http://2014.igem.org/wiki/index.php?title=Special:Contributions/Huhny&feed=atom&limit=50&target=Huhny&year=&month=2014.igem.org - User contributions [en]2024-03-19T07:52:11ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/Team:Heidelberg/ProjectTeam:Heidelberg/Project2014-10-18T03:51:27Z<p>Huhny: </p>
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<h4><img id="dropdownImg" src="/wiki/images/7/70/Heidelberg_Abstract-dropdown.png"/>&nbsp;Project overview</h4><br />
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<div id="abstract-content" class="col-lg-12" ><br />
<p>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. <br />
<br></br><br />
We have developed a comprehensive <a href=”/Team:Heidelberg/Project/Toolbox">toolbox</a> compromising five sets of <a href=”/Team:Heidelberg/Parts">standardized parts</a> that provide standard mechanisms for specific modifications of proteins: <br />
(1) A <a href=”/Team:Heidelberg/Project/Toolbox#Purification">purification</a> standard to faciliate purification of recombinant proteins. <br />
(2) A construct that allows for post-translational <a href=”/Team:Heidelberg/Project/Toolbox#Fusion_and_Tagging">fusion</a> of protein domains to produce synthetic proteins<br />
(3) A standard that allows for switching proteins <a href=”/Team:Heidelberg/Project/Toolbox#On_Off">on and off</a>.<br />
(4) One to <a href=”/Team:Heidelberg/Project/Toolbox#Oligomerization">oligomerize</a> protein monomers.<br />
And (5) perhaps the most intriguing standard construct of this toolbox that was built to produce heat stable proteins, which can be achieved by <a href=”/Team:Heidelberg/Toolbox/Circularization">circularization</a>.<br />
In order to offer a comfortable way to apply any of the above mechanisms to a protein and a biological system we designed a <a href=”/Team:Heidelberg/Toolbox_Guide”>toolbox guide</a> that provides step by step instructions for cloning based on the intein standard parts described in our <a href=”/Team:Heidelberg/Parts/RFC”>RFC</a>.<br />
<br></br><br />
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.<br />
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 <a href=”/Team:Heidelberg/Modeling/Linker_Modeling”>CRAUT</a> 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. <br />
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 <a href=”/Team:Heidelberg/Human_Practice/igemathome”>iGEM@home</a> that we also developed into a powerful science communication platform.<br />
As an evaluation of the linker software we screened linkers generated by the software by circularizing <a href=”/Team:Heidelberg/Project/Linker_Screening”>Lambda Lysozyme</a> and experimentally evaluating the heat stability of the products.<br />
<br></br><br />
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.<br />
Besides a protein with potential medical (lysozyme) and one with biotechnological (DNMT1) applications we chose the hemicellulose xylanase as a third target for circularization, that has applications in large scale production in paper and food industry.<br />
<br></br><br />
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 <a href=”/Team:Heidelberg/Project/LOV”>As LOV2</a> 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.<br />
<br></br><br />
Setting new standards also for wiki documentation we created and developed a new way of documenting collaborative lab work by bringing the MidnightDoc to life.<br />
<br></br><br />
Finally we complemented our already elaborate Human Practice activities with two events on education and religion, philosophy and ethics in synthetic biology.<br />
<br />
<br />
<br />
</p><br />
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<a href="https://2014.igem.org/Team:Heidelberg/Software/igemathome" class="block box" id="calibrated"><br />
<img style="height:60px;display: inline-block;" src="/wiki/images/e/ea/Heidelberg_Project_Computer.png"><br />
<span style="position: relative;top: 10px;display: inline-block;"><br />
calibrated<br><br />
<span class="red-text">in silico</span></span><br />
</a><br />
<a href="https://2014.igem.org/Team:Heidelberg/Project/Linker_Screening" class="block box" id="screened"><br />
<span style="position: relative;top:10px;display: inline-block;"><br />
screened <span class="red-text">in vitro</span><br><br />
with lysozyme</span><br />
<img style="height:60px; display:inline-block;" src="/wiki/images/d/df/Heidelberg_Lysozyme.png" /><br />
</a><br />
<a href="/Team:Heidelberg/Software/Linker_Software" class="block box" style="bottom:0; left:-20px; position:absolute;width:170px;"><br />
<img src="/wiki/images/4/42/Craut_small.png" alt="..." style="width:100%;"/><br />
<span class="red-text" >circularize</span> it<br><br />
with calculated linkers<br />
</a><br />
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<div id="circ-box" class="descr-box"><br />
<a href="/Team:Heidelberg/Toolbox/Circularization"><br />
<img src="/wiki/images/5/58/Heidelberg_Toolbox_Circularization.png" id="circ-icon" class="toolbox-icon toolbox-icon-scale"/><br />
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<h3>CIRCULARIZATION</h3><br />
<div><span>Create a linker with our crowd computing software and make your protein heat stable</span></div><br />
</div><br />
<div id="oligo-box" class="descr-box"><a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#Oligomerization"><img src="/wiki/images/4/40/Oligomerization.png" id="oligo-icon" class="toolbox-icon toolbox-icon-scale"/></a><h3>OLIGOMERIZATION</h3><div><span>Fuse multiple Proteins or Domains using Inteins</span></div><br />
</div><br />
<div id="fusion-box" class="descr-box"><a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#Fusion_and_Tagging"><img src="/wiki/images/8/87/Heidelberg_Toolbox_Fusion.png" id="fusion-icon" class="toolbox-icon toolbox-icon-scale"/></a><h3>FUSION</h3><div><span>Fuse two Proteins or Domains together using Inteins</span></div><br />
</div><br />
<div id="onoff-box" class="descr-box"><a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#On_Off"><img src="/wiki/images/c/c2/Heidelberg_Toolbox_On-Off.png" id="onoff-icon" class="toolbox-icon toolbox-icon-scale"/></a><h3>ON/OFF</h3><div><span>Activate or deactivate Proteins using Inteins</span></div></div><br />
<div id="purification-box" class="descr-box"><a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#Purification"><img src="/wiki/images/0/04/Heidelberg_Toolbox_Purification.png" id="purification-icon" class="toolbox-icon toolbox-icon-scale"/></a><div><h3>PURIFICATION</h3></div><span>Placeholder</span></div><br />
<div id="toolbox-text"><br />
the intein<br><br />
<span><a class="scheisslinkbleibweiss" href="/Team:Heidelberg/Project/Toolbox">toolbox</a><span><br />
</div><br />
</div><br />
<a href="https://2014.igem.org/Team:Heidelberg/Toolbox/Induction" class="block" id="lightning"><br />
<img class="toolbox-icon-scale" style="height:110px;display: inline-block;" src="/wiki/images/8/83/Heidelberg_Project_Lightning.png"><span style="position: relative;top: 20px;display: inline-block;">&nbsp;inducible via<br><br />
<span style="font-weight:bold;position: relative;left: -18px;" class="red-text">light induction</span></span><br />
</a><br />
<a href="https://2014.igem.org/Team:Heidelberg/Project/PCR_2.0" class="block box" style="width:250px;" id="dnmt1-box"><br />
<img id="dnmt1-img" src="/wiki/images/a/a6/Heidelberg_Project_Dnmt1.png"><br />
<span style="position:relative; z-index:5;" class="block"><br />
<span style="font-size:1.5em;"><br />
<span class="red-text">Heat-stable</span><br> circular <br><span style="font-weight:bold;">DNA-<br/>Methyltransferase</span><br />
</span><br><br />
<span style="font-weight:bold;font-size: 2.5em; text-align:right;line-height: 35px;"><br />
<span class="red-text">PCR 2.0</span><br />
</span><br />
</span><br />
</a><br />
<a href="/Team:Heidelberg/Toolbox_Guide" class="block" id="toolbox"><br />
<span style="position:relative; display: inline-block;height:110px; width:110px; vertical-align: middle;"><br />
<img style="height:100%; position:absolute; top:0; left:0;" id="toolbox-img" src="/wiki/images/2/24/Heidelberg_Project_Toolbox_guide.png" /><br />
<img style="height:100%; position:absolute; top:0; left:0; display:none;" id="toolbox-img-hover" src="/wiki/images/4/4c/Heidelberg_Toolbox_guide_highlighted.png" /><br />
</span><br />
<span style="position: relative;top: 20px;display: inline-block; vertical-align:middle;"><br />
<span style="font-weight:bold;" class="red-text">modify your protein</span><br/><br />
using the toolbox guide<br />
</span><br />
</a><br />
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<li class="active" style="font-size:30px"><a href="#Achievements-tab" role="tab" data-toggle="tab"><img src="https://static.igem.org/mediawiki/2014/2/22/Heidelberg_Achievements_red.png" height="50px" alt="Button"> Achievements</a></li><br />
<li style="font-size:30px"><a href="#Medal_Criteria-tab" role="tab" data-toggle="tab"> <img src="https://static.igem.org/mediawiki/2014/3/3f/Heidelberg_Gold_red.png" height="50px" alt="Button"> Medal Criteria</a></li><br />
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<p style="font-weight:light; font-size:25px">Establishing protein circularization as a NEW BIOENGINEERING TOOL in synthetic biology.</p> <br />
<p style="margin-left:50px; font-size:20px">Contributing to iGEM with a new foundational advance!</p><br />
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<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
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<p style="font-weight:light; font-size:25px">Providing a NEW COMPREHENSIVE TOOLBOX based on inteins for modifying proteins post-translationally.</p> <br />
<p style="margin-left:50px; font-size:20px">Sending 67 Biobricks to Registry of Biological parts!</p><br />
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<p style="font-weight:light; font-size:25px">Development of a NEW STANDARD to make the use of inteins easy and modular.</p> <br />
<p style="margin-left:50px; font-size:20px">Establishment of a new <a href="https://2014.igem.org/Team:Heidelberg/Parts/RFC">RFC</a>!</p><br />
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<p style="font-weight:light; font-size:25px">Showing that the toolbox WORKS: proteins are circularized and split fluorescent proteins are reconstituted.</p> <br />
<p style="margin-left:50px; font-size:20px">Making Gels, Western Blots, Fluorescence-based Assays and Mass spectrometry to prove it! </p><br />
</div><br />
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<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
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<p style="font-weight:light; font-size:25px">Creating circular DNMT1 and showing that it is ACTIVE.</p> <br />
<p style="margin-left:50px; font-size:20px">For the first time achieving the circularization of a large protein!</p><br />
<p style="margin-left:50px; font-size:20px">Circularizing LYSOZYME and XYLANASE, two very important proteins for<br />
research and industry!</p><br />
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<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
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<p style="font-weight:light; font-size:25px">Developing a NEW SOFTWARE to calculate customized linkers to circularize proteins.</p> <br />
<p style="margin-left:50px; font-size:20px">Making CRAUT open-source for the scientific community.</p><br />
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<p style="font-weight:light; font-size:25px"> Establishing a distributed computing platform called <a href="https://2014.igem.org/Team:Heidelberg/Software/igemathome">iGEM@home</a>.</p> <br />
<p style="margin-left:50px; font-size:20px">Using this platform as an entirely new way to reach out to the world with synthetic biology concepts!</p><br />
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<p style="font-weight:light; font-size:25px">Creating a NEW SOFTWARE to display the notebook on the wiki.</p> <br />
<p style="margin-left:50px; font-size:20px">Distributing MidNightDOC to the iGEM community to help future teams organize their protocols!</p><br />
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<h1>Bronze</h1><br />
<br><br />
<ul><br />
<li>Please find a comprehensive compilation of <a href="https://2014.igem.org/Team:Heidelberg/Team/Sponsoring">sponsors</a>, partners and scientific contributors on our <a href="https://2014.igem.org/Team:Heidelberg/Team/Attributions">acknowledgements page</a>. </li><br />
<br/><br />
<li>We also encourage you to take notice of the projects “Photo-intein” and “Mito-intein” by <a href="https://2014.igem.org/Team:Queens_Canada/Project">iGEM team Queens from Canada</a> that may supply you with complementary information and tools for the use of inteins in synthetic biology!</li><br />
<br/><br />
<li>A list of links to more than 60 parts in the registry submitted by our team (being or not being part of the new intein toolbox) can be found <a href="https://2014.igem.org/Team:Heidelberg/Parts#allParts">here</a>.</li><br />
</ul><br />
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<div class="col-md-9 col-xs-12"><br />
<h1>Silver</h1><br />
<br><br />
<ul><br />
<li>We experimentally validated that our biobricks <a href="http://parts.igem.org/Part:BBa_K1362000">BBa_K1362000</a>, <a href="http://parts.igem.org/Part:BBa_K1362100">BBa_K1362100 </a>and <a href="http://parts.igem.org/Part:BBa_K1362101">BBa_K1362101</a> work as expected. For more information on the <a href="https://2014.igem.org/Team:Heidelberg/Parts#Favorite Parts">parts</a> please visit the corresponding main pages in the parts registry or explore their involvement in our subprojects.</li><br />
<br/><br />
<li>Religious perceptions of synthetic biology have been part of several surveys during the past ten years of iGEM and Human Practices projects. Since religious groups cover the majority of worlds population, deliver moral values and wield power at the same time, we decided to dedicate a whole event on the topic of religion, philosophy and ethics regarding synthetic biology. Please find an <a href="https://2014.igem.org/Team:Heidelberg/Human_Practice/Ethics">evaluation of our event</a> on the corresponding Human Practices pages. <!--In order to reassure ourselves about the acceptability of our project and synthetic biology in general, we also used this opportunity to build up on the work of the iGEM Team Heidelberg 2013 and conducted a survey that addresses basic questions regarding the public reflection of our work.--><br />
</li><br />
</ul><br />
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<br />
<div class="col-md-9 col-xs-12"><br />
<h1>Gold</h1><br />
<ul><br />
<li>We improved the function of the <b>already existing</b> biobrick part <a href="http://parts.igem.org/Part:BBa_K117505">BBa_K1175005 </a>by optimizing and resubmitting the corresponding sequence of B. subtilis xylanase to the registry (Part:<a href="http://parts.igem.org/Part:BBa_K1362020"> BBa_K1362020</a>). In addition, we submitted a new part for expression of <a href="http://parts.igem.org/Part:BBa_K1362022">circularized xylanase </a>(BBa_K1362022) that might be used in future applications with need for refined enzyme stability.</li><br />
<br/><br />
<li>Despite the fact that we focused on building a set of powerful soft- and wetware tools to help future iGEM-teams developing and realizing projects in synthetic biology, we are happy to announce that we were also able to help out several team during the course of our project, aspecially with sending <a href="https://2014.igem.org/Team:Heidelberg/Parts#Backbones"> our expression vectors</a>. Read more abou in in our <a href="https://2014.igem.org/Team:Heidelberg/Team/Collaborations">Collaborations</a>.</li><br />
<br/><br />
<li>In the style of of the new iGEM community labs track that involves science amateurs “beyond the accolades of scientific publishing and economic reward”, we sought for a new way to involve laymen in actual science and build a strong community of well informed supporters and communicators of synthetic biology at the same time. Now we proudly present the crowd sourcing and communication platform <a href="https://2014.igem.org/Team:Heidelberg/Human_Practice/igemathome">iGEM@home</a>.</li><br />
</ul><br />
</div><br />
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</html></div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/backgroundTeam:Heidelberg/pages/background2014-10-18T03:42:55Z<p>Huhny: /* A detailed description of the trans-splicing reaction */</p>
<hr />
<div><br/><br />
In the variety of proteins that exist, inteins are clearly among the most mysterious.<br />
They behave in such a peculiarly way that scientists are still puzzle about their initial role in the host organisms and are even more astonished when discovering their endless seeming abilities.<br />
<br />
Inteins are integrated as extraneous polypeptide sequences into ordinary proteins. They do not contribute to the original protein function but perform an autocatalytic splicing reaction after protein translation. Analog to intron splicing on RNA level, this posttranslational modification was named protein splicing. Consequentially the protein segments were called inteins, derived from “internal protein sequence”, and the flanking protein chains exteins, “external protein sequences”. Inteins excise themselves out of the host protein while reconnecting the remaining N and C exteins via a new peptide bond. Despite this immense invasion the original protein regains its normal structure and function after splicing [[#References|[1]]]. <br />
<br />
<div><br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 1) Trans-splicing mechanism reaction by split inteins. |<br />
descr=The N-Intein is fused to the C’ terminal end of the N-extein. Complementary, C-Intein is located at the N’ end of the C-extein. After assembly of the two intein fragments, a splicing reaction takes place, where the intein removes itself from the precursor protein and simultaneously ligates the exteins via a peptide bond.|<br />
file=InteinSplicingReaction.png}}<br />
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<div class="caption"><br />
<span style="font-weight:bold;">Trans-splicing mechanism reaction by split inteins.</span><br />
<p>Animation of the split intein splicing reaction</p><br />
</div><br />
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</html><br />
<br />
==History==<br />
<br />
Inteins were discovered in 1990 when dissimilarities between the mature protein sequence of the yeast vacuolar ATPase (Vma1) and its corresponding mRNA were investigated. Surprisingly the mature protein had a lower molecular weight than expected from the encoding sequence, indicating the loss of one part of the protein after translation. <br />
Indeed, a region of 454 amino acids was found to be translated and subsequently removed from the Vma1 protein [[#References|[2]]] [[#References|[3]]]. Since then, over 600 different inteins have been reported in all three domains of life as well as in viruses [[#References|[4]]]. <br />
<br />
Dependent on the organism they belong to, the intein’s name consists out of the genus and species abbreviation followed by the host gene [[#References|[5]]]. For example, the "golden standard" split-intein, NpuDnaE has derived from the DNA polymerase III (DnaE) in <i>Nostoc punctiforme</i> PCC73102 (<i>Npu</i>).<br />
<br />
==Evolution: Inteins as parasitic genes==<br />
Despite many years of research the initial role of inteins in their host organism remains still unclear.<br />
Many natural inteins contain a homing endonuclease (HEN) domain, an enzyme that cleaves DNA and introduces new sequences at homing sites via homologous recombination or reverse transcription. This characteristic led to the perception of the intein as selfish DNA sequence - a gene generating copies of itself without an advantage for the host organism [[#References|[6]]]. This allows horizontal gene transfer of inteins. [[#References|[7]]]. Data showing that 27% of the intein's host proteins are related to DNA metabolism and involved in DNA replication or repair is further supporting the "selfish gene" hypothesis. This ensures expression of the inteins and the HEN domain during DNA replication, where they can take advantage of the DNA replication and repair system for introducing changes in the DNA [[#References|[8]]].<br />
<br />
However recent work on conditional protein splicing has shown sensitivity of some inteins to redox state, temperature and small molecules (reviewed in [[#References|[9]]], [[#References|[10]]]). <br />
This might be a sign for inteins having a role in post-translational protein regulation upon environmental signals. Inserted close to the catalytic core of an protein the intein deactivates the protein function until the splicing reaction has taken place. Evolutionary said, selfish inteins might have adapted due to positive selection pressure to provide a beneficial mechanism for the host cell, thereby becoming selfless [[#References|[8]]].<br />
<br />
==Structure and Classification==<br />
<br />
The structure of inteins contains several conserved motifs. The splicing domains are located at the N and C terminal ends. Before splicing takes place the intein rearranges itself from its initial linear structure into to a horseshoe like structure where the termini are brought in close proximity making up the catalytic core [[#References|[14]]]. <br />
Inteins are divided into three groups: bifunctional (large) inteins, mini inteins and split inteins.<br />
<br />
Large inteins carry both a splicing domain and an endonuclease (HEN) domain whereas mini inteins lack the HEN domain [[#References|[11]]]. <br />
The most promising inteins for biotechnology are split inteins, which are basically mini-inteins, just divided in two fragments and expressed separately connected different proteins. After translation, they assemble with high affinity to become catalytic active and perform a splicing reaction. Split enzymes occure naturally [[#References|[12]]] but can also be engineered artificially [[#References|[13]]]. <br />
<br />
In our project we focused on split inteins, as they present a powerful tool to insert posttranslational modifications, offering a plethora of applications in biotechnology. We have characterized the most promising inteins in our [[Team:Heidelberg/Parts#Intein_Library|Intein Library]].<br />
<br />
==A detailed description of the trans-splicing reaction==<br />
<br />
In contrast to the mini and large inteins that mediate cis-splicing reactions, split inteins are responsible for trans-splicing and fusion of protein parts. The trans-splicing reaction can be divided into the following steps:<br />
<br />
# N-Intein and C-Intein first assemble together to form a dimer like structure with a newly formed catalytic core next to the exteins.<br />
# The tertiary structure of the intein, once correctly formed, facilitates an ''N&rarr;O/S'' acyl rearrangement at its N-terminal serine or cysteine residue. The result is an ester or thioester bond, respectively between the side-chain and the peptide backbone of the N-extein.<br />
# The two exteins are then linked by trans(thio)esterification involving the N-terminal serine or cysteine residue of the C-extein. The C-terminus of the N-extein is now covalently bound to the N-terminal side-chain of the C-extein, while its backbone still retains its normal peptide bond to the intein.<br />
# The C-terminal asparagine of the intein undergoes self-cyclisation to form a succinimidyl moiety. The peptide bond between the intein and the extein is thereby broken, resolving the branched intermediate.<br />
# In a final reaction, an ''O/S&rarr;N'' acyl shift results in the two exteins now being linked by an amide bond, indistinguishable from a ribosome-assembled fusion protein.<br />
(as reviewed in [[#References|[15]]]) <br />
<br />
{{:Team:Heidelberg/templates/image-full|<br />
align=right|<br />
caption=Figure 2) Trans-splicing mechanism reaction by split inteins. |<br />
descr=Picture taken from [16]|<br />
file=Splicingmechanism.png}}<br />
<br />
For intein efficiency not only the structure of the intein domain itself is important but also the nature of the flanking extein residues, as they are heavily involved in the splicing reaction. It has been shown, that the first residue has to be a nucleophilic amino acid, preferable Cysteine, Serine or Threonine. Effectiveness of the intein can be mold by changing the these amino acids [[#References|[17]]].<br />
<br />
==Use of inteins in molecular biology and biotechnology==<br />
<br />
Due to their distinct characteristics intein are a powerful tool for molecular biology and biotechnology. Performing an autocatalytic reaction, the inteins are neither dependent on their host protein nor on any other additional substrate. This makes it them broadly applicable for in vivo and in vitro applications. [[#References|[16]]].<br />
<br />
Inteins have been used for protein purification, achieving a higher yield of protein due to more specific targeting. The production of semi-synthetic proteins and the attachment of synthetic groups is of huge interest for recombinant proteins. [[#References|[16]]]. Split Intein-mediated Circular Ligation Of Peptides a ProteinS (SICLOPPS) is a method to produce circular peptides of eight amino acids length, which could be potent therapeutic drugs [[#References|[18]]]. Inteins, fused to split fluorescent proteins as reporter and to the protein of investigation have been used to detect protein-protein interactions in vivo (reviewed in [[#References|[10]]]).<br />
Nevertheless most of this applications are performed in vitro and do not exploit the full potential of inteins as regulatory element for post-translational modification. The iGEM Team Heidelberg employs this excellent mechanism of protein splicing to specifically change whole amino-acid sequences and thereby regulate proteins via (dis-) assembly, protein cleavage or circularization of enzymes in vivo. All this and much more was embedded into our versatile intein toolbox. With this universal toolkit we provide a foundational advance for protein control - introducing the full potential of post-translational modification and therefore a new dimension of genetic engineering to Synthetic Biology. There is much more to [[Team:Heidelberg/Project|explore]]!<br />
<br />
=References=<br />
<br />
[1] Perler FB, Davis EO, Dean GE, Gimble FS, Jack WE, et al. (1994) Protein splicing elements: inteins and exteins–a definition of terms and recommended nomenclature. Nucleic Acids Res 22: 1125–1127<br />
<br />
[2] Kane P.M., et al. Protein Splicing Converts the Yeast TEPI Gene Product to the 69-kD Subunit of the Vacuolar. 13253, (1990)<br />
<br />
[3] Hirata, R. et al. Molecular structure of a gene, VMA1, encoding the catalytic subunit of H(+)-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J. Biol. Chem. 265 , 6726–6733 (1990)<br />
<br />
[4] Perler, F. B. InBase : the Intein Database. 30, 383–384 (2002)<br />
<br />
[5] Perler, F. B. et al. Protein splicing elements : inteins and exteins — a definition of terms and recommended nomenclature. 22, 1125–1127 (1994)<br />
<br />
[6] Barzel, A., Naor, A., Privman, E., Kupiec, M. & Gophna, U. Homing endonucleases residing within inteins: evolutionary puzzles awaiting genetic solutions. Biochem. Soc. Trans. 39, 169–73 (2011)<br />
<br />
[7] Pietrokovski, S. Intein spread and extinction in evolution. Trends Genet. 17, 465-472 (2001)<br />
<br />
[8] Novikova, O., Topilina, N. & Belfort, M. Enigmatic distribution, evolution and function of inteins. J. Biol. Chem. (2014)<br />
<br />
[9] Shah, N. H., and Muir, T. W. Inteins: nature's gift to protein chemists. Chem. Sci. 5, 446- 461 (2014) <br />
<br />
[10] Topilina, N. I. & Mills, K. V. Recent advances in in vivo applications of intein-mediated protein splicing. Mob. DNA 5, 5 (2014)<br />
<br />
[11] Eryilmaz, E., Shah, N., Muir, T. & Cowburn, D. Structural and Dynamical Features of Inteins and Implications on Protein Splicing. J. Biol. Chem. (2014)<br />
<br />
[12] Carvajal-Vallejos, P., Pallissé, R., Mootz, H. D. & Schmidt, S. R. Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. J. Biol. Chem. 287, 28686–96 (2012) <br />
<br />
[13] Lin, Y. et al. Protein trans-splicing of multiple atypical split inteins engineered from natural inteins. PLoS One 8, e59516 (2013). <br />
<br />
[14] Eryilmaz, E., Shah, N., Muir, T. & Cowburn, D. Structural and Dynamical Features of Inteins and Implications on Protein Splicing. J. Biol. Chem. (2014)<br />
<br />
[15] Mills, K. V, Johnson, M. a & Perler, F. B. Protein Splicing: How Inteins Escape from Precursor Proteins. J. Biol. Chem. (2014)<br />
<br />
[16] Mootz, H. D. Split inteins as versatile tools for protein semisynthesis. Chembiochem 10, 2579–89 (2009).<br />
<br />
[17] Amitai, G., Callahan, B. P., Stanger, M. J., Belfort, G. & Belfort, M. Modulation of intein activity by its neighboring extein substrates. Proc. Natl. Acad. Sci. U. S. A. 106, 11005–10 (2009).<br />
<br />
[18] Tavassoli, A. & Benkovic, S. J. Split-intein mediated circular ligation used in the synthesis of cyclic peptide libraries in E. coli. Nat. Protoc. 2, 1126–1133 (2007).</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/backgroundTeam:Heidelberg/pages/background2014-10-18T03:41:51Z<p>Huhny: /* A detailed description of the trans-splicing reaction */</p>
<hr />
<div><br/><br />
In the variety of proteins that exist, inteins are clearly among the most mysterious.<br />
They behave in such a peculiarly way that scientists are still puzzle about their initial role in the host organisms and are even more astonished when discovering their endless seeming abilities.<br />
<br />
