivo by an intein-derived protease. FEBS Letters 586 (2012) 79–84. doi:10.1016/j.febslet.2011.11.028.