Inteins are integrated as extraneous polypeptide sequences into ordinary proteins. They do not contribute to the original protein function but perform an autocatalytic splicing reaction after protein translation. Analog to intron splicing on RNA level, this posttranslational modification was named protein splicing. Consequentially the protein segments were called inteins, derived from “internal protein sequence”, and the flanking protein chains exteins, “external protein sequences”. Inteins excise themselves out of the host protein while reconnecting the remaining N and C exteins via a new peptide bond. Despite this immense invasion the original protein regains its normal structure and function after splicing [[#References|[1]]]. <br />
<br />
<div><br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 1) Trans-splicing mechanism reaction by split inteins. |<br />
descr=The N-Intein is fused to the C’ terminal end of the N-extein. Complementary, C-Intein is located at the N’ end of the C-extein. After assembly of the two intein fragments, a splicing reaction takes place, where the intein removes itself from the precursor protein and simultaneously ligates the exteins via a peptide bond.|<br />
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<span style="font-weight:bold;">Trans-splicing mechanism reaction by split inteins.</span><br />
<p>Animation of the split intein splicing reaction</p><br />
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==History==<br />
<br />
Inteins were discovered in 1990 when dissimilarities between the mature protein sequence of the yeast vacuolar ATPase (Vma1) and its corresponding mRNA were investigated. Surprisingly the mature protein had a lower molecular weight than expected from the encoding sequence, indicating the loss of one part of the protein after translation. <br />
Indeed, a region of 454 amino acids was found to be translated and subsequently removed from the Vma1 protein [[#References|[2]]] [[#References|[3]]]. Since then, over 600 different inteins have been reported in all three domains of life as well as in viruses [[#References|[4]]]. <br />
<br />
Dependent on the organism they belong to, the intein’s name consists out of the genus and species abbreviation followed by the host gene [[#References|[5]]]. For example, the "golden standard" split-intein, NpuDnaE has derived from the DNA polymerase III (DnaE) in <i>Nostoc punctiforme</i> PCC73102 (<i>Npu</i>).<br />
<br />
==Evolution: Inteins as parasitic genes==<br />
Despite many years of research the initial role of inteins in their host organism remains still unclear.<br />
Many natural inteins contain a homing endonuclease (HEN) domain, an enzyme that cleaves DNA and introduces new sequences at homing sites via homologous recombination or reverse transcription. This characteristic led to the perception of the intein as selfish DNA sequence - a gene generating copies of itself without an advantage for the host organism [[#References|[6]]]. This allows horizontal gene transfer of inteins. [[#References|[7]]]. Data showing that 27% of the intein's host proteins are related to DNA metabolism and involved in DNA replication or repair is further supporting the "selfish gene" hypothesis. This ensures expression of the inteins and the HEN domain during DNA replication, where they can take advantage of the DNA replication and repair system for introducing changes in the DNA [[#References|[8]]].<br />
<br />
However recent work on conditional protein splicing has shown sensitivity of some inteins to redox state, temperature and small molecules (reviewed in [[#References|[9]]], [[#References|[10]]]). <br />
This might be a sign for inteins having a role in post-translational protein regulation upon environmental signals. Inserted close to the catalytic core of an protein the intein deactivates the protein function until the splicing reaction has taken place. Evolutionary said, selfish inteins might have adapted due to positive selection pressure to provide a beneficial mechanism for the host cell, thereby becoming selfless [[#References|[8]]].<br />
<br />
==Structure and Classification==<br />
<br />
The structure of inteins contains several conserved motifs. The splicing domains are located at the N and C terminal ends. Before splicing takes place the intein rearranges itself from its initial linear structure into to a horseshoe like structure where the termini are brought in close proximity making up the catalytic core [[#References|[14]]]. <br />
Inteins are divided into three groups: bifunctional (large) inteins, mini inteins and split inteins.<br />
<br />
Large inteins carry both a splicing domain and an endonuclease (HEN) domain whereas mini inteins lack the HEN domain [[#References|[11]]]. <br />
The most promising inteins for biotechnology are split inteins, which are basically mini-inteins, just divided in two fragments and expressed separately connected different proteins. After translation, they assemble with high affinity to become catalytic active and perform a splicing reaction. Split enzymes occure naturally [[#References|[12]]] but can also be engineered artificially [[#References|[13]]]. <br />
<br />
In our project we focused on split inteins, as they present a powerful tool to insert posttranslational modifications, offering a plethora of applications in biotechnology. We have characterized the most promising inteins in our [[Team:Heidelberg/Parts#Intein_Library|Intein Library]].<br />
<br />
==A detailed description of the trans-splicing reaction==<br />
<br />
In contrast to the mini and large inteins that mediate cis-splicing reactions, split inteins are responsible for trans-splicing and fusion of protein parts. The trans-splicing reaction can be divided into the following steps:<br />
<br />
# N-Intein and C-Intein first assemble together to form a dimer like structure with a newly formed catalytic core next to the exteins.<br />
# The tertiary structure of the intein, once correctly formed, facilitates an ''N&rarr;O/S'' acyl rearrangement at its N-terminal serine or cysteine residue. The result is an ester or thioester bond, respectively between the side-chain and the peptide backbone of the N-extein.<br />
# The two exteins are then linked by trans(thio)esterification involving the N-terminal serine or cysteine residue of the C-extein. The C-terminus of the N-extein is now covalently bound to the N-terminal side-chain of the C-extein, while its backbone still retains its normal peptide bond to the intein.<br />
# The C-terminal asparagine of the intein undergoes self-cyclisation to form a succinimidyl moiety. The peptide bond between the intein and the extein is thereby broken, resolving the branched intermediate.<br />
# In a final reaction, an ''O/S&rarr;N'' acyl shift results in the two exteins now being linked by an amide bond, indistinguishable from a ribosome-assembled fusion protein.<br />
(as reviewed in [[#References|[15]]]) <br />
<br />
{{:Team:Heidelberg/templates/image-full|<br />
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caption=Figure 2) Trans-splicing mechanism reaction by split inteins. |<br />
descr=Picture taken from [[#References|[16]]]|<br />
file=Splicingmechanism.png}}<br />
<br />
For intein efficiency not only the structure of the intein domain itself is important but also the nature of the flanking extein residues, as they are heavily involved in the splicing reaction. It has been shown, that the first residue has to be a nucleophilic amino acid, preferable Cysteine, Serine or Threonine. Effectiveness of the intein can be mold by changing the these amino acids [[#References|[17]]].<br />
<br />
==Use of inteins in molecular biology and biotechnology==<br />
<br />
Due to their distinct characteristics intein are a powerful tool for molecular biology and biotechnology. Performing an autocatalytic reaction, the inteins are neither dependent on their host protein nor on any other additional substrate. This makes it them broadly applicable for in vivo and in vitro applications. [[#References|[16]]].<br />
<br />
Inteins have been used for protein purification, achieving a higher yield of protein due to more specific targeting. The production of semi-synthetic proteins and the attachment of synthetic groups is of huge interest for recombinant proteins. [[#References|[16]]]. Split Intein-mediated Circular Ligation Of Peptides a ProteinS (SICLOPPS) is a method to produce circular peptides of eight amino acids length, which could be potent therapeutic drugs [[#References|[18]]]. Inteins, fused to split fluorescent proteins as reporter and to the protein of investigation have been used to detect protein-protein interactions in vivo (reviewed in [[#References|[10]]]).<br />
Nevertheless most of this applications are performed in vitro and do not exploit the full potential of inteins as regulatory element for post-translational modification. The iGEM Team Heidelberg employs this excellent mechanism of protein splicing to specifically change whole amino-acid sequences and thereby regulate proteins via (dis-) assembly, protein cleavage or circularization of enzymes in vivo. All this and much more was embedded into our versatile intein toolbox. With this universal toolkit we provide a foundational advance for protein control - introducing the full potential of post-translational modification and therefore a new dimension of genetic engineering to Synthetic Biology. There is much more to [[Team:Heidelberg/Project|explore]]!<br />
<br />
=References=<br />
<br />
[1] Perler FB, Davis EO, Dean GE, Gimble FS, Jack WE, et al. (1994) Protein splicing elements: inteins and exteins–a definition of terms and recommended nomenclature. Nucleic Acids Res 22: 1125–1127<br />
<br />
[2] Kane P.M., et al. Protein Splicing Converts the Yeast TEPI Gene Product to the 69-kD Subunit of the Vacuolar. 13253, (1990)<br />
<br />
[3] Hirata, R. et al. Molecular structure of a gene, VMA1, encoding the catalytic subunit of H(+)-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J. Biol. Chem. 265 , 6726–6733 (1990)<br />
<br />
[4] Perler, F. B. InBase : the Intein Database. 30, 383–384 (2002)<br />
<br />
[5] Perler, F. B. et al. Protein splicing elements : inteins and exteins — a definition of terms and recommended nomenclature. 22, 1125–1127 (1994)<br />
<br />
[6] Barzel, A., Naor, A., Privman, E., Kupiec, M. & Gophna, U. Homing endonucleases residing within inteins: evolutionary puzzles awaiting genetic solutions. Biochem. Soc. Trans. 39, 169–73 (2011)<br />
<br />
[7] Pietrokovski, S. Intein spread and extinction in evolution. Trends Genet. 17, 465-472 (2001)<br />
<br />
[8] Novikova, O., Topilina, N. & Belfort, M. Enigmatic distribution, evolution and function of inteins. J. Biol. Chem. (2014)<br />
<br />
[9] Shah, N. H., and Muir, T. W. Inteins: nature's gift to protein chemists. Chem. Sci. 5, 446- 461 (2014) <br />
<br />
[10] Topilina, N. I. & Mills, K. V. Recent advances in in vivo applications of intein-mediated protein splicing. Mob. DNA 5, 5 (2014)<br />
<br />
[11] Eryilmaz, E., Shah, N., Muir, T. & Cowburn, D. Structural and Dynamical Features of Inteins and Implications on Protein Splicing. J. Biol. Chem. (2014)<br />
<br />
[12] Carvajal-Vallejos, P., Pallissé, R., Mootz, H. D. & Schmidt, S. R. Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. J. Biol. Chem. 287, 28686–96 (2012) <br />
<br />
[13] Lin, Y. et al. Protein trans-splicing of multiple atypical split inteins engineered from natural inteins. PLoS One 8, e59516 (2013). <br />
<br />
[14] Eryilmaz, E., Shah, N., Muir, T. & Cowburn, D. Structural and Dynamical Features of Inteins and Implications on Protein Splicing. J. Biol. Chem. (2014)<br />
<br />
[15] Mills, K. V, Johnson, M. a & Perler, F. B. Protein Splicing: How Inteins Escape from Precursor Proteins. J. Biol. Chem. (2014)<br />
<br />
[16] Mootz, H. D. Split inteins as versatile tools for protein semisynthesis. Chembiochem 10, 2579–89 (2009).<br />
<br />
[17] Amitai, G., Callahan, B. P., Stanger, M. J., Belfort, G. & Belfort, M. Modulation of intein activity by its neighboring extein substrates. Proc. Natl. Acad. Sci. U. S. A. 106, 11005–10 (2009).<br />
<br />
[18] Tavassoli, A. & Benkovic, S. J. Split-intein mediated circular ligation used in the synthesis of cyclic peptide libraries in E. coli. Nat. Protoc. 2, 1126–1133 (2007).</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/PCR_2.0Team:Heidelberg/pages/PCR 2.02014-10-17T23:05:49Z<p>Huhny: /* Thermal stability of circular mDNMT1(721-1602) */</p>
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<p style="font-size:25px; font-weight:bold">Abstract</p><br />
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<p>DNA methylation is the most abundant DNA modification and essential for embryonic development, gene regulation and genomic stability. Although several methods for the detection of methylation patterns exist, there is no easy way to amplify methylated DNA for <i>in vitro</i> or <i>in vivo</i> studies.</p><br />
<p>To empower epigenetic research, we envisioned a PCR2.0 which maintains DNA methylation patterns during amplification. The central element of this PCR2.0 is a heat-resistant DNA methyltransferase - DNMT1 (731-1602) - which we created by circularization using our intein toolbox and the CRAUT linker software.</p><br />
<p>So far, our DNMT1 represents the largest circularized protein which highlights the usefulness of our intein toolbox in combination with the CRAUT linker software. Increased heat resistance of our circular DNMT1 which was observed in initial assays smoothens the path for the establishment of a PCR2.0 and illustrates the suitability of intein-mediated circularization for the advancement of heat resistant proteins.</p><br />
<p style="font-size:25px; font-weight:bold">Highlights</p><br />
<ul><br />
<li>circularization seems to increase heat stability of mDNMT1 (731-1602): successful first step towards PCR2.0.</li><br />
<li>mDNMT1 is the largest circularized protein (105kDa) so far.</li><br />
<li>efficient expression and purification of active linear and circular mDNMT1 (731-1602) in E.coli.</li><br />
<li>successful application of CRAUT linker design software and intein toolbox for circularization.</li><br />
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=Introduction=<br />
<br />
==Motivation==<br />
<br />
The invention of the polymerase chain reaction in 1983 by Kary Mullis revolutionized our world by enabling amplification of DNA in an exponential manner. Further improvements including the use of thermo-stable DNA polymerases made the method even more efficient, allowing its widespread use in nearly every field of modern diagnostics and research. However, a major part of information is lost when using conventional PCR as DNA modifications are not copied. The most abundant modification is DNA methylation, which is a prominent regulator of gene expression in all kingdoms of life. Methylation and other modifications that influence the DNA function without changing its actual sequence are studied in the fast-growing field of epigenetics. Even though the detection and mapping of methylation patterns have become feasible in a high-throughput manner by using bisulfite sequencing and array techniques [[#References|[1]]], further functional analysis is still hindered by the small amount of primary material. Up to now, there is no method available to amplify methylated DNA without knowledge of methylation patterns and expensive de-novo synthesis.<br />
<br />
To empower epigenetics, we propose the '''“PCR 2.0”''' as an easy and efficient way to amplify DNA templates in an exponential manner while maintaining their specific methylation pattern. The pivotal element of this PCR reaction is a heat-stable DNA methyltransferase (DNMT). Although comparable enzymes exist in thermophile organisms [[#References|[2]]], until now no suitable protein has been found or synthesized that withstands the harsh conditions of a PCR.<br />
<br />
To achieve this goal of a PCR2.0 that can be used to amplify DNA including its intrinsic methylation pattern, our main aim was the generation of a heat-stable Dnmt1 that can be produced in a larger scale and shows efficient and specific methylation of DNA. It was reported that the insertion of additional intramolecular bonds can increase the stability of proteins [[#References|[3]]][[#References|[4]]], and that specifically joining the C- and N-terminus improves the thermostability of smaller peptides [[#References|[5]]]. This is why our approach to generate a heat-stable Dnmt1 applies:<br />
* protein circularization using our intein toolbox<br />
* creating a potent [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software Software] to design efficient linker peptides connecting C- and N-terminus<br />
<br />
To enable the PCR2.0 we started the circularization of the largest protein so far…<br />
<br />
==Epigenetics - there is more than just A, T, C and G…==<br />
In mammals and other vertebrates DNA methylation occurs at the C5 position of cytosine nucleotides that are followed by guanines (CpG). It is a prominent key regulator of transcription, embryonic development, X chromosome inactivation and many other cellular functions [[#References|[6]]][[#References|[7]]]. The human genome contains about 28 million CpGs of which 60-80% are methylated [[#References|[28]]]. Due to its great prevalence and the heredity of this modification, the 5-methylcytosine (5mC) is also known as the “fifth base” of eukaryotic genomes [[#References|[9]]]. DNA methylation is preferentially occurring at intergenic regions and repetitive sequences, where it is known to silence gene expression [[#References|[10]]][[#References|[11]]][[#References|[12]]]. Inappropriate cytosine methylation and silencing has been reported to play a crucial role in the development of numerous diseases, including cancer, imprinting diseases and repeat-instability based diseases such Huntington's disease [[#References|[13]]].<br />
<br />
==A closer look at DNA Methyltransferases==<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
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caption=Figure 1) Principle of mDNMT1-mediated maintenance methylation|<br />
descr=The enzyme DNMT1 is of particular interest for methylation maintenance PCR, as it allows exact duplication of the original methylation pattern to every new DNA copy.|<br />
file=methylation_principle.png}}<br />
<br />
<br />
The family of enzymes called DNA methyltransferases (DNMTs) is responsible for the establishment and maintenance of cell-type specific DNA methylation patterns. Whereas the two enzymes DNMT3a and DNMT3b contribute to de novo methylation of DNA during development, DNMT1 is preserving existing methylation patterns throughout cell divisions. To do so, DNMT1 exploits the principle of semi-conservative replication, using the parental strand as a template to create an exact copy of methylation patterns on the daughter strand (Figure 1). After DNA synthesis, the DNA consists of a methylated and an unmethylated strand. DNMT1 recognizes these so called hemi-methylated CpG sites, and transfers a methyl group from the methyl donor S-Adenosyl-methionine (SAM) to the cytosine belonging to the newly synthesized strand [[#References|[15]]][[#References|[19]]].<br />
<br />
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{{:Team:Heidelberg/templates/image-half|<br />
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caption=Figure 2) Crystal structure of full-length and truncated (731-1602) mDNMT1|<br />
descr=Structural overview of full length mDNMT1 and truncated mDNMT1(731–1602) (A) Color-coded schematic overview of domain structure and numbering of mDNMT1 sequence. (B) Ribbon presentation of full-length DNMT1 with the CXXC, BAH1, BAH2,and methyltransferase domain colored in light blue, yellow, orange and red, respectively. (C) Crystal structure of truncated DNMT1, missing the CXXC domain and schowing more adjacent protein termini that the full-length version.|<br />
file=dnmt1_structure.png}}<br />
<br />
<br />
<br />
The complex structure of DNMT1 and several truncated versions have been recently solved by X-ray crystallography [[#References|[19]]]. For our experiments we used the smallest truncated version of DNMT1 - mDNMT1 (731-1602) - that has been reported to efficiently mediate methylation maintenance. This truncated version is derived from <i>Mus musculus</i> and comprises the amino acids 731-1602 of the full length protein [[#References|[15]]][[#References|[19]]] (kindly provided by Dr. Bashtrykov after approval from Prof. Patel). Murine DNMT1 (731-1602) contains a C-terminal catalytic methyltransferase domain as well as the bromo-adjacent homology (BAH) domains, which prevent the binding of unmethylated DNA to the catalytic core. The truncated enzyme is missing the CXXC domain, which has a high affinity to hemi-methylated CpG dinucleotides, but was shown to be dispensable for protein function [[#References|[16]]]. In contrast to the full-length DNMT1, mDNMT1 (731-1602) has the great advantage of being efficiently expressed in E.coli. Another benefit of the truncated DNMT1 is that the N- and C- terminus are closer together than in the full-length DNMT1 (Figure 2). Thus circularization might cause less deformation and show lower impact on the overall activity, which makes this truncated mDNMT1 an ideal candidate for our purposes.<br />
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==Circularization - The missing link== <br />
<br />
Heat stabilization of mDNMT1 as a pivotal element of our methylation-maintaining PCR2.0 was approached by circularization of the protein. Until now, only smaller proteins have been stabilized using this method. Therefore, to our knowledge, mDNMT1 – even in its shortest truncated version of 871 amino acids and a molecular weight of approximately 100kDa – is the largest protein that has ever been tried to circularize. According to the crystal structure, the distance between the termini of the truncated DNMT1 is 48 Å. We suspected that circularization of the protein by direct fusion of both termini might cause deformation of the protein structure. Therefore we used our own [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software CRAUT Linker Software] to create a linking amino acid sequence, adapted to the structure of DNMT1. The software is able to design protein linkers with the required length to bridge the gap between the protein termini while bypassing the catalytic core of the enzyme.<br />
<br />
Since we could not estimate the impact of the linker introduction, we focused on two different kinds of linkers: a so called rigid linker that has been calculated and optimized during the establishment of our linker software and a flexible peptide connection consisting mostly of glycine and serine. To perform circularization we have used the split NpuDnaE Intein, since it is used as the <i>golden standard</i> for protein splicing and has been efficiently tested by us through circularization of GFP, Lysozyme and Xylanase. Moreover, we tried to implement protein circularization by using sortase A, which is recognizing and cleaving a carboxyterminal sorting signal followed by a transpeptidation reaction that can be exploited for protein circularization. This method has been reported to be very efficient [[#References|[4]]] and was therefore included in our study as possible candidate for large scale productions of circular proteins.<br />
<br />
=Experimental procedures=<br />
<br />
==Constructs==<br />
We designed constructs in order to characterize the efficiency of sortase A as well as intein mediated circularization of DNMT1. Both approaches are based on the comparison of the circularized protein with a corresponding linear counterpart at different temperatures and incubation times.<br />
{{:Team:Heidelberg/templates/image-half|<br />
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caption=Construct for circularization of mDNMT1 using inteins.|<br />
descr= |<br />
file=Dnmt1_construct1.png}}<br />
For circularization of DNMT1 with inteins we fused the obtained truncated version of DNMT1 (731-1602) [[#References|[15]]] with an appropriate linker and split NpuDnaE domains at either site of the construct. For efficient purification of the protein, all constructs contained a hexa-histidine tag. Moreover, we have cloned the ubiquitin-like Smt3 protein from <i> Saccharomyces cerevisiae </i> in front of the DNMT1-intein complex. This attachment is used to increase the yield when expressing large mammalian proteins like DNMT1 in <i>E.coli</i> [[#References|[17]]]. The constructs have been designed in a way that Smt3 is no longer included in the final circular protein, and therefore cannot interfere with its function. <br />
All cloning steps were carried out using our new RFC[i] standard procedure. <br />
<br />
<br />
The mDNMT1 (731-1602) constructs that have been designed to allow sortase A mediated cirularization are flanked by the sortase A recognition sequence LPETGG on the C–terminus as well as N-terminal glycines that that can be exposed through previous TEV treatment. Since the transpeptidation via sortase requires an additional <i> in vitro </i> reaction, subsequent purification is necessary. To facilitate this step and to enrich the circular product in the sample, a hexa-histidine tag is present in the initial protein that is lost during circularization. Hence, an additional affinity chromatography via His trap can be used to separate the histidine tag containing sortase and not circularized educt from the circular flow-through. <br />
All cloning steps were carried out using our sortase standard. <br />
<br />
The following linkers that had been optimized along with our [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software CRAUT Linker Software]:<br />
<br />
{| class="table table-hover"<br />
|-<br />
! Definition!! Sequence <br />
|-<br />
|Rigid linker|| GGAEAAAKEAAAKVNLTAAEAAAKEAAAKEAAAKEAAAKEAAAKAVNLTAAEAAAKAHHHHHHSGRGT <br />
|-<br />
|Flexible linker|| CWEGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSHHHHHHSGRGT<br />
|}<br />
<br />
== Expression and purification==<br />
<br />
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caption=Figure 3) Purification of recombinant mDNMT1|<br />
descr= The purification of recombinantly expressed mDNMT1 is a challenging task including large scale induction, efficient cell lysis, reduction of sample volume via ultracentrifugation, immobilized Ni ion affinity chromatography, dialysis and concentration through centrifugation. |<br />
file=Purification_procedure_new.png}}<br />
<br />
In comparison to the full length murine DNMT1, our modified mDNMT1(731-1602) can be expressed in <i>E.coli (Rosetta DE3)</i> and does not need the establishment of Baculovirus based system and the use of insect cell cultures. First experiments were conducted to determine the optimal conditions for protein induction and expression. We tested several concentrations of IPTG as well as different temperatures to maximize the yield of mDNMT1. Still, even though expression of the constructs can be simplified by using <i>E.coli</i>, purification of the recombinant protein remains a challenging task requiring multiple different steps that had to be adapted and established from published protocols [[#References|[19]]]. <br />
<br />
In a first step, we optimized the process of cell lysis, to be able to extract a maximum amount of active protein from our cultures. Therefore we compared the two commonly used methods sonication and disruption via French press. Subsequently, the sample was cleared from cell debris by ultracentrifugation. The protein of interest containing a hexa-histidine tag was further enriched via immobilized metal ion affinity chromatography (IMAC) using a His trap and eluted with imidazol. In a next step, low molecular weight solutes such as salts and imidazol that would interfere with protein function were removed from the sample by dialysis. Finally, the purified protein was concentrated by size-exclusion centrifugation using filters with an appropriate mass cutoff.<br />
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==Methylation activity assay==<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
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caption=Figure 4) Sequence of 40-mer DNA template for methylation activity assay|<br />
descr=Sequence of the 40-mer DNA template. CpG dinucleotides are shaded in gray, Sau3AI and HpaII recognition sites are depicted in red. The first CpG dinucleotide is methylated on the reverse strand.|<br />
file=DNMT1_substrate.png}}<br />
<br />
To measure the efficiency and specificity of mDNMT1 maintenance methylation activity, we used an assay that is based on the two methylation-sensitive restriction enzymes Sau3AI and HpaII. It relies on the inhibition of restriction enzymes when methyl groups are attached to the cleavage site. We used a 40-mer DNA template with one hemi-methylated and one unmethylated CpG site which are located within the Sau3AI and HpaII restriction site, respectively (adapted from Bashtrykov et colleagues [[#References|[16]]].<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
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caption=Figure 4) Principle of the mDNMT1 methylation activity assay|<br />
descr=Principle of mDNMT1 methylation assay using a 40-mer substrate with Sau3AI and HpaII cleavage sites that are blocked upon further CpG methylation. The lack of methylation activity leads to fragmentation of the DNA template with either of the two restriction enzymes. Whereas specific methylation of hemimethylated CpGs hampers only cleavage by Sau3AI, <i>de novo</i> methylation activity of mDNMT1 can be detected when HpaII cleavage is blocked as well.|<br />
file=MethylationAssay_overview.png}}<br />
<br />
The restriction enzyme Sau3AI is capable of cleaving the template despite of the hemimethylated state of the first CpG nucleotide. Similarly, HpaII can attack its unmethylated native site, leading to fragmentation of the template as long as no further methylation occurs. Impairment of Sau3AI cleavage indicates maintenance methylation activity, whereas a decrease in HpaII activity would result from de novo methylation. Therefore the assay is not only detecting activity of the enzyme but also includes its specificity for hemimethylated DNA over unmethylated DNA.<br />
To quantify maintenance and de novo methylation activity, the 40-mer fragments remaining after DNMT1 incubation and restriction digest were separated by gel electrophoresis. The intensities of the detected bands corresponding to different fragments were measured using ImageJ Gel Analyzer and quantified using gaussian fitting after background substraction. <br />
<br />
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=Results=<br />
<br />
==Optimizing expression and purification of linear mDNMT1(731-602)==<br />
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caption=Figure 5) Optimisation of induction|<br />
descr=As soon as transformed Rosetta (DE3) cultures reached an OD600 of approximately 0.6, induction of linear mDNMT1(731-1602) was perfomed over night at either 15°C or 25°C, using different concentrations of IPTG. Higest expression of DNMT1 was observed at 15°C using 0.7mM IPTG.|<br />
file=DNMT1-Induction2.png}}<br />
<br />
Before starting circularization of DNMT1, we first tested if we can actually express and purify active linear DNMT1. Since DNMT1 expression in E.coli normally results in low yields (<1mg/liter of culture)[[#References|[15]]], we started with an optimization of the expression conditions. <br />
<br />
In general, low temperatures and low IPTG concentrations are known to prevent the occurrence of insoluble inclusion bodies under conditions of overexpression. Thus, we tested different IPTG concentrations and temperatures during induction in order to maximize yields of active mDNMT1(731-1602). Generally, solubility tags such as the fused Smt3 as well as lower temperatures are known to prevent the occurrence of insoluble inclusion bodies under conditions of overexpression. Accordingly, we observed an optimal induction of our mDNMT1(731-1602) construct after incubation at 15 °C using 0.7mM IPTG (Figure 5).<br />
<br />
Even though the overall yield of recombinant protein is quite low, the presence of mDNMT1(731-1602) after induction with IPTG was detectable through Coomassie staining and specifically verified by Western blot (Figure 6A and 6B). In the western blot, several other peptides with lower molecular weight were detected (Figure 6B). These peptides possibly result from incomplete translation or degradation processes, since the construct used for initial mDNMT1 expression and purification comprised an N-terminal hexa-histidine tag. To minimize the amount of incomplete protein products that would be enriched in the following purification, we optimized the mDNMT1(731-1602) constructs for circularization by shifting the His-tag to the C-terminus.<br />
<br />
<div style="clear:both;"></div><br />
<br />
After successful expression, the next critical step for high yields of recombinant protein after purification is protein extraction. The lysis method has to be efficient but still gentle enough to preserve the proteins function - especially when dealing with sensitive and low expressed proteins like DNMT1. In order to optimize protein extraction we therefore compared cell lysis by sonification with disruption via French press (Figure 6A and 6B). Due to the low overall yield, mDNMT1 does not appear as a prominent band on the Coomassie-stained gel as we expected for an overexpressed recombinant proteins. <br />
Assuming similar amounts of overall protein that should be present in the consecutively generated samples, sonification seems to be less efficient for extraction of recombinant protein compared to usage of the French press. Moreover, the disruption of <i>E.coli</i> using French press has been reported to preserve the biological activity of susceptible proteins more efficiently than sonication [18]. Therefore we used the French press for further extraction of linear as well as circular mDNMT1.<br />
<br />
Further purification of the protein using a His-trap resulted in successful enrichment of mDNMT1f(731-1602) (Figure 6C). Corresponding eluates were pooled and further concentrated for use in functional assays. <br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
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caption=Figure 6A) Comparison of DNMT1 extraction from bacteria by French press and sonification|<br />
descr=mDNMT1(731-1602) expression in Rosetta (DE3) was performed over night at 15°C. Subsequently, the bacteria were lysed by sonification or French press and ultracentrifuged. After each step, samples were collected for analysis by SDS-PAGE.<br />
(Untransformed negative control (UnTra), intervals of cell disruption (1, 2), supernatant (SN)).|<br />
file=DNMT1-Celllysis.jpg}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 6B)Lysis via French press efficiently etracts mDNMT1(731-1602)|<br />
descr=Western Blot of lysed samples was perfomed with an anti-hexa-histidine antibody for specific detection of mDNMT1. Full length mDNMT1(731-1602) is indicated with an arrow.|<br />
file=DNMT1-Celllysis-WB.jpg}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
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caption=Figure 6C) Purification of linear mDNMT1(731-1602)|<br />
descr=Coomassie stained SDS-PAGE showing efficient purification of linear mDNMT1(731-1602) via His-column. Arrow indicates peptides corresponding to the molecular weight of mDNMT1(731-1602) (133kDa).|<br />
file=DNMT1-Purification-linear.png}}<br />
<br />
<div style="clear:both;"></div><br />
<br />
==Activity and specificity of linear mDNMT1(731-1602)==<br />
<br />
After successful expression and purification of linear DNMT1, we characterized the proteins activity and specificity for hemi-methylated DNA by performing the methylation activity assay described above. To test the maintenance methylation activity of our linear DNMT1, we performed the methylation assay with different incubation times of DNMT1. We observe an increasing amount of completely methylated template over time (Figure 7), represented by increasing amounts of template that are unsusceptible towards SauAI cleavage. <br />
The truncated DNMT1 (731-1602) showed maximal methylation of the template after approximately 1/2 to 1hour. Therefore, our data agrees with efficiencies, that have been reported in literature for wild type and truncated versions of mDNMT1 [[#References|[15]]] [[#References|[16]]] .Overall, the reaction kinetics of the linear protein seem to be promising for later use in a PCR2.0, assuming that circularization of the protein will not ablate its function. <br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
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caption=Figure 7A) Maintenance Methylation deposition of linear Dnmt1(731-1602) over time|<br />
descr= Purified linear mDNMT1(731-1602) was incubated with 40mer DNA template (see Figure4) for indicated times at 37°C. Subsequently the samples were digested with Sau3AI. Fragments were separated by PAGE and stained with ethidium bromide. (Homo me= homomethylated substrate, control for enzyme inhibition through methylation, Hemi me=hemimethylated substrate positive control for digest).|<br />
file=activityovertime.jpg}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
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caption=Figure 7B) Plot showing maintenance methylation of linear Dnmt1(731-1602) over time|<br />
descr= Amount of methylation was calculated by normalizing the intensity of the upper band to whole intensity of each line. All measurements were performed with ImageJ.|<br />
file=dnmt1_act_maint_over_time.png}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 8A) <i>De novo</i> methylation activity of mDNMT1|<br />
descr=Purified linear mDNMT1(731-1602) was incubated with 40mer DNA template (see Figure4) for indicated times at 37°C. Subsequently the samples were digested with HpaII. Fragments were separated using gel-electrophoresis and visualized with ethidiumbromid staining. (Homo me= homomethylated substrate,control for enzyme inhibition through methylation, Hemi me=hemimethylated substrate positive control for digest).|<br />
file=specificityovertime.jpg}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 8B) Plot showing <i>de novo</i> methylation activity of mDNMT1|<br />
descr=Amount of methylation was calculated by normalizing the intensity of the upper band to whole intensity of each line. All measurements were performed with ImageJ.|<br />
file=dnmt1_act_de_novo_over_time.png}}<br />
<br />
In oder to be used in the targeted PCR2.0 mDNMT1 needs to be highly specific for hemi-methylated sites and should not display <i>de novo</i> methylation activity. Therefore, we included HpaII digestion in our assay to determine <i>de novo</i> methylation of CpG sites. The first results indicate that the purified mDNMT1(731-1602) does not exhibit de novo methylation activity (Figure 8). Even with increasing DNMT1 incubation time, HpaII is not repressed which indicates absence of <i>de novo</i> methylation. <br />
However, the analysis of the gel pictures is difficult since restriction efficiency of HpaII was not 100% as it can be seen from the hemimethylated control. Therefore, uncut template which is not resulting from unspecific methylation activity of the enzyme complicates quantitative analysis. Further tests, especially with the circularized protein will have to be performed to confirm these initial data.<br />
<div style="clear:both;"></div><br />
<br />
=Circular mDNMT1(731-1602) - The pivotal element of PCR2.0=<br />
<br />
==Expression and purification of mDNMT1(731-1602) for circularization==<br />
<br />
After we succeeded in purifying active and specific linear mDNMT1, we went on with cloning, expression and purification of the circular DNMTs. <br />
<br />
Expression and purification of mDNMT1(731-1602) for the intein as well as the sortase approach were conducted as they had been established for the linear DNMT1. Elutions from the His-Trap showed an enrichment and concentration of peptides that correlate with the expected molecular weight of the truncated mDNMT1(731-1602) (Figure 9A and 9B). Nevertheless, purification with a His-Trap alone does not seem to exclude a variety of impurities that either result from degraded mDNMT1 that still contains a His-Tag or unspecific binding to the affinity column. Advanced protocols as they were used by Song and collegues [[#References|[19]]] for analysis of the proteins crystal structure are therefore including several more steps. Nevertheless, additional steps of purification increase the risk of reducing the overall protein yield and activity, necessitating greater amounts of starting material. Since the active protein should already be included and efficiently enriched in our sample despite of present impurities, we continued with the functional analysis of the sample.<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
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caption=Figure 9A) Efficiency of mDNMT1(731-1602) purification for the intein approach|<br />
descr=Induction of Rosetta (DE3) and purification were performed as described above. After each step, samples were collected for analysis by Coomassie stained SDS-PAGE. Samples are numbered according to their chronology of collection. Arrow indicates mDNMT1(731-1602).|<br />
file=DNMT1-intein-Purification3.png}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
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caption=Figure 9B) Sortase approach - Efficiency of Purification|<br />
descr=Induction of Rosetta (DE3) and purification were performed as described above. After each step, samples were collected for analysis by Coomassie stained SDS-PAGE. Samples are numbered according to their chronology of collection. Arrow indicates mDNMT1(731-1602).|<br />
file=DNMT1-sortase-Purification2.png}}<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
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caption=Figure 10) Expression and Purification of intein and sortase constructs with different linkers|<br />
descr=Expression and Purification of different constructs in Rosetta (DE3) was performed as described above. Unpurified as well as purified samples were collected for analysis by Coomassie stained SDS-PAGE. Ctrl indicates the untransformed control. D= no linker, R= Rigid linker, F=Flexible linker, L= linear. Arrow indicates peptides with molecular weight corresponding to mDNMT1(731-1602). Asterix indicates possibly spliced Intein constructs.|<br />
file=DNMT1-Purification1.png}}<br />
<br />
Our approach of heat-stabilization includes the testing of different linkers that had been calculated by our [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software CRAUT Linker Software]. We tested a flexible linker, consisting mostly of glycines and serines and a rigid linker, which was introduced to maximize stabilization of the protein through cicularization. Purification of the mDNMT1(731-1602) variants with different linkers for circularization was successful (Figure 10), but the yields were lower compared to different batches of linear mDNMT1(731-1602) that had been produced in a similar way. <br />
Interestingly, a shift between mDNMT1(731-1602) expressed from the intein construct compared to the sortase or linear versions was detected. Since protein circularization via inteins is an autocatalytic process that takes place inside the bacteria, this shift could possibly result from successful splicing activity of the inteins. The resulting product lacks Smt3 and is therefore approximately 30kDa smaller than its precursor (Figure 10, indicated by an asterix). Since intein splicing likely occurred, flexible- and rigid-linked-DNMT1 could be circularized. <br />
<br />
To prove circularization by inteins, we cooperated with the core facility Protein Analysis of the German Cancer Research Center (Dkfz.). In the facility, circularized and linear DNMT1 were extracted from a provided gel, digested with trypsin and analyzed by ESI mass spectrometry. Unfortunately, we do not have the final results yet but are confident to be able to present them at the jamboree.<br />
<br />
As against that mDNMT1(731-1602) with sortase recognition sequences needs to be further processed before a circular protein is obtained. Unfortunately, TEV cleavage of the purified protein, which is necessary for subsequent circularization with sortase was not very efficient. Therefore sortase treatment did not yield in a detectable amount of processed mDNMT1(731-1602). Therefore, we fully concentrated on the promising intein approach for further mDNMT1(731-1602) circularization.<br />
<br />
<div style="clear:both;"></div><br />
<br />
==Thermal stability of circular mDNMT1(721-1602)==<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
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caption=Figure 11A) Methylation after heat shock|<br />
descr=Circular DNMT1 with rigid linker (RC), circular DNMT1 with flexible linker (FC) and linear DNMT1 were heat shocked for 5 sec at 55°C and 60°C. |<br />
file=heatshock55-60.jpg}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 11B) Persistence of methylation activity at high temperatures|<br />
descr=Circular DNMT1 with rigid linker (RC), circular DNMT1 with flexible linker (FC) and linear DNMT1 were heat shocked for 5 sec at 65°C and 72°C.Methylation after one hour was measured. A darker upper band for FC is even visible by eye, indication higher methylation activity.|<br />
file=heatshock65-72.jpg}}<br />
<br />
<div><br />
{{:Team:Heidelberg/templates/image-full|<br />
align=right|<br />
caption=Figure 11C) Amount of methylation activity kept after heat shock |<br />
descr=Circular DNMT1 with rigid linker (RC) in blue, circular DNMT1 with flexible linker (FC) in red and linear DNMT1 (L) in blue over a temperature range from 55°C to 72°C.|<br />
file=dnmt1_heatshock.png}}<br />
</div><br />
<br />
In our final assay we examined the effect of an heat shock on the remaining activity of the different DNMT1 enzymes to proof the ability of circular enzyme to withstand higher temperatures. We wanted to produce evidence for the feasibility of a PCR 2.0 using circularization.<br />
Behavior after heat treatment of pre-used linear DNMT1 (L) was compared to DNMT1 with rigid linker (RC) and flexible linker (FC)<br />
The proteins were exposed to temperatures between 55°C and 72°C, temperatures that would also be reached in a PCR. The heat shock was conducted for 5 sec and DNA substrate was added afterwards at 37°C for methylation to occur.<br />
Looking at the gel picture (Figure 11A and B) a darker upper band indicating more methylated substrate for the circular DNMT1 with flexible Linker at all temperatures is even visible by eye, suggesting more remained stability. To confirm this results, the intensity of the gel bands were analyzed using Image J. Figure 11C highlights the remaining activity over temperature after normalization to the control at 37°C the ration of methylated DNA was normalized to activity at 37°C. Indeed activity of circular DNMT1 with the flexible linker (black) is exceeding the linear and circular with rigid linker at least double. Very surprisingly, the methylation activity of circular DNMT1 with a flexible linker seems to increase with higher temperatures. <br />
<br />
For significant evaluation, more data has to be collected. When applying Michael Menten kinetics , the methylation reaction for the controls already approaches saturation therefore the normalization to the original activity might be defective. For further improvement of the assay, less volume of DNMT1 has to be taken for the enzyme reaction.<br />
<br />
=Discussion=<br />
<br />
== Achievements and impact==<br />
Taken together, we could show that intein-mediated circularization of mDNMT1 (731-1602) conceivably increased heat stability of the enzyme without impeding specific methylation activity. We proved that circularization with inteins is an easy method to increase heat stability of complex and sensitive proteins like mDNMT1 (731-1602). In comparison, other methods used for protein stabilization such as introduction of disulfide bonds or hydrophobic amino acids, require sophisticated structure analysis or non-physiological reaction conditions [[#References|[20]]][[#References|[21]]]. In fact, mDNMT1 (731-1602) is the largest protein that has ever been circularized so far and even more might follow.<br />
<br />
Our [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software CRAUT Linker Software] was essential to achieve circularization of mDNMT1 (731-1602) while preserving its natural function. In first experiments, the two designed linker compositions resulted in divergent heat stabilities of the protein. Our software successfully narrowed down the infinite amount of possibilities to circularize mDNMT1 (731-1602). Further experimental data of enzyme-specific linker variants will help to further improve the software predictions. Circularized mDNMT1 (731-1602) represents a valuable tool for the realization of a methylation maintaining '''”PCR2.0”'''. This foundational advance aims to enable broad functional analysis of DNA methylation patterns without prior knowledge on methylation frequency or dependency on other methods. For the detection of DNA methylation using bisulfite sequencing, handling of limited amounts of primary material still represents a major challenge: <br />
<br />
{| class="table table-hover" <br />
|-<br />
| || ||| |||| ''“Future developments will undoubtedly allow information to be recovered from most genomic CpGs, the key being the ability to amplify DNA before bisulfite conversion. The capacity to capture the DNA methylome from individual cells will be critical for a full understandingof early embryonic development, cancer progression and generation of induced pluripotent stem cells.”'' - Smallwood et al., ''Nat. Methods (20014)'' [[#References|[29]]]||||| |||||| <br />
|-<br />
|}<br />
<br />
Approaches including single molecule sequencing have been reported, but have not been established in an affordable and efficient manner yet [[#References|[27]]]. Furthermore, these methods are limited to the detection of methylation patterns and do not allow functional analysis without complete analysis and cost-intensive synthesis. Therefore, our '''”PCR2.0”''' aims to revolutionize the field of epigenetics by linking complex methylation patterns to their actual function. <i>In vitro</i> as well as <i>in vivo</i> experiments, requiring large amounts of methylated DNA will be feasible by using our '''”PCR2.0”'''. Those include for example the detection of DNA methylation binding proteins using Chromatin Immunoprecipitation or Electrophoretic Mobility Shift Assays. Further functional studies could investigate cellular regulation upon transfection with methylated template. <br />
<br />
A very interesting application for the pharmaceutical use of our '''"PCR2.0"''' could be the large scale production of methylated gene therapy vectors. Since methylation of gene transfer vectors has been reported to extend transgene product expression by lowering cellular immunogenicity, the efficiency gene therapies might be improved with our method.[[#References|[22]]]<br />
<br />
==Considerations for further PCR2.0 development==<br />
Since significance of the obtained data still has to be proven in biological replicates, we could not quantify the effect of stabilization in a significant way yet. Therefore additionally to the circularization of mDNMT1 (731-1602), further steps could be necessary to obtain optimal heat stability and specificity for our methylation maintaining '''"PCR2.0"'''. Those for example include further stabilization of the protein, either by enhancing the hydrophobicity of the proteins core or introducing direct links to limit possible conformations [[#References|[23]]].<br />
<br />
Nevertheless, it is unlikely that mDNMT1 (731-1602) can be stabilized in a way to bear temperatures of 95°C, as they would be essential for conventional PCR. Therefore, another upcoming topic is the method of low temperature PCRs that exploit the function of helicases to segregate DNA strands. Even though these PCRs can be performed at lower temperatures than conventional PCRs, none of them has reached widespread establishment yet. Main reasons are that isothermal PCRs require either too long incubation times per cycle [[#References|[24]]] or still exceed physiological temperatures of most enzymes, introducing new candidates for circularization... [[#References|[25]]].<br />
<br />
Despite enduring activity of mDNMT1 (731-1602) at high temperatures, the heat-stability of its cofactors also plays a crucial role for establishing a '''”PCR2.0”'''. Here, a still challenging factor is the methyl-donor S-adenosyl methionine (SAM) that exhibits only low stability at high temperatures [[#References|[30]]]. During our experiments, there has not been observed any limitation due to this instability. Nevertheless, addition of the quite cheap methyl-donor after each cycle of replication could circumvent this problem until a more stable variant of the cofactor is available.<br />
<br />
If there are occurring problems with the specificity of the enzyme at higher temperatures which have not been assessable from our data, the '''“PCR2.0”''' could be complemented with enzymes such as UHRF1, that have been reported to increase the specificity of mDNMT1 (731-1602) [[#References|[31]]]. The wild type DNMT1 is known to be associated to the replication complex for direction of its activity. Design of an appropriate Polymerase-DNMT-fusion protein could imitate this connection and therefore further reduce stochastic methylation events [[#References|[26]]].<br />
<br />
Overall, the iGEM Team Heidelberg has achieved important milestones in establishing a methylation maintaining '''"PCR2.0"''' by expressing and purifying the largest circular protein ever. Despite of its use in a '''"PCR2.0"''', the stabilized mDNMT1 (731-1602) can also be used in other methylation assays.<br />
<br />
=References=<br />
[1] Meissner, A. et al. Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature 454, 766–70 (2008).<br />
<br />
[2] Watanabe, M., Yuzawa, H., Handa, N., & Kobayashi, I.. Hyperthermophilic DNA methyltransferase M.PabI from the archaeon Pyrococcus abyssi. Applied and Environmental Microbiology, 72(8), 5367–75 (2006).<br />
<br />
[3] Vieille, C. & Zeikus, G. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65, 1–43 (2001).<br />
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[4] Antos, J. M., Popp, M. W.-L., Ernst, R., Chew, G.-L., Spooner, E., & Ploegh, H. L.. A straight path to circular proteins. The Journal of Biological Chemistry, 284(23), 16028–36 (2009).<br />
<br />
[5] Tam, J. P. & Wong, C. T. T. Chemical synthesis of circular proteins. J. Biol. Chem. 287, 27020–5 (2012).<br />
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[6] Robertson, K. D. DNA methylation and human disease. Nat. Rev. Genet. 6, 597–610 (2005).<br />
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[7] Jaenisch, R. & Bird, A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat. Genet. 33 Suppl, 245–54 (2003).<br />
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[8] Ehrlich, M.. Amount and distribution of 5-methycytosine in human DNA from different types of tissues or cells. Nucleic Acids Res. 10: 2709–2721 (1982).<br />
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[9] Lister, R. & Ecker, J. Finding the fifth base: genome-wide sequencing of cytosine methylation. Genome Res. 959–966 (2009).<br />
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[10] Bird, A.. DNA methylation patterns and epigenetic memory. Genes & Development 16, 6-21 (2002).<br />
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[11] Jaenisch, R., and Bird, A.. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet 33, 245-254 (2003).<br />
<br />
[12] Weichenhan, D., and Plass, C.. The evolving epigenome. Human Molecular Genetics 22, R1 (2013).<br />
<br />
[13] Robertson, K. D. DNA methylation and human disease. Nat. Rev. Genet. 6, 597–610 (2005).<br />
<br />
[14] Jurkowska, R. Z., Jurkowski, T. P. & Jeltsch, A. Structure and function of mammalian DNA methyltransferases. Chembiochem 12, 206–22 (2011).<br />
<br />
[15] Song, J., Teplova, M., Ishibe-Murakami, S. & Patel, D. J. Structure-based mechanistic insights into DNMT1-mediated maintenance DNA methylation. Science 335, 709–12 (2012).<br />
<br />
[16] Bashtrykov, P. et al. Specificity of Dnmt1 for methylation of hemimethylated CpG sites resides in its catalytic domain. Chem. Biol. 19, 572–8 (2012).<br />
<br />
[17] Lee, C.-D., Sun, H.-C., Hu, S.-M., Chiu, C.-F., Homhuan, A., Liang, S.-M., Wang, T.-F. An improved SUMO fusion protein system for effective production of native proteins. Protein Science : A Publication of the Protein Society, 17(7), 1241–8 (2008).<br />
<br />
[18] Benov, L. & Al-ibraheem, J. Disrupting Escherichia coli : A Comparison of Methods. 35, 428–431 (2002).<br />
<br />
[19] Song, J., Rechkoblit, O., Bestor, T. H. & Patel, D. J. Structure of DNMT1-DNA complex reveals a role for autoinhibition in maintenance DNA methylation. Science 331, 1036–40 (2011).<br />
<br />
[20] Betz, S. F. Disulfide bonds and the stability of globular proteins. 1551–1558 (1993).<br />
<br />
[21] Bulaj, G. Formation of disulfide bonds in proteins and peptides. Biotechnol. Adv. 23, 87–92 (2005).<br />
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[22] Reyes-Sandoval, a & Ertl, H. C. J. CpG methylation of a plasmid vector results in extended transgene product expression by circumventing induction of immune responses. Mol. Ther. 9, 249–61 (2004).<br />
<br />
[23] Vieille, C. & Zeikus, G. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65, 1–43 (2001).<br />
<br />
[24] Vincent, M., Xu, Y. & Kong, H. Helicase-dependent isothermal DNA amplification. EMBO Rep. 5, 795–800 (2004).<br />
<br />
[25] Xu, G. et al. Cross priming amplification: mechanism and optimization for isothermal DNA amplification. Sci. Rep. 2, 246 (2012).<br />
<br />
[26] Vilkaitis, G., Suetake, I., Klimasauskas, S. & Tajima, S. Processive methylation of hemimethylated CpG sites by mouse Dnmt1 DNA methyltransferase. J. Biol. Chem. 280, 64–72 (2005).<br />
<br />
[27] Krueger, F., Kreck, B., Franke, A. & Andrews, S. R. DNA methylome analysis using short bisulfite sequencing data. Nat. Methods 9, 145–51 (2012).<br />
<br />
[28] Smith, Z. D. & Meissner, A. DNA methylation: roles in mammalian development. Nat. Rev. Genet. 14, 204–20 (2013).<br />
<br />
[29] Smallwood, S. a et al. Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity. Nat. Methods 11, 6–11 (2014).<br />
<br />
[30] Matos, J. & Wong, C. S-adenosylmethionine: Stability and stabilization. Bioorg. Chem. 80, 71–80 (1987). <br />
<br />
[31] Bashtrykov, P., Jankevicius, G., Jurkowska, R. Z., Ragozin, S. & Jeltsch, A. The UHRF1 protein stimulates the activity and specificity of the maintenance DNA methyltransferase DNMT1 by an allosteric mechanism. J. Biol. Chem. 289, 4106–15 (2014).</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/PCR_2.0Team:Heidelberg/pages/PCR 2.02014-10-17T23:02:07Z<p>Huhny: /* Activity and specificity of linear mDNMT1(731-1602) */</p>
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<p style="font-size:25px; font-weight:bold">Abstract</p><br />
<br/><br />
<p>DNA methylation is the most abundant DNA modification and essential for embryonic development, gene regulation and genomic stability. Although several methods for the detection of methylation patterns exist, there is no easy way to amplify methylated DNA for <i>in vitro</i> or <i>in vivo</i> studies.</p><br />
<p>To empower epigenetic research, we envisioned a PCR2.0 which maintains DNA methylation patterns during amplification. The central element of this PCR2.0 is a heat-resistant DNA methyltransferase - DNMT1 (731-1602) - which we created by circularization using our intein toolbox and the CRAUT linker software.</p><br />
<p>So far, our DNMT1 represents the largest circularized protein which highlights the usefulness of our intein toolbox in combination with the CRAUT linker software. Increased heat resistance of our circular DNMT1 which was observed in initial assays smoothens the path for the establishment of a PCR2.0 and illustrates the suitability of intein-mediated circularization for the advancement of heat resistant proteins.</p><br />
<p style="font-size:25px; font-weight:bold">Highlights</p><br />
<ul><br />
<li>circularization seems to increase heat stability of mDNMT1 (731-1602): successful first step towards PCR2.0.</li><br />
<li>mDNMT1 is the largest circularized protein (105kDa) so far.</li><br />
<li>efficient expression and purification of active linear and circular mDNMT1 (731-1602) in E.coli.</li><br />
<li>successful application of CRAUT linker design software and intein toolbox for circularization.</li><br />
</ul><br />
</div><br />
</div><br />
</div><br />
</div><br />
<br />
</html><br />
<br />
=Introduction=<br />
<br />
==Motivation==<br />
<br />
The invention of the polymerase chain reaction in 1983 by Kary Mullis revolutionized our world by enabling amplification of DNA in an exponential manner. Further improvements including the use of thermo-stable DNA polymerases made the method even more efficient, allowing its widespread use in nearly every field of modern diagnostics and research. However, a major part of information is lost when using conventional PCR as DNA modifications are not copied. The most abundant modification is DNA methylation, which is a prominent regulator of gene expression in all kingdoms of life. Methylation and other modifications that influence the DNA function without changing its actual sequence are studied in the fast-growing field of epigenetics. Even though the detection and mapping of methylation patterns have become feasible in a high-throughput manner by using bisulfite sequencing and array techniques [[#References|[1]]], further functional analysis is still hindered by the small amount of primary material. Up to now, there is no method available to amplify methylated DNA without knowledge of methylation patterns and expensive de-novo synthesis.<br />
<br />
To empower epigenetics, we propose the '''“PCR 2.0”''' as an easy and efficient way to amplify DNA templates in an exponential manner while maintaining their specific methylation pattern. The pivotal element of this PCR reaction is a heat-stable DNA methyltransferase (DNMT). Although comparable enzymes exist in thermophile organisms [[#References|[2]]], until now no suitable protein has been found or synthesized that withstands the harsh conditions of a PCR.<br />
<br />
To achieve this goal of a PCR2.0 that can be used to amplify DNA including its intrinsic methylation pattern, our main aim was the generation of a heat-stable Dnmt1 that can be produced in a larger scale and shows efficient and specific methylation of DNA. It was reported that the insertion of additional intramolecular bonds can increase the stability of proteins [[#References|[3]]][[#References|[4]]], and that specifically joining the C- and N-terminus improves the thermostability of smaller peptides [[#References|[5]]]. This is why our approach to generate a heat-stable Dnmt1 applies:<br />
* protein circularization using our intein toolbox<br />
* creating a potent [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software Software] to design efficient linker peptides connecting C- and N-terminus<br />
<br />
To enable the PCR2.0 we started the circularization of the largest protein so far…<br />
<br />
==Epigenetics - there is more than just A, T, C and G…==<br />
In mammals and other vertebrates DNA methylation occurs at the C5 position of cytosine nucleotides that are followed by guanines (CpG). It is a prominent key regulator of transcription, embryonic development, X chromosome inactivation and many other cellular functions [[#References|[6]]][[#References|[7]]]. The human genome contains about 28 million CpGs of which 60-80% are methylated [[#References|[28]]]. Due to its great prevalence and the heredity of this modification, the 5-methylcytosine (5mC) is also known as the “fifth base” of eukaryotic genomes [[#References|[9]]]. DNA methylation is preferentially occurring at intergenic regions and repetitive sequences, where it is known to silence gene expression [[#References|[10]]][[#References|[11]]][[#References|[12]]]. Inappropriate cytosine methylation and silencing has been reported to play a crucial role in the development of numerous diseases, including cancer, imprinting diseases and repeat-instability based diseases such Huntington's disease [[#References|[13]]].<br />
<br />
==A closer look at DNA Methyltransferases==<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 1) Principle of mDNMT1-mediated maintenance methylation|<br />
descr=The enzyme DNMT1 is of particular interest for methylation maintenance PCR, as it allows exact duplication of the original methylation pattern to every new DNA copy.|<br />
file=methylation_principle.png}}<br />
<br />
<br />
The family of enzymes called DNA methyltransferases (DNMTs) is responsible for the establishment and maintenance of cell-type specific DNA methylation patterns. Whereas the two enzymes DNMT3a and DNMT3b contribute to de novo methylation of DNA during development, DNMT1 is preserving existing methylation patterns throughout cell divisions. To do so, DNMT1 exploits the principle of semi-conservative replication, using the parental strand as a template to create an exact copy of methylation patterns on the daughter strand (Figure 1). After DNA synthesis, the DNA consists of a methylated and an unmethylated strand. DNMT1 recognizes these so called hemi-methylated CpG sites, and transfers a methyl group from the methyl donor S-Adenosyl-methionine (SAM) to the cytosine belonging to the newly synthesized strand [[#References|[15]]][[#References|[19]]].<br />
<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 2) Crystal structure of full-length and truncated (731-1602) mDNMT1|<br />
descr=Structural overview of full length mDNMT1 and truncated mDNMT1(731–1602) (A) Color-coded schematic overview of domain structure and numbering of mDNMT1 sequence. (B) Ribbon presentation of full-length DNMT1 with the CXXC, BAH1, BAH2,and methyltransferase domain colored in light blue, yellow, orange and red, respectively. (C) Crystal structure of truncated DNMT1, missing the CXXC domain and schowing more adjacent protein termini that the full-length version.|<br />
file=dnmt1_structure.png}}<br />
<br />
<br />
<br />
The complex structure of DNMT1 and several truncated versions have been recently solved by X-ray crystallography [[#References|[19]]]. For our experiments we used the smallest truncated version of DNMT1 - mDNMT1 (731-1602) - that has been reported to efficiently mediate methylation maintenance. This truncated version is derived from <i>Mus musculus</i> and comprises the amino acids 731-1602 of the full length protein [[#References|[15]]][[#References|[19]]] (kindly provided by Dr. Bashtrykov after approval from Prof. Patel). Murine DNMT1 (731-1602) contains a C-terminal catalytic methyltransferase domain as well as the bromo-adjacent homology (BAH) domains, which prevent the binding of unmethylated DNA to the catalytic core. The truncated enzyme is missing the CXXC domain, which has a high affinity to hemi-methylated CpG dinucleotides, but was shown to be dispensable for protein function [[#References|[16]]]. In contrast to the full-length DNMT1, mDNMT1 (731-1602) has the great advantage of being efficiently expressed in E.coli. Another benefit of the truncated DNMT1 is that the N- and C- terminus are closer together than in the full-length DNMT1 (Figure 2). Thus circularization might cause less deformation and show lower impact on the overall activity, which makes this truncated mDNMT1 an ideal candidate for our purposes.<br />
<br />
<div style="clear:both;"></div><br />
<br />
==Circularization - The missing link== <br />
<br />
Heat stabilization of mDNMT1 as a pivotal element of our methylation-maintaining PCR2.0 was approached by circularization of the protein. Until now, only smaller proteins have been stabilized using this method. Therefore, to our knowledge, mDNMT1 – even in its shortest truncated version of 871 amino acids and a molecular weight of approximately 100kDa – is the largest protein that has ever been tried to circularize. According to the crystal structure, the distance between the termini of the truncated DNMT1 is 48 Å. We suspected that circularization of the protein by direct fusion of both termini might cause deformation of the protein structure. Therefore we used our own [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software CRAUT Linker Software] to create a linking amino acid sequence, adapted to the structure of DNMT1. The software is able to design protein linkers with the required length to bridge the gap between the protein termini while bypassing the catalytic core of the enzyme.<br />
<br />
Since we could not estimate the impact of the linker introduction, we focused on two different kinds of linkers: a so called rigid linker that has been calculated and optimized during the establishment of our linker software and a flexible peptide connection consisting mostly of glycine and serine. To perform circularization we have used the split NpuDnaE Intein, since it is used as the <i>golden standard</i> for protein splicing and has been efficiently tested by us through circularization of GFP, Lysozyme and Xylanase. Moreover, we tried to implement protein circularization by using sortase A, which is recognizing and cleaving a carboxyterminal sorting signal followed by a transpeptidation reaction that can be exploited for protein circularization. This method has been reported to be very efficient [[#References|[4]]] and was therefore included in our study as possible candidate for large scale productions of circular proteins.<br />
<br />
=Experimental procedures=<br />
<br />
==Constructs==<br />
We designed constructs in order to characterize the efficiency of sortase A as well as intein mediated circularization of DNMT1. Both approaches are based on the comparison of the circularized protein with a corresponding linear counterpart at different temperatures and incubation times.<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Construct for circularization of mDNMT1 using inteins.|<br />
descr= |<br />
file=Dnmt1_construct1.png}}<br />
For circularization of DNMT1 with inteins we fused the obtained truncated version of DNMT1 (731-1602) [[#References|[15]]] with an appropriate linker and split NpuDnaE domains at either site of the construct. For efficient purification of the protein, all constructs contained a hexa-histidine tag. Moreover, we have cloned the ubiquitin-like Smt3 protein from <i> Saccharomyces cerevisiae </i> in front of the DNMT1-intein complex. This attachment is used to increase the yield when expressing large mammalian proteins like DNMT1 in <i>E.coli</i> [[#References|[17]]]. The constructs have been designed in a way that Smt3 is no longer included in the final circular protein, and therefore cannot interfere with its function. <br />
All cloning steps were carried out using our new RFC[i] standard procedure. <br />
<br />
<br />
The mDNMT1 (731-1602) constructs that have been designed to allow sortase A mediated cirularization are flanked by the sortase A recognition sequence LPETGG on the C–terminus as well as N-terminal glycines that that can be exposed through previous TEV treatment. Since the transpeptidation via sortase requires an additional <i> in vitro </i> reaction, subsequent purification is necessary. To facilitate this step and to enrich the circular product in the sample, a hexa-histidine tag is present in the initial protein that is lost during circularization. Hence, an additional affinity chromatography via His trap can be used to separate the histidine tag containing sortase and not circularized educt from the circular flow-through. <br />
All cloning steps were carried out using our sortase standard. <br />
<br />
The following linkers that had been optimized along with our [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software CRAUT Linker Software]:<br />
<br />
{| class="table table-hover"<br />
|-<br />
! Definition!! Sequence <br />
|-<br />
|Rigid linker|| GGAEAAAKEAAAKVNLTAAEAAAKEAAAKEAAAKEAAAKEAAAKAVNLTAAEAAAKAHHHHHHSGRGT <br />
|-<br />
|Flexible linker|| CWEGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSHHHHHHSGRGT<br />
|}<br />
<br />
== Expression and purification==<br />
<br />
{{:Team:Heidelberg/templates/image-quarter|<br />
align=right|<br />
caption=Figure 3) Purification of recombinant mDNMT1|<br />
descr= The purification of recombinantly expressed mDNMT1 is a challenging task including large scale induction, efficient cell lysis, reduction of sample volume via ultracentrifugation, immobilized Ni ion affinity chromatography, dialysis and concentration through centrifugation. |<br />
file=Purification_procedure_new.png}}<br />
<br />
In comparison to the full length murine DNMT1, our modified mDNMT1(731-1602) can be expressed in <i>E.coli (Rosetta DE3)</i> and does not need the establishment of Baculovirus based system and the use of insect cell cultures. First experiments were conducted to determine the optimal conditions for protein induction and expression. We tested several concentrations of IPTG as well as different temperatures to maximize the yield of mDNMT1. Still, even though expression of the constructs can be simplified by using <i>E.coli</i>, purification of the recombinant protein remains a challenging task requiring multiple different steps that had to be adapted and established from published protocols [[#References|[19]]]. <br />
<br />
In a first step, we optimized the process of cell lysis, to be able to extract a maximum amount of active protein from our cultures. Therefore we compared the two commonly used methods sonication and disruption via French press. Subsequently, the sample was cleared from cell debris by ultracentrifugation. The protein of interest containing a hexa-histidine tag was further enriched via immobilized metal ion affinity chromatography (IMAC) using a His trap and eluted with imidazol. In a next step, low molecular weight solutes such as salts and imidazol that would interfere with protein function were removed from the sample by dialysis. Finally, the purified protein was concentrated by size-exclusion centrifugation using filters with an appropriate mass cutoff.<br />
<br />
<div style="clear:both;"></div><br />
<br />
==Methylation activity assay==<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 4) Sequence of 40-mer DNA template for methylation activity assay|<br />
descr=Sequence of the 40-mer DNA template. CpG dinucleotides are shaded in gray, Sau3AI and HpaII recognition sites are depicted in red. The first CpG dinucleotide is methylated on the reverse strand.|<br />
file=DNMT1_substrate.png}}<br />
<br />
To measure the efficiency and specificity of mDNMT1 maintenance methylation activity, we used an assay that is based on the two methylation-sensitive restriction enzymes Sau3AI and HpaII. It relies on the inhibition of restriction enzymes when methyl groups are attached to the cleavage site. We used a 40-mer DNA template with one hemi-methylated and one unmethylated CpG site which are located within the Sau3AI and HpaII restriction site, respectively (adapted from Bashtrykov et colleagues [[#References|[16]]].<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 4) Principle of the mDNMT1 methylation activity assay|<br />
descr=Principle of mDNMT1 methylation assay using a 40-mer substrate with Sau3AI and HpaII cleavage sites that are blocked upon further CpG methylation. The lack of methylation activity leads to fragmentation of the DNA template with either of the two restriction enzymes. Whereas specific methylation of hemimethylated CpGs hampers only cleavage by Sau3AI, <i>de novo</i> methylation activity of mDNMT1 can be detected when HpaII cleavage is blocked as well.|<br />
file=MethylationAssay_overview.png}}<br />
<br />
The restriction enzyme Sau3AI is capable of cleaving the template despite of the hemimethylated state of the first CpG nucleotide. Similarly, HpaII can attack its unmethylated native site, leading to fragmentation of the template as long as no further methylation occurs. Impairment of Sau3AI cleavage indicates maintenance methylation activity, whereas a decrease in HpaII activity would result from de novo methylation. Therefore the assay is not only detecting activity of the enzyme but also includes its specificity for hemimethylated DNA over unmethylated DNA.<br />
To quantify maintenance and de novo methylation activity, the 40-mer fragments remaining after DNMT1 incubation and restriction digest were separated by gel electrophoresis. The intensities of the detected bands corresponding to different fragments were measured using ImageJ Gel Analyzer and quantified using gaussian fitting after background substraction. <br />
<br />
<br />
<div style="clear:both;"></div><br />
<br />
=Results=<br />
<br />
==Optimizing expression and purification of linear mDNMT1(731-602)==<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 5) Optimisation of induction|<br />
descr=As soon as transformed Rosetta (DE3) cultures reached an OD600 of approximately 0.6, induction of linear mDNMT1(731-1602) was perfomed over night at either 15°C or 25°C, using different concentrations of IPTG. Higest expression of DNMT1 was observed at 15°C using 0.7mM IPTG.|<br />
file=DNMT1-Induction2.png}}<br />
<br />
Before starting circularization of DNMT1, we first tested if we can actually express and purify active linear DNMT1. Since DNMT1 expression in E.coli normally results in low yields (<1mg/liter of culture)[[#References|[15]]], we started with an optimization of the expression conditions. <br />
<br />
In general, low temperatures and low IPTG concentrations are known to prevent the occurrence of insoluble inclusion bodies under conditions of overexpression. Thus, we tested different IPTG concentrations and temperatures during induction in order to maximize yields of active mDNMT1(731-1602). Generally, solubility tags such as the fused Smt3 as well as lower temperatures are known to prevent the occurrence of insoluble inclusion bodies under conditions of overexpression. Accordingly, we observed an optimal induction of our mDNMT1(731-1602) construct after incubation at 15 °C using 0.7mM IPTG (Figure 5).<br />
<br />
Even though the overall yield of recombinant protein is quite low, the presence of mDNMT1(731-1602) after induction with IPTG was detectable through Coomassie staining and specifically verified by Western blot (Figure 6A and 6B). In the western blot, several other peptides with lower molecular weight were detected (Figure 6B). These peptides possibly result from incomplete translation or degradation processes, since the construct used for initial mDNMT1 expression and purification comprised an N-terminal hexa-histidine tag. To minimize the amount of incomplete protein products that would be enriched in the following purification, we optimized the mDNMT1(731-1602) constructs for circularization by shifting the His-tag to the C-terminus.<br />
<br />
<div style="clear:both;"></div><br />
<br />
After successful expression, the next critical step for high yields of recombinant protein after purification is protein extraction. The lysis method has to be efficient but still gentle enough to preserve the proteins function - especially when dealing with sensitive and low expressed proteins like DNMT1. In order to optimize protein extraction we therefore compared cell lysis by sonification with disruption via French press (Figure 6A and 6B). Due to the low overall yield, mDNMT1 does not appear as a prominent band on the Coomassie-stained gel as we expected for an overexpressed recombinant proteins. <br />
Assuming similar amounts of overall protein that should be present in the consecutively generated samples, sonification seems to be less efficient for extraction of recombinant protein compared to usage of the French press. Moreover, the disruption of <i>E.coli</i> using French press has been reported to preserve the biological activity of susceptible proteins more efficiently than sonication [18]. Therefore we used the French press for further extraction of linear as well as circular mDNMT1.<br />
<br />
Further purification of the protein using a His-trap resulted in successful enrichment of mDNMT1f(731-1602) (Figure 6C). Corresponding eluates were pooled and further concentrated for use in functional assays. <br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 6A) Comparison of DNMT1 extraction from bacteria by French press and sonification|<br />
descr=mDNMT1(731-1602) expression in Rosetta (DE3) was performed over night at 15°C. Subsequently, the bacteria were lysed by sonification or French press and ultracentrifuged. After each step, samples were collected for analysis by SDS-PAGE.<br />
(Untransformed negative control (UnTra), intervals of cell disruption (1, 2), supernatant (SN)).|<br />
file=DNMT1-Celllysis.jpg}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 6B)Lysis via French press efficiently etracts mDNMT1(731-1602)|<br />
descr=Western Blot of lysed samples was perfomed with an anti-hexa-histidine antibody for specific detection of mDNMT1. Full length mDNMT1(731-1602) is indicated with an arrow.|<br />
file=DNMT1-Celllysis-WB.jpg}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 6C) Purification of linear mDNMT1(731-1602)|<br />
descr=Coomassie stained SDS-PAGE showing efficient purification of linear mDNMT1(731-1602) via His-column. Arrow indicates peptides corresponding to the molecular weight of mDNMT1(731-1602) (133kDa).|<br />
file=DNMT1-Purification-linear.png}}<br />
<br />
<div style="clear:both;"></div><br />
<br />
==Activity and specificity of linear mDNMT1(731-1602)==<br />
<br />
After successful expression and purification of linear DNMT1, we characterized the proteins activity and specificity for hemi-methylated DNA by performing the methylation activity assay described above. To test the maintenance methylation activity of our linear DNMT1, we performed the methylation assay with different incubation times of DNMT1. We observe an increasing amount of completely methylated template over time (Figure 7), represented by increasing amounts of template that are unsusceptible towards SauAI cleavage. <br />
The truncated DNMT1 (731-1602) showed maximal methylation of the template after approximately 1/2 to 1hour. Therefore, our data agrees with efficiencies, that have been reported in literature for wild type and truncated versions of mDNMT1 [[#References|[15]]] [[#References|[16]]] .Overall, the reaction kinetics of the linear protein seem to be promising for later use in a PCR2.0, assuming that circularization of the protein will not ablate its function. <br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 7A) Maintenance Methylation deposition of linear Dnmt1(731-1602) over time|<br />
descr= Purified linear mDNMT1(731-1602) was incubated with 40mer DNA template (see Figure4) for indicated times at 37°C. Subsequently the samples were digested with Sau3AI. Fragments were separated by PAGE and stained with ethidium bromide. (Homo me= homomethylated substrate, control for enzyme inhibition through methylation, Hemi me=hemimethylated substrate positive control for digest).|<br />
file=activityovertime.jpg}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 7B) Plot showing maintenance methylation of linear Dnmt1(731-1602) over time|<br />
descr= Amount of methylation was calculated by normalizing the intensity of the upper band to whole intensity of each line. All measurements were performed with ImageJ.|<br />
file=dnmt1_act_maint_over_time.png}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 8A) <i>De novo</i> methylation activity of mDNMT1|<br />
descr=Purified linear mDNMT1(731-1602) was incubated with 40mer DNA template (see Figure4) for indicated times at 37°C. Subsequently the samples were digested with HpaII. Fragments were separated using gel-electrophoresis and visualized with ethidiumbromid staining. (Homo me= homomethylated substrate,control for enzyme inhibition through methylation, Hemi me=hemimethylated substrate positive control for digest).|<br />
file=specificityovertime.jpg}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 8B) Plot showing <i>de novo</i> methylation activity of mDNMT1|<br />
descr=Amount of methylation was calculated by normalizing the intensity of the upper band to whole intensity of each line. All measurements were performed with ImageJ.|<br />
file=dnmt1_act_de_novo_over_time.png}}<br />
<br />
In oder to be used in the targeted PCR2.0 mDNMT1 needs to be highly specific for hemi-methylated sites and should not display <i>de novo</i> methylation activity. Therefore, we included HpaII digestion in our assay to determine <i>de novo</i> methylation of CpG sites. The first results indicate that the purified mDNMT1(731-1602) does not exhibit de novo methylation activity (Figure 8). Even with increasing DNMT1 incubation time, HpaII is not repressed which indicates absence of <i>de novo</i> methylation. <br />
However, the analysis of the gel pictures is difficult since restriction efficiency of HpaII was not 100% as it can be seen from the hemimethylated control. Therefore, uncut template which is not resulting from unspecific methylation activity of the enzyme complicates quantitative analysis. Further tests, especially with the circularized protein will have to be performed to confirm these initial data.<br />
<div style="clear:both;"></div><br />
<br />
=Circular mDNMT1(731-1602) - The pivotal element of PCR2.0=<br />
<br />
==Expression and purification of mDNMT1(731-1602) for circularization==<br />
<br />
After we succeeded in purifying active and specific linear mDNMT1, we went on with cloning, expression and purification of the circular DNMTs. <br />
<br />
Expression and purification of mDNMT1(731-1602) for the intein as well as the sortase approach were conducted as they had been established for the linear DNMT1. Elutions from the His-Trap showed an enrichment and concentration of peptides that correlate with the expected molecular weight of the truncated mDNMT1(731-1602) (Figure 9A and 9B). Nevertheless, purification with a His-Trap alone does not seem to exclude a variety of impurities that either result from degraded mDNMT1 that still contains a His-Tag or unspecific binding to the affinity column. Advanced protocols as they were used by Song and collegues [[#References|[19]]] for analysis of the proteins crystal structure are therefore including several more steps. Nevertheless, additional steps of purification increase the risk of reducing the overall protein yield and activity, necessitating greater amounts of starting material. Since the active protein should already be included and efficiently enriched in our sample despite of present impurities, we continued with the functional analysis of the sample.<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 9A) Efficiency of mDNMT1(731-1602) purification for the intein approach|<br />
descr=Induction of Rosetta (DE3) and purification were performed as described above. After each step, samples were collected for analysis by Coomassie stained SDS-PAGE. Samples are numbered according to their chronology of collection. Arrow indicates mDNMT1(731-1602).|<br />
file=DNMT1-intein-Purification3.png}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 9B) Sortase approach - Efficiency of Purification|<br />
descr=Induction of Rosetta (DE3) and purification were performed as described above. After each step, samples were collected for analysis by Coomassie stained SDS-PAGE. Samples are numbered according to their chronology of collection. Arrow indicates mDNMT1(731-1602).|<br />
file=DNMT1-sortase-Purification2.png}}<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 10) Expression and Purification of intein and sortase constructs with different linkers|<br />
descr=Expression and Purification of different constructs in Rosetta (DE3) was performed as described above. Unpurified as well as purified samples were collected for analysis by Coomassie stained SDS-PAGE. Ctrl indicates the untransformed control. D= no linker, R= Rigid linker, F=Flexible linker, L= linear. Arrow indicates peptides with molecular weight corresponding to mDNMT1(731-1602). Asterix indicates possibly spliced Intein constructs.|<br />
file=DNMT1-Purification1.png}}<br />
<br />
Our approach of heat-stabilization includes the testing of different linkers that had been calculated by our [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software CRAUT Linker Software]. We tested a flexible linker, consisting mostly of glycines and serines and a rigid linker, which was introduced to maximize stabilization of the protein through cicularization. Purification of the mDNMT1(731-1602) variants with different linkers for circularization was successful (Figure 10), but the yields were lower compared to different batches of linear mDNMT1(731-1602) that had been produced in a similar way. <br />
Interestingly, a shift between mDNMT1(731-1602) expressed from the intein construct compared to the sortase or linear versions was detected. Since protein circularization via inteins is an autocatalytic process that takes place inside the bacteria, this shift could possibly result from successful splicing activity of the inteins. The resulting product lacks Smt3 and is therefore approximately 30kDa smaller than its precursor (Figure 10, indicated by an asterix). Since intein splicing likely occurred, flexible- and rigid-linked-DNMT1 could be circularized. <br />
<br />
To prove circularization by inteins, we cooperated with the core facility Protein Analysis of the German Cancer Research Center (Dkfz.). In the facility, circularized and linear DNMT1 were extracted from a provided gel, digested with trypsin and analyzed by ESI mass spectrometry. Unfortunately, we do not have the final results yet but are confident to be able to present them at the jamboree.<br />
<br />
As against that mDNMT1(731-1602) with sortase recognition sequences needs to be further processed before a circular protein is obtained. Unfortunately, TEV cleavage of the purified protein, which is necessary for subsequent circularization with sortase was not very efficient. Therefore sortase treatment did not yield in a detectable amount of processed mDNMT1(731-1602). Therefore, we fully concentrated on the promising intein approach for further mDNMT1(731-1602) circularization.<br />
<br />
<div style="clear:both;"></div><br />
<br />
==Thermal stability of circular mDNMT1(721-1602)==<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 8) Methylation after heat shock|<br />
descr=Circular DNMT1 with rigid linker (RC), circular DNMT1 with flexible linker (FC) and linear DNMT1 were heat shocked for 5 sec at 55°C and 60°C. |<br />
file=heatshock55-60.jpg}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 9) Persistance of methylation activity at high temperatures|<br />
descr=Circular DNMT1 with rigid linker (RC), circular DNMT1 with flexible linker (FC) and linear DNMT1 were heat shocked for 5 sec at 65°C and 72°C.Methylation after one hour was measured. A darker upper band for FC is even visible by eye, indication higher methylation activity.|<br />
file=heatshock65-72.jpg}}<br />
<br />
<div><br />
{{:Team:Heidelberg/templates/image-full|<br />
align=right|<br />
caption=Figure 10) Amoun of methylation activity kept after heat shock |<br />
descr=Circular DNMT1 with rigid linker (RC) in blue, circular DNMT1 with flexible linker (FC) in red and linear DNMT1 (L) in blue over a temperature range from 55°C to 72°C.|<br />
file=dnmt1_heatshock.png}}<br />
</div><br />
<br />
In our final assay we examined the effect of an heat shock on the remaining activity of the different Dnmt1 enzymes to proof the ability of circular enzyme to withstand higher temperatures. We wanted to produce evidence for the feasibility of a PCR 2.0 using circularisation.<br />
Behaviour after heat treatment of pre-used linear Dnmt1 (L) was compared to Dnmt1 with rigid linker (RC) and flexible linker (FC)<br />
The proteins were exposed to temperatures between 55°C and 72°C, temperatures that would also be reached in a PCR. The heat shock was conducted for 5 sec and DNA substrate was added afterwards at 37°C for methylation to occur.<br />
Looking at the gel picture (Figure?) a darker upper band indicating more methylated substrate for the circular Dnmt1 with flexible Linker at all temperatures is even visible by eye, suggesting more remained stability. To confirm this results, the intensity of the gel bands were analysed using Image J. Figure ? highlights the remaining activity over temperature after normalisation to the control at 37°C the ration of methylated DNA was normalised to activity at 37°C. Indeed activity of circular DNMT1 with the flexible linker (black) is exciding the linear and ciruclar with rigid linker at least double. Very surprisingly, the methylation activity of circular DNMT1 with a flexible linker seems to increase with higher temperatures. <br />
<br />
For significant evaluation, more data has to be collected. When applying Michael Menten kinetics , the methylation reaction for the controls already approaches saturation therefore the normalisation to the original activity might be defective. For further improvement of the assay, less volume of DNMT1 has to be taken for the enzyme reaction.<br />
<br />
=Discussion=<br />
<br />
== Achievements and impact==<br />
Taken together, we could show that intein-mediated circularization of mDNMT1 (731-1602) conceivably increased heat stability of the enzyme without impeding specific methylation activity. We proved that circularization with inteins is an easy method to increase heat stability of complex and sensitive proteins like mDNMT1 (731-1602). In comparison, other methods used for protein stabilization such as introduction of disulfide bonds or hydrophobic amino acids, require sophisticated structure analysis or non-physiological reaction conditions [[#References|[20]]][[#References|[21]]]. In fact, mDNMT1 (731-1602) is the largest protein that has ever been circularized so far and even more might follow.<br />
<br />
Our [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software CRAUT Linker Software] was essential to achieve circularization of mDNMT1 (731-1602) while preserving its natural function. In first experiments, the two designed linker compositions resulted in divergent heat stabilities of the protein. Our software successfully narrowed down the infinite amount of possibilities to circularize mDNMT1 (731-1602). Further experimental data of enzyme-specific linker variants will help to further improve the software predictions. Circularized mDNMT1 (731-1602) represents a valuable tool for the realization of a methylation maintaining '''”PCR2.0”'''. This foundational advance aims to enable broad functional analysis of DNA methylation patterns without prior knowledge on methylation frequency or dependency on other methods. For the detection of DNA methylation using bisulfite sequencing, handling of limited amounts of primary material still represents a major challenge: <br />
<br />
{| class="table table-hover" <br />
|-<br />
| || ||| |||| ''“Future developments will undoubtedly allow information to be recovered from most genomic CpGs, the key being the ability to amplify DNA before bisulfite conversion. The capacity to capture the DNA methylome from individual cells will be critical for a full understandingof early embryonic development, cancer progression and generation of induced pluripotent stem cells.”'' - Smallwood et al., ''Nat. Methods (20014)'' [[#References|[29]]]||||| |||||| <br />
|-<br />
|}<br />
<br />
Approaches including single molecule sequencing have been reported, but have not been established in an affordable and efficient manner yet [[#References|[27]]]. Furthermore, these methods are limited to the detection of methylation patterns and do not allow functional analysis without complete analysis and cost-intensive synthesis. Therefore, our '''”PCR2.0”''' aims to revolutionize the field of epigenetics by linking complex methylation patterns to their actual function. <i>In vitro</i> as well as <i>in vivo</i> experiments, requiring large amounts of methylated DNA will be feasible by using our '''”PCR2.0”'''. Those include for example the detection of DNA methylation binding proteins using Chromatin Immunoprecipitation or Electrophoretic Mobility Shift Assays. Further functional studies could investigate cellular regulation upon transfection with methylated template. <br />
<br />
A very interesting application for the pharmaceutical use of our '''"PCR2.0"''' could be the large scale production of methylated gene therapy vectors. Since methylation of gene transfer vectors has been reported to extend transgene product expression by lowering cellular immunogenicity, the efficiency gene therapies might be improved with our method.[[#References|[22]]]<br />
<br />
==Considerations for further PCR2.0 development==<br />
Since significance of the obtained data still has to be proven in biological replicates, we could not quantify the effect of stabilization in a significant way yet. Therefore additionally to the circularization of mDNMT1 (731-1602), further steps could be necessary to obtain optimal heat stability and specificity for our methylation maintaining '''"PCR2.0"'''. Those for example include further stabilization of the protein, either by enhancing the hydrophobicity of the proteins core or introducing direct links to limit possible conformations [[#References|[23]]].<br />
<br />
Nevertheless, it is unlikely that mDNMT1 (731-1602) can be stabilized in a way to bear temperatures of 95°C, as they would be essential for conventional PCR. Therefore, another upcoming topic is the method of low temperature PCRs that exploit the function of helicases to segregate DNA strands. Even though these PCRs can be performed at lower temperatures than conventional PCRs, none of them has reached widespread establishment yet. Main reasons are that isothermal PCRs require either too long incubation times per cycle [[#References|[24]]] or still exceed physiological temperatures of most enzymes, introducing new candidates for circularization... [[#References|[25]]].<br />
<br />
Despite enduring activity of mDNMT1 (731-1602) at high temperatures, the heat-stability of its cofactors also plays a crucial role for establishing a '''”PCR2.0”'''. Here, a still challenging factor is the methyl-donor S-adenosyl methionine (SAM) that exhibits only low stability at high temperatures [[#References|[30]]]. During our experiments, there has not been observed any limitation due to this instability. Nevertheless, addition of the quite cheap methyl-donor after each cycle of replication could circumvent this problem until a more stable variant of the cofactor is available.<br />
<br />
If there are occurring problems with the specificity of the enzyme at higher temperatures which have not been assessable from our data, the '''“PCR2.0”''' could be complemented with enzymes such as UHRF1, that have been reported to increase the specificity of mDNMT1 (731-1602) [[#References|[31]]]. The wild type DNMT1 is known to be associated to the replication complex for direction of its activity. Design of an appropriate Polymerase-DNMT-fusion protein could imitate this connection and therefore further reduce stochastic methylation events [[#References|[26]]].<br />
<br />
Overall, the iGEM Team Heidelberg has achieved important milestones in establishing a methylation maintaining '''"PCR2.0"''' by expressing and purifying the largest circular protein ever. Despite of its use in a '''"PCR2.0"''', the stabilized mDNMT1 (731-1602) can also be used in other methylation assays.<br />
<br />
=References=<br />
[1] Meissner, A. et al. Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature 454, 766–70 (2008).<br />
<br />
[2] Watanabe, M., Yuzawa, H., Handa, N., & Kobayashi, I.. Hyperthermophilic DNA methyltransferase M.PabI from the archaeon Pyrococcus abyssi. Applied and Environmental Microbiology, 72(8), 5367–75 (2006).<br />
<br />
[3] Vieille, C. & Zeikus, G. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65, 1–43 (2001).<br />
<br />
[4] Antos, J. M., Popp, M. W.-L., Ernst, R., Chew, G.-L., Spooner, E., & Ploegh, H. L.. A straight path to circular proteins. The Journal of Biological Chemistry, 284(23), 16028–36 (2009).<br />
<br />
[5] Tam, J. P. & Wong, C. T. T. Chemical synthesis of circular proteins. J. Biol. Chem. 287, 27020–5 (2012).<br />
<br />
[6] Robertson, K. D. DNA methylation and human disease. Nat. Rev. Genet. 6, 597–610 (2005).<br />
<br />
[7] Jaenisch, R. & Bird, A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat. Genet. 33 Suppl, 245–54 (2003).<br />
<br />
[8] Ehrlich, M.. Amount and distribution of 5-methycytosine in human DNA from different types of tissues or cells. Nucleic Acids Res. 10: 2709–2721 (1982).<br />
<br />
[9] Lister, R. & Ecker, J. Finding the fifth base: genome-wide sequencing of cytosine methylation. Genome Res. 959–966 (2009).<br />
<br />
[10] Bird, A.. DNA methylation patterns and epigenetic memory. Genes & Development 16, 6-21 (2002).<br />
<br />
[11] Jaenisch, R., and Bird, A.. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet 33, 245-254 (2003).<br />
<br />
[12] Weichenhan, D., and Plass, C.. The evolving epigenome. Human Molecular Genetics 22, R1 (2013).<br />
<br />
[13] Robertson, K. D. DNA methylation and human disease. Nat. Rev. Genet. 6, 597–610 (2005).<br />
<br />
[14] Jurkowska, R. Z., Jurkowski, T. P. & Jeltsch, A. Structure and function of mammalian DNA methyltransferases. Chembiochem 12, 206–22 (2011).<br />
<br />
[15] Song, J., Teplova, M., Ishibe-Murakami, S. & Patel, D. J. Structure-based mechanistic insights into DNMT1-mediated maintenance DNA methylation. Science 335, 709–12 (2012).<br />
<br />
[16] Bashtrykov, P. et al. Specificity of Dnmt1 for methylation of hemimethylated CpG sites resides in its catalytic domain. Chem. Biol. 19, 572–8 (2012).<br />
<br />
[17] Lee, C.-D., Sun, H.-C., Hu, S.-M., Chiu, C.-F., Homhuan, A., Liang, S.-M., Wang, T.-F. An improved SUMO fusion protein system for effective production of native proteins. Protein Science : A Publication of the Protein Society, 17(7), 1241–8 (2008).<br />
<br />
[18] Benov, L. & Al-ibraheem, J. Disrupting Escherichia coli : A Comparison of Methods. 35, 428–431 (2002).<br />
<br />
[19] Song, J., Rechkoblit, O., Bestor, T. H. & Patel, D. J. Structure of DNMT1-DNA complex reveals a role for autoinhibition in maintenance DNA methylation. Science 331, 1036–40 (2011).<br />
<br />
[20] Betz, S. F. Disulfide bonds and the stability of globular proteins. 1551–1558 (1993).<br />
<br />
[21] Bulaj, G. Formation of disulfide bonds in proteins and peptides. Biotechnol. Adv. 23, 87–92 (2005).<br />
<br />
[22] Reyes-Sandoval, a & Ertl, H. C. J. CpG methylation of a plasmid vector results in extended transgene product expression by circumventing induction of immune responses. Mol. Ther. 9, 249–61 (2004).<br />
<br />
[23] Vieille, C. & Zeikus, G. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65, 1–43 (2001).<br />
<br />
[24] Vincent, M., Xu, Y. & Kong, H. Helicase-dependent isothermal DNA amplification. EMBO Rep. 5, 795–800 (2004).<br />
<br />
[25] Xu, G. et al. Cross priming amplification: mechanism and optimization for isothermal DNA amplification. Sci. Rep. 2, 246 (2012).<br />
<br />
[26] Vilkaitis, G., Suetake, I., Klimasauskas, S. & Tajima, S. Processive methylation of hemimethylated CpG sites by mouse Dnmt1 DNA methyltransferase. J. Biol. Chem. 280, 64–72 (2005).<br />
<br />
[27] Krueger, F., Kreck, B., Franke, A. & Andrews, S. R. DNA methylome analysis using short bisulfite sequencing data. Nat. Methods 9, 145–51 (2012).<br />
<br />
[28] Smith, Z. D. & Meissner, A. DNA methylation: roles in mammalian development. Nat. Rev. Genet. 14, 204–20 (2013).<br />
<br />
[29] Smallwood, S. a et al. Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity. Nat. Methods 11, 6–11 (2014).<br />
<br />
[30] Matos, J. & Wong, C. S-adenosylmethionine: Stability and stabilization. Bioorg. Chem. 80, 71–80 (1987). <br />
<br />
[31] Bashtrykov, P., Jankevicius, G., Jurkowska, R. Z., Ragozin, S. & Jeltsch, A. The UHRF1 protein stimulates the activity and specificity of the maintenance DNA methyltransferase DNMT1 by an allosteric mechanism. J. Biol. Chem. 289, 4106–15 (2014).</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/PCR_2.0Team:Heidelberg/pages/PCR 2.02014-10-17T23:01:07Z<p>Huhny: /* Activity and specificity of linear mDNMT1(731-1602) */</p>
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<p style="font-size:25px; font-weight:bold">Abstract</p><br />
<br/><br />
<p>DNA methylation is the most abundant DNA modification and essential for embryonic development, gene regulation and genomic stability. Although several methods for the detection of methylation patterns exist, there is no easy way to amplify methylated DNA for <i>in vitro</i> or <i>in vivo</i> studies.</p><br />
<p>To empower epigenetic research, we envisioned a PCR2.0 which maintains DNA methylation patterns during amplification. The central element of this PCR2.0 is a heat-resistant DNA methyltransferase - DNMT1 (731-1602) - which we created by circularization using our intein toolbox and the CRAUT linker software.</p><br />
<p>So far, our DNMT1 represents the largest circularized protein which highlights the usefulness of our intein toolbox in combination with the CRAUT linker software. Increased heat resistance of our circular DNMT1 which was observed in initial assays smoothens the path for the establishment of a PCR2.0 and illustrates the suitability of intein-mediated circularization for the advancement of heat resistant proteins.</p><br />
<p style="font-size:25px; font-weight:bold">Highlights</p><br />
<ul><br />
<li>circularization seems to increase heat stability of mDNMT1 (731-1602): successful first step towards PCR2.0.</li><br />
<li>mDNMT1 is the largest circularized protein (105kDa) so far.</li><br />
<li>efficient expression and purification of active linear and circular mDNMT1 (731-1602) in E.coli.</li><br />
<li>successful application of CRAUT linker design software and intein toolbox for circularization.</li><br />
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<br />
=Introduction=<br />
<br />
==Motivation==<br />
<br />
The invention of the polymerase chain reaction in 1983 by Kary Mullis revolutionized our world by enabling amplification of DNA in an exponential manner. Further improvements including the use of thermo-stable DNA polymerases made the method even more efficient, allowing its widespread use in nearly every field of modern diagnostics and research. However, a major part of information is lost when using conventional PCR as DNA modifications are not copied. The most abundant modification is DNA methylation, which is a prominent regulator of gene expression in all kingdoms of life. Methylation and other modifications that influence the DNA function without changing its actual sequence are studied in the fast-growing field of epigenetics. Even though the detection and mapping of methylation patterns have become feasible in a high-throughput manner by using bisulfite sequencing and array techniques [[#References|[1]]], further functional analysis is still hindered by the small amount of primary material. Up to now, there is no method available to amplify methylated DNA without knowledge of methylation patterns and expensive de-novo synthesis.<br />
<br />
To empower epigenetics, we propose the '''“PCR 2.0”''' as an easy and efficient way to amplify DNA templates in an exponential manner while maintaining their specific methylation pattern. The pivotal element of this PCR reaction is a heat-stable DNA methyltransferase (DNMT). Although comparable enzymes exist in thermophile organisms [[#References|[2]]], until now no suitable protein has been found or synthesized that withstands the harsh conditions of a PCR.<br />
<br />
To achieve this goal of a PCR2.0 that can be used to amplify DNA including its intrinsic methylation pattern, our main aim was the generation of a heat-stable Dnmt1 that can be produced in a larger scale and shows efficient and specific methylation of DNA. It was reported that the insertion of additional intramolecular bonds can increase the stability of proteins [[#References|[3]]][[#References|[4]]], and that specifically joining the C- and N-terminus improves the thermostability of smaller peptides [[#References|[5]]]. This is why our approach to generate a heat-stable Dnmt1 applies:<br />
* protein circularization using our intein toolbox<br />
* creating a potent [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software Software] to design efficient linker peptides connecting C- and N-terminus<br />
<br />
To enable the PCR2.0 we started the circularization of the largest protein so far…<br />
<br />
==Epigenetics - there is more than just A, T, C and G…==<br />
In mammals and other vertebrates DNA methylation occurs at the C5 position of cytosine nucleotides that are followed by guanines (CpG). It is a prominent key regulator of transcription, embryonic development, X chromosome inactivation and many other cellular functions [[#References|[6]]][[#References|[7]]]. The human genome contains about 28 million CpGs of which 60-80% are methylated [[#References|[28]]]. Due to its great prevalence and the heredity of this modification, the 5-methylcytosine (5mC) is also known as the “fifth base” of eukaryotic genomes [[#References|[9]]]. DNA methylation is preferentially occurring at intergenic regions and repetitive sequences, where it is known to silence gene expression [[#References|[10]]][[#References|[11]]][[#References|[12]]]. Inappropriate cytosine methylation and silencing has been reported to play a crucial role in the development of numerous diseases, including cancer, imprinting diseases and repeat-instability based diseases such Huntington's disease [[#References|[13]]].<br />
<br />
==A closer look at DNA Methyltransferases==<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 1) Principle of mDNMT1-mediated maintenance methylation|<br />
descr=The enzyme DNMT1 is of particular interest for methylation maintenance PCR, as it allows exact duplication of the original methylation pattern to every new DNA copy.|<br />
file=methylation_principle.png}}<br />
<br />
<br />
The family of enzymes called DNA methyltransferases (DNMTs) is responsible for the establishment and maintenance of cell-type specific DNA methylation patterns. Whereas the two enzymes DNMT3a and DNMT3b contribute to de novo methylation of DNA during development, DNMT1 is preserving existing methylation patterns throughout cell divisions. To do so, DNMT1 exploits the principle of semi-conservative replication, using the parental strand as a template to create an exact copy of methylation patterns on the daughter strand (Figure 1). After DNA synthesis, the DNA consists of a methylated and an unmethylated strand. DNMT1 recognizes these so called hemi-methylated CpG sites, and transfers a methyl group from the methyl donor S-Adenosyl-methionine (SAM) to the cytosine belonging to the newly synthesized strand [[#References|[15]]][[#References|[19]]].<br />
<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 2) Crystal structure of full-length and truncated (731-1602) mDNMT1|<br />
descr=Structural overview of full length mDNMT1 and truncated mDNMT1(731–1602) (A) Color-coded schematic overview of domain structure and numbering of mDNMT1 sequence. (B) Ribbon presentation of full-length DNMT1 with the CXXC, BAH1, BAH2,and methyltransferase domain colored in light blue, yellow, orange and red, respectively. (C) Crystal structure of truncated DNMT1, missing the CXXC domain and schowing more adjacent protein termini that the full-length version.|<br />
file=dnmt1_structure.png}}<br />
<br />
<br />
<br />
The complex structure of DNMT1 and several truncated versions have been recently solved by X-ray crystallography [[#References|[19]]]. For our experiments we used the smallest truncated version of DNMT1 - mDNMT1 (731-1602) - that has been reported to efficiently mediate methylation maintenance. This truncated version is derived from <i>Mus musculus</i> and comprises the amino acids 731-1602 of the full length protein [[#References|[15]]][[#References|[19]]] (kindly provided by Dr. Bashtrykov after approval from Prof. Patel). Murine DNMT1 (731-1602) contains a C-terminal catalytic methyltransferase domain as well as the bromo-adjacent homology (BAH) domains, which prevent the binding of unmethylated DNA to the catalytic core. The truncated enzyme is missing the CXXC domain, which has a high affinity to hemi-methylated CpG dinucleotides, but was shown to be dispensable for protein function [[#References|[16]]]. In contrast to the full-length DNMT1, mDNMT1 (731-1602) has the great advantage of being efficiently expressed in E.coli. Another benefit of the truncated DNMT1 is that the N- and C- terminus are closer together than in the full-length DNMT1 (Figure 2). Thus circularization might cause less deformation and show lower impact on the overall activity, which makes this truncated mDNMT1 an ideal candidate for our purposes.<br />
<br />
<div style="clear:both;"></div><br />
<br />
==Circularization - The missing link== <br />
<br />
Heat stabilization of mDNMT1 as a pivotal element of our methylation-maintaining PCR2.0 was approached by circularization of the protein. Until now, only smaller proteins have been stabilized using this method. Therefore, to our knowledge, mDNMT1 – even in its shortest truncated version of 871 amino acids and a molecular weight of approximately 100kDa – is the largest protein that has ever been tried to circularize. According to the crystal structure, the distance between the termini of the truncated DNMT1 is 48 Å. We suspected that circularization of the protein by direct fusion of both termini might cause deformation of the protein structure. Therefore we used our own [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software CRAUT Linker Software] to create a linking amino acid sequence, adapted to the structure of DNMT1. The software is able to design protein linkers with the required length to bridge the gap between the protein termini while bypassing the catalytic core of the enzyme.<br />
<br />
Since we could not estimate the impact of the linker introduction, we focused on two different kinds of linkers: a so called rigid linker that has been calculated and optimized during the establishment of our linker software and a flexible peptide connection consisting mostly of glycine and serine. To perform circularization we have used the split NpuDnaE Intein, since it is used as the <i>golden standard</i> for protein splicing and has been efficiently tested by us through circularization of GFP, Lysozyme and Xylanase. Moreover, we tried to implement protein circularization by using sortase A, which is recognizing and cleaving a carboxyterminal sorting signal followed by a transpeptidation reaction that can be exploited for protein circularization. This method has been reported to be very efficient [[#References|[4]]] and was therefore included in our study as possible candidate for large scale productions of circular proteins.<br />
<br />
=Experimental procedures=<br />
<br />
==Constructs==<br />
We designed constructs in order to characterize the efficiency of sortase A as well as intein mediated circularization of DNMT1. Both approaches are based on the comparison of the circularized protein with a corresponding linear counterpart at different temperatures and incubation times.<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Construct for circularization of mDNMT1 using inteins.|<br />
descr= |<br />
file=Dnmt1_construct1.png}}<br />
For circularization of DNMT1 with inteins we fused the obtained truncated version of DNMT1 (731-1602) [[#References|[15]]] with an appropriate linker and split NpuDnaE domains at either site of the construct. For efficient purification of the protein, all constructs contained a hexa-histidine tag. Moreover, we have cloned the ubiquitin-like Smt3 protein from <i> Saccharomyces cerevisiae </i> in front of the DNMT1-intein complex. This attachment is used to increase the yield when expressing large mammalian proteins like DNMT1 in <i>E.coli</i> [[#References|[17]]]. The constructs have been designed in a way that Smt3 is no longer included in the final circular protein, and therefore cannot interfere with its function. <br />
All cloning steps were carried out using our new RFC[i] standard procedure. <br />
<br />
<br />
The mDNMT1 (731-1602) constructs that have been designed to allow sortase A mediated cirularization are flanked by the sortase A recognition sequence LPETGG on the C–terminus as well as N-terminal glycines that that can be exposed through previous TEV treatment. Since the transpeptidation via sortase requires an additional <i> in vitro </i> reaction, subsequent purification is necessary. To facilitate this step and to enrich the circular product in the sample, a hexa-histidine tag is present in the initial protein that is lost during circularization. Hence, an additional affinity chromatography via His trap can be used to separate the histidine tag containing sortase and not circularized educt from the circular flow-through. <br />
All cloning steps were carried out using our sortase standard. <br />
<br />
The following linkers that had been optimized along with our [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software CRAUT Linker Software]:<br />
<br />
{| class="table table-hover"<br />
|-<br />
! Definition!! Sequence <br />
|-<br />
|Rigid linker|| GGAEAAAKEAAAKVNLTAAEAAAKEAAAKEAAAKEAAAKEAAAKAVNLTAAEAAAKAHHHHHHSGRGT <br />
|-<br />
|Flexible linker|| CWEGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSHHHHHHSGRGT<br />
|}<br />
<br />
== Expression and purification==<br />
<br />
{{:Team:Heidelberg/templates/image-quarter|<br />
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caption=Figure 3) Purification of recombinant mDNMT1|<br />
descr= The purification of recombinantly expressed mDNMT1 is a challenging task including large scale induction, efficient cell lysis, reduction of sample volume via ultracentrifugation, immobilized Ni ion affinity chromatography, dialysis and concentration through centrifugation. |<br />
file=Purification_procedure_new.png}}<br />
<br />
In comparison to the full length murine DNMT1, our modified mDNMT1(731-1602) can be expressed in <i>E.coli (Rosetta DE3)</i> and does not need the establishment of Baculovirus based system and the use of insect cell cultures. First experiments were conducted to determine the optimal conditions for protein induction and expression. We tested several concentrations of IPTG as well as different temperatures to maximize the yield of mDNMT1. Still, even though expression of the constructs can be simplified by using <i>E.coli</i>, purification of the recombinant protein remains a challenging task requiring multiple different steps that had to be adapted and established from published protocols [[#References|[19]]]. <br />
<br />
In a first step, we optimized the process of cell lysis, to be able to extract a maximum amount of active protein from our cultures. Therefore we compared the two commonly used methods sonication and disruption via French press. Subsequently, the sample was cleared from cell debris by ultracentrifugation. The protein of interest containing a hexa-histidine tag was further enriched via immobilized metal ion affinity chromatography (IMAC) using a His trap and eluted with imidazol. In a next step, low molecular weight solutes such as salts and imidazol that would interfere with protein function were removed from the sample by dialysis. Finally, the purified protein was concentrated by size-exclusion centrifugation using filters with an appropriate mass cutoff.<br />
<br />
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<br />
==Methylation activity assay==<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 4) Sequence of 40-mer DNA template for methylation activity assay|<br />
descr=Sequence of the 40-mer DNA template. CpG dinucleotides are shaded in gray, Sau3AI and HpaII recognition sites are depicted in red. The first CpG dinucleotide is methylated on the reverse strand.|<br />
file=DNMT1_substrate.png}}<br />
<br />
To measure the efficiency and specificity of mDNMT1 maintenance methylation activity, we used an assay that is based on the two methylation-sensitive restriction enzymes Sau3AI and HpaII. It relies on the inhibition of restriction enzymes when methyl groups are attached to the cleavage site. We used a 40-mer DNA template with one hemi-methylated and one unmethylated CpG site which are located within the Sau3AI and HpaII restriction site, respectively (adapted from Bashtrykov et colleagues [[#References|[16]]].<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 4) Principle of the mDNMT1 methylation activity assay|<br />
descr=Principle of mDNMT1 methylation assay using a 40-mer substrate with Sau3AI and HpaII cleavage sites that are blocked upon further CpG methylation. The lack of methylation activity leads to fragmentation of the DNA template with either of the two restriction enzymes. Whereas specific methylation of hemimethylated CpGs hampers only cleavage by Sau3AI, <i>de novo</i> methylation activity of mDNMT1 can be detected when HpaII cleavage is blocked as well.|<br />
file=MethylationAssay_overview.png}}<br />
<br />
The restriction enzyme Sau3AI is capable of cleaving the template despite of the hemimethylated state of the first CpG nucleotide. Similarly, HpaII can attack its unmethylated native site, leading to fragmentation of the template as long as no further methylation occurs. Impairment of Sau3AI cleavage indicates maintenance methylation activity, whereas a decrease in HpaII activity would result from de novo methylation. Therefore the assay is not only detecting activity of the enzyme but also includes its specificity for hemimethylated DNA over unmethylated DNA.<br />
To quantify maintenance and de novo methylation activity, the 40-mer fragments remaining after DNMT1 incubation and restriction digest were separated by gel electrophoresis. The intensities of the detected bands corresponding to different fragments were measured using ImageJ Gel Analyzer and quantified using gaussian fitting after background substraction. <br />
<br />
<br />
<div style="clear:both;"></div><br />
<br />
=Results=<br />
<br />
==Optimizing expression and purification of linear mDNMT1(731-602)==<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 5) Optimisation of induction|<br />
descr=As soon as transformed Rosetta (DE3) cultures reached an OD600 of approximately 0.6, induction of linear mDNMT1(731-1602) was perfomed over night at either 15°C or 25°C, using different concentrations of IPTG. Higest expression of DNMT1 was observed at 15°C using 0.7mM IPTG.|<br />
file=DNMT1-Induction2.png}}<br />
<br />
Before starting circularization of DNMT1, we first tested if we can actually express and purify active linear DNMT1. Since DNMT1 expression in E.coli normally results in low yields (<1mg/liter of culture)[[#References|[15]]], we started with an optimization of the expression conditions. <br />
<br />
In general, low temperatures and low IPTG concentrations are known to prevent the occurrence of insoluble inclusion bodies under conditions of overexpression. Thus, we tested different IPTG concentrations and temperatures during induction in order to maximize yields of active mDNMT1(731-1602). Generally, solubility tags such as the fused Smt3 as well as lower temperatures are known to prevent the occurrence of insoluble inclusion bodies under conditions of overexpression. Accordingly, we observed an optimal induction of our mDNMT1(731-1602) construct after incubation at 15 °C using 0.7mM IPTG (Figure 5).<br />
<br />
Even though the overall yield of recombinant protein is quite low, the presence of mDNMT1(731-1602) after induction with IPTG was detectable through Coomassie staining and specifically verified by Western blot (Figure 6A and 6B). In the western blot, several other peptides with lower molecular weight were detected (Figure 6B). These peptides possibly result from incomplete translation or degradation processes, since the construct used for initial mDNMT1 expression and purification comprised an N-terminal hexa-histidine tag. To minimize the amount of incomplete protein products that would be enriched in the following purification, we optimized the mDNMT1(731-1602) constructs for circularization by shifting the His-tag to the C-terminus.<br />
<br />
<div style="clear:both;"></div><br />
<br />
After successful expression, the next critical step for high yields of recombinant protein after purification is protein extraction. The lysis method has to be efficient but still gentle enough to preserve the proteins function - especially when dealing with sensitive and low expressed proteins like DNMT1. In order to optimize protein extraction we therefore compared cell lysis by sonification with disruption via French press (Figure 6A and 6B). Due to the low overall yield, mDNMT1 does not appear as a prominent band on the Coomassie-stained gel as we expected for an overexpressed recombinant proteins. <br />
Assuming similar amounts of overall protein that should be present in the consecutively generated samples, sonification seems to be less efficient for extraction of recombinant protein compared to usage of the French press. Moreover, the disruption of <i>E.coli</i> using French press has been reported to preserve the biological activity of susceptible proteins more efficiently than sonication [18]. Therefore we used the French press for further extraction of linear as well as circular mDNMT1.<br />
<br />
Further purification of the protein using a His-trap resulted in successful enrichment of mDNMT1f(731-1602) (Figure 6C). Corresponding eluates were pooled and further concentrated for use in functional assays. <br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 6A) Comparison of DNMT1 extraction from bacteria by French press and sonification|<br />
descr=mDNMT1(731-1602) expression in Rosetta (DE3) was performed over night at 15°C. Subsequently, the bacteria were lysed by sonification or French press and ultracentrifuged. After each step, samples were collected for analysis by SDS-PAGE.<br />
(Untransformed negative control (UnTra), intervals of cell disruption (1, 2), supernatant (SN)).|<br />
file=DNMT1-Celllysis.jpg}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 6B)Lysis via French press efficiently etracts mDNMT1(731-1602)|<br />
descr=Western Blot of lysed samples was perfomed with an anti-hexa-histidine antibody for specific detection of mDNMT1. Full length mDNMT1(731-1602) is indicated with an arrow.|<br />
file=DNMT1-Celllysis-WB.jpg}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 6C) Purification of linear mDNMT1(731-1602)|<br />
descr=Coomassie stained SDS-PAGE showing efficient purification of linear mDNMT1(731-1602) via His-column. Arrow indicates peptides corresponding to the molecular weight of mDNMT1(731-1602) (133kDa).|<br />
file=DNMT1-Purification-linear.png}}<br />
<br />
<div style="clear:both;"></div><br />
<br />
==Activity and specificity of linear mDNMT1(731-1602)==<br />
<br />
After successful expression and purification of linear DNMT1, we characterized the proteins activity and specificity for hemi-methylated DNA by performing the methylation activity assay described above. To test the maintenance methylation activity of our linear DNMT1, we performed the methylation assay with different incubation times of DNMT1. We observe an increasing amount of completely methylated template over time (Figure 7), represented by increasing amounts of template that are unsusceptible towards SauAI cleavage. <br />
The truncated DNMT1 (731-1602) showed maximal methylation of the template after approximately 1/2 to 1hour. Therefore, our data agrees with efficiencies, that have been reported in literature for wild type and truncated versions of mDNMT1 [[#References|[15]]] [[#References|[16]]] .Overall, the reaction kinetics of the linear protein seem to be promising for later use in a PCR2.0, assuming that circularization of the protein will not ablate its function. <br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 7) Maintenance Methylation deposition of linear Dnmt1(731-1602) over time|<br />
descr= Purified linear mDNMT1(731-1602) was incubated with 40mer DNA template (see Figure4) for indicated times at 37°C. Subsequently the samples were digested with Sau3AI. Fragments were separated by PAGE and stained with ethidium bromide. (Homo me= homomethylated substrate, control for enzyme inhibition through methylation, Hemi me=hemimethylated substrate positive control for digest).|<br />
file=activityovertime.jpg}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 8) Plot showing maintenance methylation of linear Dnmt1(731-1602) over time|<br />
descr= Amount of methylation was calculated by normalizing the intensity of the upper band to whole intensity of each line. All measurements were performed with ImageJ.|<br />
file=dnmt1_act_maint_over_time.png}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 9) <i>De novo</i> methylation activity of mDNMT1|<br />
descr=Purified linear mDNMT1(731-1602) was incubated with 40mer DNA template (see Figure4) for indicated times at 37°C. Subsequently the samples were digested with HpaII. Fragments were separated using gel-electrophoresis and visualized with ethidiumbromid staining. (Homo me= homomethylated substrate,control for enzyme inhibition through methylation, Hemi me=hemimethylated substrate positive control for digest).|<br />
file=specificityovertime.jpg}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 10) Plot showing <i>de novo</i> methylation activity of mDNMT1|<br />
descr=Amount of methylation was calculated by normalizing the intensity of the upper band to whole intensity of each line. All measurements were performed with ImageJ.|<br />
file=dnmt1_act_de_novo_over_time.png}}<br />
<br />
In oder to be used in the targeted PCR2.0 mDNMT1 needs to be highly specific for hemi-methylated sites and should not display <i>de novo</i> methylation activity. Therefore, we included HpaII digestion in our assay to determine <i>de novo</i> methylation of CpG sites. The first results indicate that the purified mDNMT1(731-1602) does not exhibit de novo methylation activity (Figure 8). Even with increasing DNMT1 incubation time, HpaII is not repressed which indicates absence of <i>de novo</i> methylation. <br />
However, the analysis of the gel pictures is difficult since restriction efficiency of HpaII was not 100% as it can be seen from the hemimethylated control. Therefore, uncut template which is not resulting from unspecific methylation activity of the enzyme complicates quantitative analysis. Further tests, especially with the circularized protein will have to be performed to confirm these initial data.<br />
<div style="clear:both;"></div><br />
<br />
=Circular mDNMT1(731-1602) - The pivotal element of PCR2.0=<br />
<br />
==Expression and purification of mDNMT1(731-1602) for circularization==<br />
<br />
After we succeeded in purifying active and specific linear mDNMT1, we went on with cloning, expression and purification of the circular DNMTs. <br />
<br />
Expression and purification of mDNMT1(731-1602) for the intein as well as the sortase approach were conducted as they had been established for the linear DNMT1. Elutions from the His-Trap showed an enrichment and concentration of peptides that correlate with the expected molecular weight of the truncated mDNMT1(731-1602) (Figure 9A and 9B). Nevertheless, purification with a His-Trap alone does not seem to exclude a variety of impurities that either result from degraded mDNMT1 that still contains a His-Tag or unspecific binding to the affinity column. Advanced protocols as they were used by Song and collegues [[#References|[19]]] for analysis of the proteins crystal structure are therefore including several more steps. Nevertheless, additional steps of purification increase the risk of reducing the overall protein yield and activity, necessitating greater amounts of starting material. Since the active protein should already be included and efficiently enriched in our sample despite of present impurities, we continued with the functional analysis of the sample.<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 9A) Efficiency of mDNMT1(731-1602) purification for the intein approach|<br />
descr=Induction of Rosetta (DE3) and purification were performed as described above. After each step, samples were collected for analysis by Coomassie stained SDS-PAGE. Samples are numbered according to their chronology of collection. Arrow indicates mDNMT1(731-1602).|<br />
file=DNMT1-intein-Purification3.png}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 9B) Sortase approach - Efficiency of Purification|<br />
descr=Induction of Rosetta (DE3) and purification were performed as described above. After each step, samples were collected for analysis by Coomassie stained SDS-PAGE. Samples are numbered according to their chronology of collection. Arrow indicates mDNMT1(731-1602).|<br />
file=DNMT1-sortase-Purification2.png}}<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 10) Expression and Purification of intein and sortase constructs with different linkers|<br />
descr=Expression and Purification of different constructs in Rosetta (DE3) was performed as described above. Unpurified as well as purified samples were collected for analysis by Coomassie stained SDS-PAGE. Ctrl indicates the untransformed control. D= no linker, R= Rigid linker, F=Flexible linker, L= linear. Arrow indicates peptides with molecular weight corresponding to mDNMT1(731-1602). Asterix indicates possibly spliced Intein constructs.|<br />
file=DNMT1-Purification1.png}}<br />
<br />
Our approach of heat-stabilization includes the testing of different linkers that had been calculated by our [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software CRAUT Linker Software]. We tested a flexible linker, consisting mostly of glycines and serines and a rigid linker, which was introduced to maximize stabilization of the protein through cicularization. Purification of the mDNMT1(731-1602) variants with different linkers for circularization was successful (Figure 10), but the yields were lower compared to different batches of linear mDNMT1(731-1602) that had been produced in a similar way. <br />
Interestingly, a shift between mDNMT1(731-1602) expressed from the intein construct compared to the sortase or linear versions was detected. Since protein circularization via inteins is an autocatalytic process that takes place inside the bacteria, this shift could possibly result from successful splicing activity of the inteins. The resulting product lacks Smt3 and is therefore approximately 30kDa smaller than its precursor (Figure 10, indicated by an asterix). Since intein splicing likely occurred, flexible- and rigid-linked-DNMT1 could be circularized. <br />
<br />
To prove circularization by inteins, we cooperated with the core facility Protein Analysis of the German Cancer Research Center (Dkfz.). In the facility, circularized and linear DNMT1 were extracted from a provided gel, digested with trypsin and analyzed by ESI mass spectrometry. Unfortunately, we do not have the final results yet but are confident to be able to present them at the jamboree.<br />
<br />
As against that mDNMT1(731-1602) with sortase recognition sequences needs to be further processed before a circular protein is obtained. Unfortunately, TEV cleavage of the purified protein, which is necessary for subsequent circularization with sortase was not very efficient. Therefore sortase treatment did not yield in a detectable amount of processed mDNMT1(731-1602). Therefore, we fully concentrated on the promising intein approach for further mDNMT1(731-1602) circularization.<br />
<br />
<div style="clear:both;"></div><br />
<br />
==Thermal stability of circular mDNMT1(721-1602)==<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 8) Methylation after heat shock|<br />
descr=Circular DNMT1 with rigid linker (RC), circular DNMT1 with flexible linker (FC) and linear DNMT1 were heat shocked for 5 sec at 55°C and 60°C. |<br />
file=heatshock55-60.jpg}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 9) Persistance of methylation activity at high temperatures|<br />
descr=Circular DNMT1 with rigid linker (RC), circular DNMT1 with flexible linker (FC) and linear DNMT1 were heat shocked for 5 sec at 65°C and 72°C.Methylation after one hour was measured. A darker upper band for FC is even visible by eye, indication higher methylation activity.|<br />
file=heatshock65-72.jpg}}<br />
<br />
<div><br />
{{:Team:Heidelberg/templates/image-full|<br />
align=right|<br />
caption=Figure 10) Amoun of methylation activity kept after heat shock |<br />
descr=Circular DNMT1 with rigid linker (RC) in blue, circular DNMT1 with flexible linker (FC) in red and linear DNMT1 (L) in blue over a temperature range from 55°C to 72°C.|<br />
file=dnmt1_heatshock.png}}<br />
</div><br />
<br />
In our final assay we examined the effect of an heat shock on the remaining activity of the different Dnmt1 enzymes to proof the ability of circular enzyme to withstand higher temperatures. We wanted to produce evidence for the feasibility of a PCR 2.0 using circularisation.<br />
Behaviour after heat treatment of pre-used linear Dnmt1 (L) was compared to Dnmt1 with rigid linker (RC) and flexible linker (FC)<br />
The proteins were exposed to temperatures between 55°C and 72°C, temperatures that would also be reached in a PCR. The heat shock was conducted for 5 sec and DNA substrate was added afterwards at 37°C for methylation to occur.<br />
Looking at the gel picture (Figure?) a darker upper band indicating more methylated substrate for the circular Dnmt1 with flexible Linker at all temperatures is even visible by eye, suggesting more remained stability. To confirm this results, the intensity of the gel bands were analysed using Image J. Figure ? highlights the remaining activity over temperature after normalisation to the control at 37°C the ration of methylated DNA was normalised to activity at 37°C. Indeed activity of circular DNMT1 with the flexible linker (black) is exciding the linear and ciruclar with rigid linker at least double. Very surprisingly, the methylation activity of circular DNMT1 with a flexible linker seems to increase with higher temperatures. <br />
<br />
For significant evaluation, more data has to be collected. When applying Michael Menten kinetics , the methylation reaction for the controls already approaches saturation therefore the normalisation to the original activity might be defective. For further improvement of the assay, less volume of DNMT1 has to be taken for the enzyme reaction.<br />
<br />
=Discussion=<br />
<br />
== Achievements and impact==<br />
Taken together, we could show that intein-mediated circularization of mDNMT1 (731-1602) conceivably increased heat stability of the enzyme without impeding specific methylation activity. We proved that circularization with inteins is an easy method to increase heat stability of complex and sensitive proteins like mDNMT1 (731-1602). In comparison, other methods used for protein stabilization such as introduction of disulfide bonds or hydrophobic amino acids, require sophisticated structure analysis or non-physiological reaction conditions [[#References|[20]]][[#References|[21]]]. In fact, mDNMT1 (731-1602) is the largest protein that has ever been circularized so far and even more might follow.<br />
<br />
Our [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software CRAUT Linker Software] was essential to achieve circularization of mDNMT1 (731-1602) while preserving its natural function. In first experiments, the two designed linker compositions resulted in divergent heat stabilities of the protein. Our software successfully narrowed down the infinite amount of possibilities to circularize mDNMT1 (731-1602). Further experimental data of enzyme-specific linker variants will help to further improve the software predictions. Circularized mDNMT1 (731-1602) represents a valuable tool for the realization of a methylation maintaining '''”PCR2.0”'''. This foundational advance aims to enable broad functional analysis of DNA methylation patterns without prior knowledge on methylation frequency or dependency on other methods. For the detection of DNA methylation using bisulfite sequencing, handling of limited amounts of primary material still represents a major challenge: <br />
<br />
{| class="table table-hover" <br />
|-<br />
| || ||| |||| ''“Future developments will undoubtedly allow information to be recovered from most genomic CpGs, the key being the ability to amplify DNA before bisulfite conversion. The capacity to capture the DNA methylome from individual cells will be critical for a full understandingof early embryonic development, cancer progression and generation of induced pluripotent stem cells.”'' - Smallwood et al., ''Nat. Methods (20014)'' [[#References|[29]]]||||| |||||| <br />
|-<br />
|}<br />
<br />
Approaches including single molecule sequencing have been reported, but have not been established in an affordable and efficient manner yet [[#References|[27]]]. Furthermore, these methods are limited to the detection of methylation patterns and do not allow functional analysis without complete analysis and cost-intensive synthesis. Therefore, our '''”PCR2.0”''' aims to revolutionize the field of epigenetics by linking complex methylation patterns to their actual function. <i>In vitro</i> as well as <i>in vivo</i> experiments, requiring large amounts of methylated DNA will be feasible by using our '''”PCR2.0”'''. Those include for example the detection of DNA methylation binding proteins using Chromatin Immunoprecipitation or Electrophoretic Mobility Shift Assays. Further functional studies could investigate cellular regulation upon transfection with methylated template. <br />
<br />
A very interesting application for the pharmaceutical use of our '''"PCR2.0"''' could be the large scale production of methylated gene therapy vectors. Since methylation of gene transfer vectors has been reported to extend transgene product expression by lowering cellular immunogenicity, the efficiency gene therapies might be improved with our method.[[#References|[22]]]<br />
<br />
==Considerations for further PCR2.0 development==<br />
Since significance of the obtained data still has to be proven in biological replicates, we could not quantify the effect of stabilization in a significant way yet. Therefore additionally to the circularization of mDNMT1 (731-1602), further steps could be necessary to obtain optimal heat stability and specificity for our methylation maintaining '''"PCR2.0"'''. Those for example include further stabilization of the protein, either by enhancing the hydrophobicity of the proteins core or introducing direct links to limit possible conformations [[#References|[23]]].<br />
<br />
Nevertheless, it is unlikely that mDNMT1 (731-1602) can be stabilized in a way to bear temperatures of 95°C, as they would be essential for conventional PCR. Therefore, another upcoming topic is the method of low temperature PCRs that exploit the function of helicases to segregate DNA strands. Even though these PCRs can be performed at lower temperatures than conventional PCRs, none of them has reached widespread establishment yet. Main reasons are that isothermal PCRs require either too long incubation times per cycle [[#References|[24]]] or still exceed physiological temperatures of most enzymes, introducing new candidates for circularization... [[#References|[25]]].<br />
<br />
Despite enduring activity of mDNMT1 (731-1602) at high temperatures, the heat-stability of its cofactors also plays a crucial role for establishing a '''”PCR2.0”'''. Here, a still challenging factor is the methyl-donor S-adenosyl methionine (SAM) that exhibits only low stability at high temperatures [[#References|[30]]]. During our experiments, there has not been observed any limitation due to this instability. Nevertheless, addition of the quite cheap methyl-donor after each cycle of replication could circumvent this problem until a more stable variant of the cofactor is available.<br />
<br />
If there are occurring problems with the specificity of the enzyme at higher temperatures which have not been assessable from our data, the '''“PCR2.0”''' could be complemented with enzymes such as UHRF1, that have been reported to increase the specificity of mDNMT1 (731-1602) [[#References|[31]]]. The wild type DNMT1 is known to be associated to the replication complex for direction of its activity. Design of an appropriate Polymerase-DNMT-fusion protein could imitate this connection and therefore further reduce stochastic methylation events [[#References|[26]]].<br />
<br />
Overall, the iGEM Team Heidelberg has achieved important milestones in establishing a methylation maintaining '''"PCR2.0"''' by expressing and purifying the largest circular protein ever. Despite of its use in a '''"PCR2.0"''', the stabilized mDNMT1 (731-1602) can also be used in other methylation assays.<br />
<br />
=References=<br />
[1] Meissner, A. et al. Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature 454, 766–70 (2008).<br />
<br />
[2] Watanabe, M., Yuzawa, H., Handa, N., & Kobayashi, I.. Hyperthermophilic DNA methyltransferase M.PabI from the archaeon Pyrococcus abyssi. Applied and Environmental Microbiology, 72(8), 5367–75 (2006).<br />
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[3] Vieille, C. & Zeikus, G. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65, 1–43 (2001).<br />
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[4] Antos, J. M., Popp, M. W.-L., Ernst, R., Chew, G.-L., Spooner, E., & Ploegh, H. L.. A straight path to circular proteins. The Journal of Biological Chemistry, 284(23), 16028–36 (2009).<br />
<br />
[5] Tam, J. P. & Wong, C. T. T. Chemical synthesis of circular proteins. J. Biol. Chem. 287, 27020–5 (2012).<br />
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[6] Robertson, K. D. DNA methylation and human disease. Nat. Rev. Genet. 6, 597–610 (2005).<br />
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[7] Jaenisch, R. & Bird, A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat. Genet. 33 Suppl, 245–54 (2003).<br />
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[8] Ehrlich, M.. Amount and distribution of 5-methycytosine in human DNA from different types of tissues or cells. Nucleic Acids Res. 10: 2709–2721 (1982).<br />
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[9] Lister, R. & Ecker, J. Finding the fifth base: genome-wide sequencing of cytosine methylation. Genome Res. 959–966 (2009).<br />
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[10] Bird, A.. DNA methylation patterns and epigenetic memory. Genes & Development 16, 6-21 (2002).<br />
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[11] Jaenisch, R., and Bird, A.. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet 33, 245-254 (2003).<br />
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[12] Weichenhan, D., and Plass, C.. The evolving epigenome. Human Molecular Genetics 22, R1 (2013).<br />
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[13] Robertson, K. D. DNA methylation and human disease. Nat. Rev. Genet. 6, 597–610 (2005).<br />
<br />
[14] Jurkowska, R. Z., Jurkowski, T. P. & Jeltsch, A. Structure and function of mammalian DNA methyltransferases. Chembiochem 12, 206–22 (2011).<br />
<br />
[15] Song, J., Teplova, M., Ishibe-Murakami, S. & Patel, D. J. Structure-based mechanistic insights into DNMT1-mediated maintenance DNA methylation. Science 335, 709–12 (2012).<br />
<br />
[16] Bashtrykov, P. et al. Specificity of Dnmt1 for methylation of hemimethylated CpG sites resides in its catalytic domain. Chem. Biol. 19, 572–8 (2012).<br />
<br />
[17] Lee, C.-D., Sun, H.-C., Hu, S.-M., Chiu, C.-F., Homhuan, A., Liang, S.-M., Wang, T.-F. An improved SUMO fusion protein system for effective production of native proteins. Protein Science : A Publication of the Protein Society, 17(7), 1241–8 (2008).<br />
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[18] Benov, L. & Al-ibraheem, J. Disrupting Escherichia coli : A Comparison of Methods. 35, 428–431 (2002).<br />
<br />
[19] Song, J., Rechkoblit, O., Bestor, T. H. & Patel, D. J. Structure of DNMT1-DNA complex reveals a role for autoinhibition in maintenance DNA methylation. Science 331, 1036–40 (2011).<br />
<br />
[20] Betz, S. F. Disulfide bonds and the stability of globular proteins. 1551–1558 (1993).<br />
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[21] Bulaj, G. Formation of disulfide bonds in proteins and peptides. Biotechnol. Adv. 23, 87–92 (2005).<br />
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[22] Reyes-Sandoval, a & Ertl, H. C. J. CpG methylation of a plasmid vector results in extended transgene product expression by circumventing induction of immune responses. Mol. Ther. 9, 249–61 (2004).<br />
<br />
[23] Vieille, C. & Zeikus, G. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65, 1–43 (2001).<br />
<br />
[24] Vincent, M., Xu, Y. & Kong, H. Helicase-dependent isothermal DNA amplification. EMBO Rep. 5, 795–800 (2004).<br />
<br />
[25] Xu, G. et al. Cross priming amplification: mechanism and optimization for isothermal DNA amplification. Sci. Rep. 2, 246 (2012).<br />
<br />
[26] Vilkaitis, G., Suetake, I., Klimasauskas, S. & Tajima, S. Processive methylation of hemimethylated CpG sites by mouse Dnmt1 DNA methyltransferase. J. Biol. Chem. 280, 64–72 (2005).<br />
<br />
[27] Krueger, F., Kreck, B., Franke, A. & Andrews, S. R. DNA methylome analysis using short bisulfite sequencing data. Nat. Methods 9, 145–51 (2012).<br />
<br />
[28] Smith, Z. D. & Meissner, A. DNA methylation: roles in mammalian development. Nat. Rev. Genet. 14, 204–20 (2013).<br />
<br />
[29] Smallwood, S. a et al. Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity. Nat. Methods 11, 6–11 (2014).<br />
<br />
[30] Matos, J. & Wong, C. S-adenosylmethionine: Stability and stabilization. Bioorg. Chem. 80, 71–80 (1987). <br />
<br />
[31] Bashtrykov, P., Jankevicius, G., Jurkowska, R. Z., Ragozin, S. & Jeltsch, A. The UHRF1 protein stimulates the activity and specificity of the maintenance DNA methyltransferase DNMT1 by an allosteric mechanism. J. Biol. Chem. 289, 4106–15 (2014).</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/PCR_2.0Team:Heidelberg/pages/PCR 2.02014-10-17T22:53:26Z<p>Huhny: /* Thermal stability of circular mDNMT1(721-1602) */</p>
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<p style="font-size:25px; font-weight:bold">Abstract</p><br />
<br/><br />
<p>DNA methylation is the most abundant DNA modification and essential for embryonic development, gene regulation and genomic stability. Although several methods for the detection of methylation patterns exist, there is no easy way to amplify methylated DNA for <i>in vitro</i> or <i>in vivo</i> studies.</p><br />
<p>To empower epigenetic research, we envisioned a PCR2.0 which maintains DNA methylation patterns during amplification. The central element of this PCR2.0 is a heat-resistant DNA methyltransferase - DNMT1 (731-1602) - which we created by circularization using our intein toolbox and the CRAUT linker software.</p><br />
<p>So far, our DNMT1 represents the largest circularized protein which highlights the usefulness of our intein toolbox in combination with the CRAUT linker software. Increased heat resistance of our circular DNMT1 which was observed in initial assays smoothens the path for the establishment of a PCR2.0 and illustrates the suitability of intein-mediated circularization for the advancement of heat resistant proteins.</p><br />
<p style="font-size:25px; font-weight:bold">Highlights</p><br />
<ul><br />
<li>circularization seems to increase heat stability of mDNMT1 (731-1602): successful first step towards PCR2.0.</li><br />
<li>mDNMT1 is the largest circularized protein (105kDa) so far.</li><br />
<li>efficient expression and purification of active linear and circular mDNMT1 (731-1602) in E.coli.</li><br />
<li>successful application of CRAUT linker design software and intein toolbox for circularization.</li><br />
</ul><br />
</div><br />
</div><br />
</div><br />
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<br />
=Introduction=<br />
<br />
==Motivation==<br />
<br />
The invention of the polymerase chain reaction in 1983 by Kary Mullis revolutionized our world by enabling amplification of DNA in an exponential manner. Further improvements including the use of thermo-stable DNA polymerases made the method even more efficient, allowing its widespread use in nearly every field of modern diagnostics and research. However, a major part of information is lost when using conventional PCR as DNA modifications are not copied. The most abundant modification is DNA methylation, which is a prominent regulator of gene expression in all kingdoms of life. Methylation and other modifications that influence the DNA function without changing its actual sequence are studied in the fast-growing field of epigenetics. Even though the detection and mapping of methylation patterns have become feasible in a high-throughput manner by using bisulfite sequencing and array techniques [[#References|[1]]], further functional analysis is still hindered by the small amount of primary material. Up to now, there is no method available to amplify methylated DNA without knowledge of methylation patterns and expensive de-novo synthesis.<br />
<br />
To empower epigenetics, we propose the '''“PCR 2.0”''' as an easy and efficient way to amplify DNA templates in an exponential manner while maintaining their specific methylation pattern. The pivotal element of this PCR reaction is a heat-stable DNA methyltransferase (DNMT). Although comparable enzymes exist in thermophile organisms [[#References|[2]]], until now no suitable protein has been found or synthesized that withstands the harsh conditions of a PCR.<br />
<br />
To achieve this goal of a PCR2.0 that can be used to amplify DNA including its intrinsic methylation pattern, our main aim was the generation of a heat-stable Dnmt1 that can be produced in a larger scale and shows efficient and specific methylation of DNA. It was reported that the insertion of additional intramolecular bonds can increase the stability of proteins [[#References|[3]]][[#References|[4]]], and that specifically joining the C- and N-terminus improves the thermostability of smaller peptides [[#References|[5]]]. This is why our approach to generate a heat-stable Dnmt1 applies:<br />
* protein circularization using our intein toolbox<br />
* creating a potent [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software Software] to design efficient linker peptides connecting C- and N-terminus<br />
<br />
To enable the PCR2.0 we started the circularization of the largest protein so far…<br />
<br />
==Epigenetics - there is more than just A, T, C and G…==<br />
In mammals and other vertebrates DNA methylation occurs at the C5 position of cytosine nucleotides that are followed by guanines (CpG). It is a prominent key regulator of transcription, embryonic development, X chromosome inactivation and many other cellular functions [[#References|[6]]][[#References|[7]]]. The human genome contains about 28 million CpGs of which 60-80% are methylated [[#References|[28]]]. Due to its great prevalence and the heredity of this modification, the 5-methylcytosine (5mC) is also known as the “fifth base” of eukaryotic genomes [[#References|[9]]]. DNA methylation is preferentially occurring at intergenic regions and repetitive sequences, where it is known to silence gene expression [[#References|[10]]][[#References|[11]]][[#References|[12]]]. Inappropriate cytosine methylation and silencing has been reported to play a crucial role in the development of numerous diseases, including cancer, imprinting diseases and repeat-instability based diseases such Huntington's disease [[#References|[13]]].<br />
<br />
==A closer look at DNA Methyltransferases==<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 1) Principle of mDNMT1-mediated maintenance methylation|<br />
descr=The enzyme DNMT1 is of particular interest for methylation maintenance PCR, as it allows exact duplication of the original methylation pattern to every new DNA copy.|<br />
file=methylation_principle.png}}<br />
<br />
<br />
The family of enzymes called DNA methyltransferases (DNMTs) is responsible for the establishment and maintenance of cell-type specific DNA methylation patterns. Whereas the two enzymes DNMT3a and DNMT3b contribute to de novo methylation of DNA during development, DNMT1 is preserving existing methylation patterns throughout cell divisions. To do so, DNMT1 exploits the principle of semi-conservative replication, using the parental strand as a template to create an exact copy of methylation patterns on the daughter strand (Figure 1). After DNA synthesis, the DNA consists of a methylated and an unmethylated strand. DNMT1 recognizes these so called hemi-methylated CpG sites, and transfers a methyl group from the methyl donor S-Adenosyl-methionine (SAM) to the cytosine belonging to the newly synthesized strand [[#References|[15]]][[#References|[19]]].<br />
<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 2) Crystal structure of full-length and truncated (731-1602) mDNMT1|<br />
descr=Structural overview of full length mDNMT1 and truncated mDNMT1(731–1602) (A) Color-coded schematic overview of domain structure and numbering of mDNMT1 sequence. (B) Ribbon presentation of full-length DNMT1 with the CXXC, BAH1, BAH2,and methyltransferase domain colored in light blue, yellow, orange and red, respectively. (C) Crystal structure of truncated DNMT1, missing the CXXC domain and schowing more adjacent protein termini that the full-length version.|<br />
file=dnmt1_structure.png}}<br />
<br />
<br />
<br />
The complex structure of DNMT1 and several truncated versions have been recently solved by X-ray crystallography [[#References|[19]]]. For our experiments we used the smallest truncated version of DNMT1 - mDNMT1 (731-1602) - that has been reported to efficiently mediate methylation maintenance. This truncated version is derived from <i>Mus musculus</i> and comprises the amino acids 731-1602 of the full length protein [[#References|[15]]][[#References|[19]]] (kindly provided by Dr. Bashtrykov after approval from Prof. Patel). Murine DNMT1 (731-1602) contains a C-terminal catalytic methyltransferase domain as well as the bromo-adjacent homology (BAH) domains, which prevent the binding of unmethylated DNA to the catalytic core. The truncated enzyme is missing the CXXC domain, which has a high affinity to hemi-methylated CpG dinucleotides, but was shown to be dispensable for protein function [[#References|[16]]]. In contrast to the full-length DNMT1, mDNMT1 (731-1602) has the great advantage of being efficiently expressed in E.coli. Another benefit of the truncated DNMT1 is that the N- and C- terminus are closer together than in the full-length DNMT1 (Figure 2). Thus circularization might cause less deformation and show lower impact on the overall activity, which makes this truncated mDNMT1 an ideal candidate for our purposes.<br />
<br />
<div style="clear:both;"></div><br />
<br />
==Circularization - The missing link== <br />
<br />
Heat stabilization of mDNMT1 as a pivotal element of our methylation-maintaining PCR2.0 was approached by circularization of the protein. Until now, only smaller proteins have been stabilized using this method. Therefore, to our knowledge, mDNMT1 – even in its shortest truncated version of 871 amino acids and a molecular weight of approximately 100kDa – is the largest protein that has ever been tried to circularize. According to the crystal structure, the distance between the termini of the truncated DNMT1 is 48 Å. We suspected that circularization of the protein by direct fusion of both termini might cause deformation of the protein structure. Therefore we used our own [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software CRAUT Linker Software] to create a linking amino acid sequence, adapted to the structure of DNMT1. The software is able to design protein linkers with the required length to bridge the gap between the protein termini while bypassing the catalytic core of the enzyme.<br />
<br />
Since we could not estimate the impact of the linker introduction, we focused on two different kinds of linkers: a so called rigid linker that has been calculated and optimized during the establishment of our linker software and a flexible peptide connection consisting mostly of glycine and serine. To perform circularization we have used the split NpuDnaE Intein, since it is used as the <i>golden standard</i> for protein splicing and has been efficiently tested by us through circularization of GFP, Lysozyme and Xylanase. Moreover, we tried to implement protein circularization by using sortase A, which is recognizing and cleaving a carboxyterminal sorting signal followed by a transpeptidation reaction that can be exploited for protein circularization. This method has been reported to be very efficient [[#References|[4]]] and was therefore included in our study as possible candidate for large scale productions of circular proteins.<br />
<br />
=Experimental procedures=<br />
<br />
==Constructs==<br />
We designed constructs in order to characterize the efficiency of sortase A as well as intein mediated circularization of DNMT1. Both approaches are based on the comparison of the circularized protein with a corresponding linear counterpart at different temperatures and incubation times.<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Construct for circularization of mDNMT1 using inteins.|<br />
descr= |<br />
file=Dnmt1_construct1.png}}<br />
For circularization of DNMT1 with inteins we fused the obtained truncated version of DNMT1 (731-1602) [[#References|[15]]] with an appropriate linker and split NpuDnaE domains at either site of the construct. For efficient purification of the protein, all constructs contained a hexa-histidine tag. Moreover, we have cloned the ubiquitin-like Smt3 protein from <i> Saccharomyces cerevisiae </i> in front of the DNMT1-intein complex. This attachment is used to increase the yield when expressing large mammalian proteins like DNMT1 in <i>E.coli</i> [[#References|[17]]]. The constructs have been designed in a way that Smt3 is no longer included in the final circular protein, and therefore cannot interfere with its function. <br />
All cloning steps were carried out using our new RFC[i] standard procedure. <br />
<br />
<br />
The mDNMT1 (731-1602) constructs that have been designed to allow sortase A mediated cirularization are flanked by the sortase A recognition sequence LPETGG on the C–terminus as well as N-terminal glycines that that can be exposed through previous TEV treatment. Since the transpeptidation via sortase requires an additional <i> in vitro </i> reaction, subsequent purification is necessary. To facilitate this step and to enrich the circular product in the sample, a hexa-histidine tag is present in the initial protein that is lost during circularization. Hence, an additional affinity chromatography via His trap can be used to separate the histidine tag containing sortase and not circularized educt from the circular flow-through. <br />
All cloning steps were carried out using our sortase standard. <br />
<br />
The following linkers that had been optimized along with our [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software CRAUT Linker Software]:<br />
<br />
{| class="table table-hover"<br />
|-<br />
! Definition!! Sequence <br />
|-<br />
|Rigid linker|| GGAEAAAKEAAAKVNLTAAEAAAKEAAAKEAAAKEAAAKEAAAKAVNLTAAEAAAKAHHHHHHSGRGT <br />
|-<br />
|Flexible linker|| CWEGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSHHHHHHSGRGT<br />
|}<br />
<br />
== Expression and purification==<br />
<br />
{{:Team:Heidelberg/templates/image-quarter|<br />
align=right|<br />
caption=Figure 3) Purification of recombinant mDNMT1|<br />
descr= The purification of recombinantly expressed mDNMT1 is a challenging task including large scale induction, efficient cell lysis, reduction of sample volume via ultracentrifugation, immobilized Ni ion affinity chromatography, dialysis and concentration through centrifugation. |<br />
file=Purification_procedure_new.png}}<br />
<br />
In comparison to the full length murine DNMT1, our modified mDNMT1(731-1602) can be expressed in <i>E.coli (Rosetta DE3)</i> and does not need the establishment of Baculovirus based system and the use of insect cell cultures. First experiments were conducted to determine the optimal conditions for protein induction and expression. We tested several concentrations of IPTG as well as different temperatures to maximize the yield of mDNMT1. Still, even though expression of the constructs can be simplified by using <i>E.coli</i>, purification of the recombinant protein remains a challenging task requiring multiple different steps that had to be adapted and established from published protocols [[#References|[19]]]. <br />
<br />
In a first step, we optimized the process of cell lysis, to be able to extract a maximum amount of active protein from our cultures. Therefore we compared the two commonly used methods sonication and disruption via French press. Subsequently, the sample was cleared from cell debris by ultracentrifugation. The protein of interest containing a hexa-histidine tag was further enriched via immobilized metal ion affinity chromatography (IMAC) using a His trap and eluted with imidazol. In a next step, low molecular weight solutes such as salts and imidazol that would interfere with protein function were removed from the sample by dialysis. Finally, the purified protein was concentrated by size-exclusion centrifugation using filters with an appropriate mass cutoff.<br />
<br />
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<br />
==Methylation activity assay==<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 4) Sequence of 40-mer DNA template for methylation activity assay|<br />
descr=Sequence of the 40-mer DNA template. CpG dinucleotides are shaded in gray, Sau3AI and HpaII recognition sites are depicted in red. The first CpG dinucleotide is methylated on the reverse strand.|<br />
file=DNMT1_substrate.png}}<br />
<br />
To measure the efficiency of mDNMT1 maintenance methylation, we used an assay that is based on two methylation-sensitive restriction enzymes Sau3AI and HpaII. It relies on the inhibition of restriction enzymes when methyl groups are attached to the cleavage site. We used a 40-mer DNA template with one hemi-methylated and one unmethylated CpG site which are located within the Sau3AI and HpaII restriction site, respectively (adapted from Bashtrykov et colleagues [[#References|[16]]].<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 4) Principle of the mDNMT1 methylation activity assay|<br />
descr=Principle of mDNMT1 methylation assay using a 40-mer substrate with Sau3AI and HpaII cleavage sites that are blocked upon further CpG methylation. The lack of methylation activity leads to fragmentation of the DNA template with either of the two restriction enzymes. Whereas specific methylation of hemimethylated CpGs hampers only cleavage by Sau3AI, <i>de novo</i> methylation activity of mDNMT1 can be detected when HpaII cleavage is blocked as well.|<br />
file=MethylationAssay_overview.png}}<br />
<br />
The restriction enzyme Sau3AI is capable of cleaving the template despite of the hemimethylated state of the first CpG nucleotide. Similarly, HpaII can attack its unmethylated native site, leading to fragmentation of the template as long as no further methylation occurs. Accordingly, the methylation activity of circular and linear mDNMT1 can be measured by quantifying the cleavage efficiency of the restriction enzymes after incubation with the protein. Impairment of Sau3AI cleavage indicates maintenance methylation activity, whereas a decrease in HpaII activity would result from <i>de novo </i> methylation. Therefore the assay is not only detecting activity of the enzyme but also includes its specificity for hemimethylated DNA over unmethylated DNA. To quantify maintenance and de novo methylation activity, the 40-mer fragments remaining after DNMT1 incubation and restriction digest were separated by gel electrophoresis. The intensities of the detected bands corresponding to different fragments were measured using ImageJ Gel Analyzer and quantified using gaussian fitting after background substraction. <br />
<br />
<br />
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<br />
=Results=<br />
<br />
==Optimizing expression and purification of linear mDNMT1(731-602)==<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 5) Optimisation of induction|<br />
descr=As soon as transformed Rosetta (DE3) cultures reached an OD600 of approximately 0.6, induction of linear mDNMT1(731-1602) was perfomed over night at either 15°C or 25°C, using different concentrations of IPTG. Higest expression of DNMT1 was observed at 15°C using 0.7mM IPTG.|<br />
file=DNMT1-Induction2.png}}<br />
<br />
Before starting circularization of DNMT1, we first tested if we can actually express and purify active linear DNMT1. Since DNMT1 expression in E.coli normally results in low yields (<1mg/liter of culture)[[#References|[15]]], we started with an optimization of the expression conditions. <br />
<br />
In general, low temperatures and low IPTG concentrations are known to prevent the occurrence of insoluble inclusion bodies under conditions of overexpression. Thus, we tested different IPTG concentrations and temperatures during induction in order to maximize yields of active mDNMT1(731-1602). Generally, solubility tags such as the fused Smt3 as well as lower temperatures are known to prevent the occurrence of insoluble inclusion bodies under conditions of overexpression. Accordingly, we observed an optimal induction of our mDNMT1(731-1602) construct after incubation at 15 °C using 0.7mM IPTG (Figure 5).<br />
<br />
Even though the overall yield of recombinant protein is quite low, the presence of mDNMT1(731-1602) after induction with IPTG was detectable through Coomassie staining and specifically verified by Western blot (Figure 6A and 6B). In the western blot, several other peptides with lower molecular weight were detected (Figure 6B). These peptides possibly result from incomplete translation or degradation processes, since the construct used for initial mDNMT1 expression and purification comprised an N-terminal hexa-histidine tag. To minimize the amount of incomplete protein products that would be enriched in the following purification, we optimized the mDNMT1(731-1602) constructs for circularization by shifting the His-tag to the C-terminus.<br />
<br />
<div style="clear:both;"></div><br />
<br />
After successful expression, the next critical step for high yields of recombinant protein after purification is protein extraction. The lysis method has to be efficient but still gentle enough to preserve the proteins function - especially when dealing with sensitive and low expressed proteins like DNMT1. In order to optimize protein extraction we therefore compared cell lysis by sonification with disruption via French press (Figure 6A and 6B). Due to the low overall yield, mDNMT1 does not appear as a prominent band on the Coomassie-stained gel as we expected for an overexpressed recombinant proteins. <br />
Assuming similar amounts of overall protein that should be present in the consecutively generated samples, sonification seems to be less efficient for extraction of recombinant protein compared to usage of the French press. Moreover, the disruption of <i>E.coli</i> using French press has been reported to preserve the biological activity of susceptible proteins more efficiently than sonication [18]. Therefore we used the French press for further extraction of linear as well as circular mDNMT1.<br />
<br />
Further purification of the protein using a His-trap resulted in successful enrichment of mDNMT1f(731-1602) (Figure 6C). Corresponding eluates were pooled and further concentrated for use in functional assays. <br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 6A) Comparison of DNMT1 extraction from bacteria by French press and sonification|<br />
descr=mDNMT1(731-1602) expression in Rosetta (DE3) was performed over night at 15°C. Subsequently, the bacteria were lysed by sonification or French press and ultracentrifuged. After each step, samples were collected for analysis by SDS-PAGE.<br />
(Untransformed negative control (UnTra), intervals of cell disruption (1, 2), supernatant (SN)).|<br />
file=DNMT1-Celllysis.jpg}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 6B)Lysis via French press efficiently etracts mDNMT1(731-1602)|<br />
descr=Western Blot of lysed samples was perfomed with an anti-hexa-histidine antibody for specific detection of mDNMT1. Full length mDNMT1(731-1602) is indicated with an arrow.|<br />
file=DNMT1-Celllysis-WB.jpg}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 6C) Purification of linear mDNMT1(731-1602)|<br />
descr=Coomassie stained SDS-PAGE showing efficient purification of linear mDNMT1(731-1602) via His-column. Arrow indicates peptides corresponding to the molecular weight of mDNMT1(731-1602) (133kDa).|<br />
file=DNMT1-Purification-linear.png}}<br />
<br />
<div style="clear:both;"></div><br />
<br />
==Activity and specificity of linear mDNMT1(731-1602)==<br />
<br />
After successful expression and purification of linear DNMT1, we characterized the proteins activity and specificity for hemi-methylated DNA by performing the methylation activity assay described above. To test the maintenance methylation activity of our linear DNMT1, we performed the methylation assay with different incubation times of DNMT1. We observe an increasing amount of completely methylated template over time (Figure 7), represented by increasing amounts of template that are unsusceptible towards SauAI cleavage. <br />
The truncated DNMT1 (731-1602) showed maximal methylation of the template after approximately 1/2 to 1hour. Therefore, our data agrees with efficiencies, that have been reported in literature for wild type and truncated versions of mDNMT1 [[#References|[15]]] [[#References|[16]]] .Overall, the reaction kinetics of the linear protein seem to be promising for later use in a PCR2.0, assuming that circularization of the protein will not ablate its function. <br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 7) Maintenance Methylation deposition of linear Dnmt1(731-1602) over time|<br />
descr= Purified linear mDNMT1(731-1602) was incubated with 40mer DNA template (see Figure4) for indicated times at 37°C. Subsequently the samples were digested with Sau3AI. Fragments were separated by PAGE and stained with ethidium bromide. (Homo me= homomethylated substrate, control for enzyme inhibition through methylation, Hemi me=hemimethylated substrate positive control for digest).|<br />
file=activityovertime.jpg}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 8) <i>De novo</i> methylation activity of mDNMT1|<br />
descr=Purified linear mDNMT1(731-1602) was incubated with 40mer DNA template (see Figure4) for indicated times at 37°C. Subsequently the samples were digested with HpaII. Fragments were separated using gel-electrophoresis and visualized with ethidiumbromid staining. (Homo me= homomethylated substrate,control for enzyme inhibition through methylation, Hemi me=hemimethylated substrate positive control for digest).|<br />
file=specificityovertime.jpg}}<br />
<br />
In oder to be used in the targeted PCR2.0 mDNMT1 needs to be highly specific for hemi-methylated sites and should not display <i>de novo</i> methylation activity. Therefore, we included HpaII digestion in our assay to determine <i>de novo</i> methylation of CpG sites. The first results indicate that the purified mDNMT1(731-1602) does not exhibit de novo methylation activity (Figure 8). Even with increasing DNMT1 incubation time, HpaII is not repressed which indicates absence of <i>de novo</i> methylation. <br />
However, the analysis of the gel pictures is difficult since restriction efficiency of HpaII was not 100% as it can be seen from the hemimethylated control. Therefore, uncut template which is not resulting from unspecific methylation activity of the enzyme complicates quantitative analysis. Further tests, especially with the circularized protein will have to be performed to confirm these initial data.<br />
<div style="clear:both;"></div><br />
<br />
=Circular mDNMT1(731-1602) - The pivotal element of PCR2.0=<br />
<br />
==Expression and purification of mDNMT1(731-1602) for circularization==<br />
<br />
After we succeeded in purifying active and specific linear mDNMT1, we went on with cloning, expression and purification of the circular DNMTs. <br />
<br />
Expression and purification of mDNMT1(731-1602) for the intein as well as the sortase approach were conducted as they had been established for the linear DNMT1. Elutions from the His-Trap showed an enrichment and concentration of peptides that correlate with the expected molecular weight of the truncated mDNMT1(731-1602) (Figure 9A and 9B). Nevertheless, purification with a His-Trap alone does not seem to exclude a variety of impurities that either result from degraded mDNMT1 that still contains a His-Tag or unspecific binding to the affinity column. Advanced protocols as they were used by Song and collegues [[#References|[19]]] for analysis of the proteins crystal structure are therefore including several more steps. Nevertheless, additional steps of purification increase the risk of reducing the overall protein yield and activity, necessitating greater amounts of starting material. Since the active protein should already be included and efficiently enriched in our sample despite of present impurities, we continued with the functional analysis of the sample.<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 9A) Efficiency of mDNMT1(731-1602) purification for the intein approach|<br />
descr=Induction of Rosetta (DE3) and purification were performed as described above. After each step, samples were collected for analysis by Coomassie stained SDS-PAGE. Samples are numbered according to their chronology of collection. Arrow indicates mDNMT1(731-1602).|<br />
file=DNMT1-intein-Purification3.png}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 9B) Sortase approach - Efficiency of Purification|<br />
descr=Induction of Rosetta (DE3) and purification were performed as described above. After each step, samples were collected for analysis by Coomassie stained SDS-PAGE. Samples are numbered according to their chronology of collection. Arrow indicates mDNMT1(731-1602).|<br />
file=DNMT1-sortase-Purification2.png}}<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 10) Expression and Purification of intein and sortase constructs with different linkers|<br />
descr=Expression and Purification of different constructs in Rosetta (DE3) was performed as described above. Unpurified as well as purified samples were collected for analysis by Coomassie stained SDS-PAGE. Ctrl indicates the untransformed control. D= no linker, R= Rigid linker, F=Flexible linker, L= linear. Arrow indicates peptides with molecular weight corresponding to mDNMT1(731-1602). Asterix indicates possibly spliced Intein constructs.|<br />
file=DNMT1-Purification1.png}}<br />
<br />
Our approach of heat-stabilization includes the testing of different linkers that had been calculated by our [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software CRAUT Linker Software]. We tested a flexible linker, consisting mostly of glycines and serines and a rigid linker, which was introduced to maximize stabilization of the protein through cicularization. Purification of the mDNMT1(731-1602) variants with different linkers for circularization was successful (Figure 10), but the yields were lower compared to different batches of linear mDNMT1(731-1602) that had been produced in a similar way. <br />
Interestingly, a shift between mDNMT1(731-1602) expressed from the intein construct compared to the sortase or linear versions was detected. Since protein circularization via inteins is an autocatalytic process that takes place inside the bacteria, this shift could possibly result from successful splicing activity of the inteins. The resulting product lacks Smt3 and is therefore approximately 30kDa smaller than its precursor (Figure 10, indicated by an asterix). Since intein splicing likely occurred, flexible- and rigid-linked-DNMT1 could be circularized. <br />
<br />
To prove circularization by inteins, we cooperated with the core facility Protein Analysis of the German Cancer Research Center (Dkfz.). In the facility, circularized and linear DNMT1 were extracted from a provided gel, digested with trypsin and analyzed by ESI mass spectrometry. Unfortunately, we do not have the final results yet but are confident to be able to present them at the jamboree.<br />
<br />
As against that mDNMT1(731-1602) with sortase recognition sequences needs to be further processed before a circular protein is obtained. Unfortunately, TEV cleavage of the purified protein, which is necessary for subsequent circularization with sortase was not very efficient. Therefore sortase treatment did not yield in a detectable amount of processed mDNMT1(731-1602). Therefore, we fully concentrated on the promising intein approach for further mDNMT1(731-1602) circularization.<br />
<br />
<div style="clear:both;"></div><br />
<br />
==Thermal stability of circular mDNMT1(721-1602)==<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 8) Methylation after heat shock|<br />
descr=Circular DNMT1 with rigid linker (RC), circular DNMT1 with flexible linker (FC) and linear DNMT1 were heat shocked for 5 sec at 55°C and 60°C. |<br />
file=heatshock55-60.jpg}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 9) Persistance of methylation activity at high temperatures|<br />
descr=Circular DNMT1 with rigid linker (RC), circular DNMT1 with flexible linker (FC) and linear DNMT1 were heat shocked for 5 sec at 65°C and 72°C.Methylation after one hour was measured. A darker upper band for FC is even visible by eye, indication higher methylation activity.|<br />
file=heatshock65-72.jpg}}<br />
<br />
<div><br />
{{:Team:Heidelberg/templates/image-full|<br />
align=right|<br />
caption=Figure 10) Amoun of methylation activity kept after heat shock |<br />
descr=Circular DNMT1 with rigid linker (RC) in blue, circular DNMT1 with flexible linker (FC) in red and linear DNMT1 (L) in blue over a temperature range from 55°C to 72°C.|<br />
file=dnmt1_heatshock.png}}<br />
</div><br />
<br />
In our final assay we examined the effect of an heat shock on the remaining activity of the different Dnmt1 enzymes to proof the ability of circular enzyme to withstand higher temperatures. We wanted to produce evidence for the feasibility of a PCR 2.0 using circularisation.<br />
Behaviour after heat treatment of pre-used linear Dnmt1 (L) was compared to Dnmt1 with rigid linker (RC) and flexible linker (FC)<br />
The proteins were exposed to temperatures between 55°C and 72°C, temperatures that would also be reached in a PCR. The heat shock was conducted for 5 sec and DNA substrate was added afterwards at 37°C for methylation to occur.<br />
Looking at the gel picture (Figure?) a darker upper band indicating more methylated substrate for the circular Dnmt1 with flexible Linker at all temperatures is even visible by eye, suggesting more remained stability. To confirm this results, the intensity of the gel bands were analysed using Image J. Figure ? highlights the remaining activity over temperature after normalisation to the control at 37°C the ration of methylated DNA was normalised to activity at 37°C. Indeed activity of circular DNMT1 with the flexible linker (black) is exciding the linear and ciruclar with rigid linker at least double. Very surprisingly, the methylation activity of circular DNMT1 with a flexible linker seems to increase with higher temperatures. <br />
<br />
For significant evaluation, more data has to be collected. When applying Michael Menten kinetics , the methylation reaction for the controls already approaches saturation therefore the normalisation to the original activity might be defective. For further improvement of the assay, less volume of DNMT1 has to be taken for the enzyme reaction.<br />
<br />
=Discussion=<br />
<br />
== Achievements and impact==<br />
Taken together, we could show that intein-mediated circularization of mDNMT1 (731-1602) conceivably increased heat stability of the enzyme without impeding specific methylation activity. We proved that circularization with inteins is an easy method to increase heat stability of complex and sensitive proteins like mDNMT1 (731-1602). In comparison, other methods used for protein stabilization such as introduction of disulfide bonds or hydrophobic amino acids, require sophisticated structure analysis or non-physiological reaction conditions [[#References|[20]]][[#References|[21]]]. In fact, mDNMT1 (731-1602) is the largest protein that has ever been circularized so far and even more might follow.<br />
<br />
Our [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software CRAUT Linker Software] was essential to achieve circularization of mDNMT1 (731-1602) while preserving its natural function. In first experiments, the two designed linker compositions resulted in divergent heat stabilities of the protein. Our software successfully narrowed down the infinite amount of possibilities to circularize mDNMT1 (731-1602). Further experimental data of enzyme-specific linker variants will help to further improve the software predictions. Circularized mDNMT1 (731-1602) represents a valuable tool for the realization of a methylation maintaining '''”PCR2.0”'''. This foundational advance aims to enable broad functional analysis of DNA methylation patterns without prior knowledge on methylation frequency or dependency on other methods. For the detection of DNA methylation using bisulfite sequencing, handling of limited amounts of primary material still represents a major challenge: <br />
<br />
{| class="table table-hover" <br />
|-<br />
| || ||| |||| ''“Future developments will undoubtedly allow information to be recovered from most genomic CpGs, the key being the ability to amplify DNA before bisulfite conversion. The capacity to capture the DNA methylome from individual cells will be critical for a full understandingof early embryonic development, cancer progression and generation of induced pluripotent stem cells.”'' - Smallwood et al., ''Nat. Methods (20014)'' [[#References|[29]]]||||| |||||| <br />
|-<br />
|}<br />
<br />
Approaches including single molecule sequencing have been reported, but have not been established in an affordable and efficient manner yet [[#References|[27]]]. Furthermore, these methods are limited to the detection of methylation patterns and do not allow functional analysis without complete analysis and cost-intensive synthesis. Therefore, our '''”PCR2.0”''' aims to revolutionize the field of epigenetics by linking complex methylation patterns to their actual function. <i>In vitro</i> as well as <i>in vivo</i> experiments, requiring large amounts of methylated DNA will be feasible by using our '''”PCR2.0”'''. Those include for example the detection of DNA methylation binding proteins using Chromatin Immunoprecipitation or Electrophoretic Mobility Shift Assays. Further functional studies could investigate cellular regulation upon transfection with methylated template. <br />
<br />
A very interesting application for the pharmaceutical use of our '''"PCR2.0"''' could be the large scale production of methylated gene therapy vectors. Since methylation of gene transfer vectors has been reported to extend transgene product expression by lowering cellular immunogenicity, the efficiency gene therapies might be improved with our method.[[#References|[22]]]<br />
<br />
==Considerations for further PCR2.0 development==<br />
Since significance of the obtained data still has to be proven in biological replicates, we could not quantify the effect of stabilization in a significant way yet. Therefore additionally to the circularization of mDNMT1 (731-1602), further steps could be necessary to obtain optimal heat stability and specificity for our methylation maintaining '''"PCR2.0"'''. Those for example include further stabilization of the protein, either by enhancing the hydrophobicity of the proteins core or introducing direct links to limit possible conformations [[#References|[23]]].<br />
<br />
Nevertheless, it is unlikely that mDNMT1 (731-1602) can be stabilized in a way to bear temperatures of 95°C, as they would be essential for conventional PCR. Therefore, another upcoming topic is the method of low temperature PCRs that exploit the function of helicases to segregate DNA strands. Even though these PCRs can be performed at lower temperatures than conventional PCRs, none of them has reached widespread establishment yet. Main reasons are that isothermal PCRs require either too long incubation times per cycle [[#References|[24]]] or still exceed physiological temperatures of most enzymes, introducing new candidates for circularization... [[#References|[25]]].<br />
<br />
Despite enduring activity of mDNMT1 (731-1602) at high temperatures, the heat-stability of its cofactors also plays a crucial role for establishing a '''”PCR2.0”'''. Here, a still challenging factor is the methyl-donor S-adenosyl methionine (SAM) that exhibits only low stability at high temperatures [[#References|[30]]]. During our experiments, there has not been observed any limitation due to this instability. Nevertheless, addition of the quite cheap methyl-donor after each cycle of replication could circumvent this problem until a more stable variant of the cofactor is available.<br />
<br />
If there are occurring problems with the specificity of the enzyme at higher temperatures which have not been assessable from our data, the '''“PCR2.0”''' could be complemented with enzymes such as UHRF1, that have been reported to increase the specificity of mDNMT1 (731-1602) [[#References|[31]]]. The wild type DNMT1 is known to be associated to the replication complex for direction of its activity. Design of an appropriate Polymerase-DNMT-fusion protein could imitate this connection and therefore further reduce stochastic methylation events [[#References|[26]]].<br />
<br />
Overall, the iGEM Team Heidelberg has achieved important milestones in establishing a methylation maintaining '''"PCR2.0"''' by expressing and purifying the largest circular protein ever. Despite of its use in a '''"PCR2.0"''', the stabilized mDNMT1 (731-1602) can also be used in other methylation assays.<br />
<br />
=References=<br />
[1] Meissner, A. et al. Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature 454, 766–70 (2008).<br />
<br />
[2] Watanabe, M., Yuzawa, H., Handa, N., & Kobayashi, I.. Hyperthermophilic DNA methyltransferase M.PabI from the archaeon Pyrococcus abyssi. Applied and Environmental Microbiology, 72(8), 5367–75 (2006).<br />
<br />
[3] Vieille, C. & Zeikus, G. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65, 1–43 (2001).<br />
<br />
[4] Antos, J. M., Popp, M. W.-L., Ernst, R., Chew, G.-L., Spooner, E., & Ploegh, H. L.. A straight path to circular proteins. The Journal of Biological Chemistry, 284(23), 16028–36 (2009).<br />
<br />
[5] Tam, J. P. & Wong, C. T. T. Chemical synthesis of circular proteins. J. Biol. Chem. 287, 27020–5 (2012).<br />
<br />
[6] Robertson, K. D. DNA methylation and human disease. Nat. Rev. Genet. 6, 597–610 (2005).<br />
<br />
[7] Jaenisch, R. & Bird, A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat. Genet. 33 Suppl, 245–54 (2003).<br />
<br />
[8] Ehrlich, M.. Amount and distribution of 5-methycytosine in human DNA from different types of tissues or cells. Nucleic Acids Res. 10: 2709–2721 (1982).<br />
<br />
[9] Lister, R. & Ecker, J. Finding the fifth base: genome-wide sequencing of cytosine methylation. Genome Res. 959–966 (2009).<br />
<br />
[10] Bird, A.. DNA methylation patterns and epigenetic memory. Genes & Development 16, 6-21 (2002).<br />
<br />
[11] Jaenisch, R., and Bird, A.. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet 33, 245-254 (2003).<br />
<br />
[12] Weichenhan, D., and Plass, C.. The evolving epigenome. Human Molecular Genetics 22, R1 (2013).<br />
<br />
[13] Robertson, K. D. DNA methylation and human disease. Nat. Rev. Genet. 6, 597–610 (2005).<br />
<br />
[14] Jurkowska, R. Z., Jurkowski, T. P. & Jeltsch, A. Structure and function of mammalian DNA methyltransferases. Chembiochem 12, 206–22 (2011).<br />
<br />
[15] Song, J., Teplova, M., Ishibe-Murakami, S. & Patel, D. J. Structure-based mechanistic insights into DNMT1-mediated maintenance DNA methylation. Science 335, 709–12 (2012).<br />
<br />
[16] Bashtrykov, P. et al. Specificity of Dnmt1 for methylation of hemimethylated CpG sites resides in its catalytic domain. Chem. Biol. 19, 572–8 (2012).<br />
<br />
[17] Lee, C.-D., Sun, H.-C., Hu, S.-M., Chiu, C.-F., Homhuan, A., Liang, S.-M., Wang, T.-F. An improved SUMO fusion protein system for effective production of native proteins. Protein Science : A Publication of the Protein Society, 17(7), 1241–8 (2008).<br />
<br />
[18] Benov, L. & Al-ibraheem, J. Disrupting Escherichia coli : A Comparison of Methods. 35, 428–431 (2002).<br />
<br />
[19] Song, J., Rechkoblit, O., Bestor, T. H. & Patel, D. J. Structure of DNMT1-DNA complex reveals a role for autoinhibition in maintenance DNA methylation. Science 331, 1036–40 (2011).<br />
<br />
[20] Betz, S. F. Disulfide bonds and the stability of globular proteins. 1551–1558 (1993).<br />
<br />
[21] Bulaj, G. Formation of disulfide bonds in proteins and peptides. Biotechnol. Adv. 23, 87–92 (2005).<br />
<br />
[22] Reyes-Sandoval, a & Ertl, H. C. J. CpG methylation of a plasmid vector results in extended transgene product expression by circumventing induction of immune responses. Mol. Ther. 9, 249–61 (2004).<br />
<br />
[23] Vieille, C. & Zeikus, G. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65, 1–43 (2001).<br />
<br />
[24] Vincent, M., Xu, Y. & Kong, H. Helicase-dependent isothermal DNA amplification. EMBO Rep. 5, 795–800 (2004).<br />
<br />
[25] Xu, G. et al. Cross priming amplification: mechanism and optimization for isothermal DNA amplification. Sci. Rep. 2, 246 (2012).<br />
<br />
[26] Vilkaitis, G., Suetake, I., Klimasauskas, S. & Tajima, S. Processive methylation of hemimethylated CpG sites by mouse Dnmt1 DNA methyltransferase. J. Biol. Chem. 280, 64–72 (2005).<br />
<br />
[27] Krueger, F., Kreck, B., Franke, A. & Andrews, S. R. DNA methylome analysis using short bisulfite sequencing data. Nat. Methods 9, 145–51 (2012).<br />
<br />
[28] Smith, Z. D. & Meissner, A. DNA methylation: roles in mammalian development. Nat. Rev. Genet. 14, 204–20 (2013).<br />
<br />
[29] Smallwood, S. a et al. Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity. Nat. Methods 11, 6–11 (2014).<br />
<br />
[30] Matos, J. & Wong, C. S-adenosylmethionine: Stability and stabilization. Bioorg. Chem. 80, 71–80 (1987). <br />
<br />
[31] Bashtrykov, P., Jankevicius, G., Jurkowska, R. Z., Ragozin, S. & Jeltsch, A. The UHRF1 protein stimulates the activity and specificity of the maintenance DNA methyltransferase DNMT1 by an allosteric mechanism. J. Biol. Chem. 289, 4106–15 (2014).</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/backgroundTeam:Heidelberg/pages/background2014-10-17T22:35:00Z<p>Huhny: /* A detailed description of the trans-splicing reaction */</p>
<hr />
<div><br/><br />
In the variety of proteins that exist, inteins are clearly among the most mysterious.<br />
They behave in such a peculiarly way that scientists are still puzzle about their initial role in the host organisms and are even more astonished when discovering their endless seeming abilities.<br />
<br />
Inteins are integrated as extraneous polypeptide sequences into ordinary proteins. They do not contribute to the original protein function but perform an autocatalytic splicing reaction after protein translation. Analog to intron splicing on RNA level, this posttranslational modification was named protein splicing. Consequentially the protein segments were called inteins, derived from “internal protein sequence”, and the flanking protein chains exteins, “external protein sequences”. Inteins excise themselves out of the host protein while reconnecting the remaining N and C exteins via a new peptide bond. Despite this immense invasion the original protein regains its normal structure and function after splicing [[#References|[1]]]. <br />
<br />
<div><br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 1) Trans-splicing mechanism reaction by split inteins. |<br />
descr=The N-Intein is fused to the C’ terminal end of the N-extein. Complementary, C-Intein is located at the N’ end of the C-extein. After assembly of the two intein fragments, a splicing reaction takes place, where the intein removes itself from the precursor protein and simultaneously ligates the exteins via a peptide bond.|<br />
file=InteinSplicingReaction.png}}<br />
</div><br />
<br />
==History==<br />
<br />
Inteins were discovered in 1990 when dissimilarities between the mature protein sequence of the yeast vacuolar ATPase (Vma1) and its corresponding mRNA were investigated. Surprisingly the mature protein had a lower molecular weight than expected from the encoding sequence, indicating the loss of one part of the protein after translation. <br />
Indeed, a region of 454 amino acids was found to be translated and subsequently removed from the Vma1 protein [[#References|[2]]] [[#References|[3]]]. Since then, over 600 different inteins have been reported in all three domains of life as well as in viruses [[#References|[4]]]. <br />
<br />
Dependent on the organism they belong to, the intein’s name consists out of the genus and species abbreviation followed by the host gene [[#References|[5]]]. For example, the "golden standard" split-intein, NpuDnaE has derived from the DNA polymerase III (DnaE) in <i>Nostoc punctiforme</i> PCC73102 (<i>Npu</i>).<br />
<br />
==Evolution: Inteins as parasitic genes==<br />
Despite many years of research the initial role of inteins in their host organism remains still unclear.<br />
Many natural inteins contain a homing endonuclease (HEN) domain, an enzyme that cleaves DNA and introduces new sequences at homing sites via homologous recombination or reverse transcription. This characteristic led to the perception of the intein as selfish DNA sequence - a gene generating copies of itself without an advantage for the host organism [[#References|[6]]]. This allows horizontal gene transfer of inteins. [[#References|[7]]]. Data showing that 27% of the intein's host proteins are related to DNA metabolism and involved in DNA replication or repair is further supporting the "selfish gene" hypothesis. This ensures expression of the inteins and the HEN domain during DNA replication, where they can take advantage of the DNA replication and repair system for introducing changes in the DNA [[#References|[8]]].<br />
<br />
However recent work on conditional protein splicing has shown sensitivity of some inteins to redox state, temperature and small molecules (reviewed in [[#References|[9]]], [[#References|[10]]]). <br />
This might be a sign for inteins having a role in post-translational protein regulation upon environmental signals. Inserted close to the catalytic core of an protein the intein deactivates the protein function until the splicing reaction has taken place. Evolutionary said, selfish inteins might have adapted due to positive selection pressure to provide a beneficial mechanism for the host cell, thereby becoming selfless [[#References|[8]]].<br />
<br />
==Structure and Classification==<br />
<br />
The structure of inteins contains several conserved motifs. The splicing domains are located at the N and C terminal ends. Before splicing takes place the intein rearranges itself from its initial linear structure into to a horseshoe like structure where the termini are brought in close proximity making up the catalytic core [[#References|[14]]]. <br />
Inteins are divided into three groups: bifunctional (large) inteins, mini inteins and split inteins.<br />
<br />
Large inteins carry both a splicing domain and an endonuclease (HEN) domain whereas mini inteins lack the HEN domain [[#References|[11]]]. <br />
The most promising inteins for biotechnology are split inteins, which are basically mini-inteins, just divided in two fragments and expressed separately connected different proteins. After translation, they assemble with high affinity to become catalytic active and perform a splicing reaction. Split enzymes occure naturally [[#References|[12]]] but can also be engineered artificially [[#References|[13]]]. <br />
<br />
In our project we focused on split inteins, as they present a powerful tool to insert posttranslational modifications, offering a plethora of applications in biotechnology. We have characterized the most promising inteins in our [[Team:Heidelberg/Parts#Intein_Library|Intein Library]].<br />
<br />
==A detailed description of the trans-splicing reaction==<br />
<br />
In contrast to the mini and large inteins that mediate cis-splicing reactions, split inteins are responsible for trans-splicing and fusion of protein parts. The trans-splicing reaction can be divided into the following steps:<br />
<br />
# N-Intein and C-Intein first assemble together to form a dimer like structure with a newly formed catalytic core next to the exteins.<br />
# The tertiary structure of the intein, once correctly formed, facilitates an ''N&rarr;O/S'' acyl rearrangement at its N-terminal serine or cysteine residue. The result is an ester or thioester bond, respectively between the side-chain and the peptide backbone of the N-extein.<br />
# The two exteins are then linked by trans(thio)esterification involving the N-terminal serine or cysteine residue of the C-extein. The C-terminus of the N-extein is now covalently bound to the N-terminal side-chain of the C-extein, while its backbone still retains its normal peptide bond to the intein.<br />
# The C-terminal asparagine of the intein undergoes self-cyclisation to form a succinimidyl moiety. The peptide bond between the intein and the extein is thereby broken, resolving the branched intermediate.<br />
# In a final reaction, an ''O/S&rarr;N'' acyl shift results in the two exteins now being linked by an amide bond, indistinguishable from a ribosome-assembled fusion protein.<br />
(as reviewed in [[#References|[15]]]) <br />
<br />
{{:Team:Heidelberg/templates/image-full|<br />
align=right|<br />
caption=Figure 2) Trans-splicing mechanism reaction by split inteins. |<br />
descr=Picture taken from [[#References|[16]]]|<br />
file=Trans_splicing_OCreaction}}<br />
<br />
For intein efficiency not only the structure of the intein domain itself is important but also the nature of the flanking extein residues, as they are heavily involved in the splicing reaction. It has been shown, that the first residue has to be a nucleophilic amino acid, preferable Cysteine, Serine or Threonine. Effectiveness of the intein can be mold by changing the these amino acids [[#References|[17]]].<br />
<br />
==Use of inteins in molecular biology and biotechnology==<br />
<br />
Due to their distinct characteristics intein are a powerful tool for molecular biology and biotechnology. Performing an autocatalytic reaction, the inteins are neither dependent on their host protein nor on any other additional substrate. This makes it them broadly applicable for in vivo and in vitro applications. [[#References|[16]]].<br />
<br />
Inteins have been used for protein purification, achieving a higher yield of protein due to more specific targeting. The production of semi-synthetic proteins and the attachment of synthetic groups is of huge interest for recombinant proteins. [[#References|[16]]]. Split Intein-mediated Circular Ligation Of Peptides a ProteinS (SICLOPPS) is a method to produce circular peptides of eight amino acids length, which could be potent therapeutic drugs [[#References|[18]]]. Inteins, fused to split fluorescent proteins as reporter and to the protein of investigation have been used to detect protein-protein interactions in vivo (reviewed in [[#References|[10]]]).<br />
Nevertheless most of this applications are performed in vitro and do not exploit the full potential of inteins as regulatory element for post-translational modification. The iGEM Team Heidelberg employs this excellent mechanism of protein splicing to specifically change whole amino-acid sequences and thereby regulate proteins via (dis-) assembly, protein cleavage or circularization of enzymes in vivo. All this and much more was embedded into our versatile intein toolbox. With this universal toolkit we provide a foundational advance for protein control - introducing the full potential of post-translational modification and therefore a new dimension of genetic engineering to Synthetic Biology. There is much more to [[Team:Heidelberg/Project|explore]]!<br />
<br />
=References=<br />
<br />
[1] Perler FB, Davis EO, Dean GE, Gimble FS, Jack WE, et al. (1994) Protein splicing elements: inteins and exteins–a definition of terms and recommended nomenclature. Nucleic Acids Res 22: 1125–1127<br />
<br />
[2] Kane P.M., et al. Protein Splicing Converts the Yeast TEPI Gene Product to the 69-kD Subunit of the Vacuolar. 13253, (1990)<br />
<br />
[3] Hirata, R. et al. Molecular structure of a gene, VMA1, encoding the catalytic subunit of H(+)-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J. Biol. Chem. 265 , 6726–6733 (1990)<br />
<br />
[4] Perler, F. B. InBase : the Intein Database. 30, 383–384 (2002)<br />
<br />
[5] Perler, F. B. et al. Protein splicing elements : inteins and exteins — a definition of terms and recommended nomenclature. 22, 1125–1127 (1994)<br />
<br />
[6] Barzel, A., Naor, A., Privman, E., Kupiec, M. & Gophna, U. Homing endonucleases residing within inteins: evolutionary puzzles awaiting genetic solutions. Biochem. Soc. Trans. 39, 169–73 (2011)<br />
<br />
[7] Pietrokovski, S. Intein spread and extinction in evolution. Trends Genet. 17, 465-472 (2001)<br />
<br />
[8] Novikova, O., Topilina, N. & Belfort, M. Enigmatic distribution, evolution and function of inteins. J. Biol. Chem. (2014)<br />
<br />
[9] Shah, N. H., and Muir, T. W. Inteins: nature's gift to protein chemists. Chem. Sci. 5, 446- 461 (2014) <br />
<br />
[10] Topilina, N. I. & Mills, K. V. Recent advances in in vivo applications of intein-mediated protein splicing. Mob. DNA 5, 5 (2014)<br />
<br />
[11] Eryilmaz, E., Shah, N., Muir, T. & Cowburn, D. Structural and Dynamical Features of Inteins and Implications on Protein Splicing. J. Biol. Chem. (2014)<br />
<br />
[12] Carvajal-Vallejos, P., Pallissé, R., Mootz, H. D. & Schmidt, S. R. Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. J. Biol. Chem. 287, 28686–96 (2012) <br />
<br />
[13] Lin, Y. et al. Protein trans-splicing of multiple atypical split inteins engineered from natural inteins. PLoS One 8, e59516 (2013). <br />
<br />
[14] Eryilmaz, E., Shah, N., Muir, T. & Cowburn, D. Structural and Dynamical Features of Inteins and Implications on Protein Splicing. J. Biol. Chem. (2014)<br />
<br />
[15] Mills, K. V, Johnson, M. a & Perler, F. B. Protein Splicing: How Inteins Escape from Precursor Proteins. J. Biol. Chem. (2014)<br />
<br />
[16] Mootz, H. D. Split inteins as versatile tools for protein semisynthesis. Chembiochem 10, 2579–89 (2009).<br />
<br />
[17] Amitai, G., Callahan, B. P., Stanger, M. J., Belfort, G. & Belfort, M. Modulation of intein activity by its neighboring extein substrates. Proc. Natl. Acad. Sci. U. S. A. 106, 11005–10 (2009).<br />
<br />
[18] Tavassoli, A. & Benkovic, S. J. Split-intein mediated circular ligation used in the synthesis of cyclic peptide libraries in E. coli. Nat. Protoc. 2, 1126–1133 (2007).</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/CollaborationsTeam:Heidelberg/pages/Collaborations2014-10-17T22:26:46Z<p>Huhny: </p>
<hr />
<div><br/><br />
<br />
Besides spending a whole summer in the lab and developing an own project, iGEM is about meeting new people, people with the same interests, the same motivation, the same dedication. Collaborations between team ensure the exchange of ideas, therefore helping the field of Synthetic Biology to be interdisciplinary and creative - the best conditions to give rise to revolutionary projects. We are very happy to met some of the team members from Freiburg, Aachen, Tuebingen, Marburg and London. <br />
<br />
=iGEM Team Aachen=<br />
<br />
<br />
Michael from the iGEM team Aachen visited us in Heidelberg. Their team worked on the development of a real-time pathogen detection technique for what they have build their own fluorescence measurement camera. We provided our expression vectors to the iGEM team Aachen, which they used to express their part [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1319003 E1010-K1319003], the human galectin-3 protein fused to mRFP. Nicely seen in Figure 1 and 2 the the intensity of the red color is significantly higher for the induced sample with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362091 pSBX1A30] compared to the uninduced ones. To ensure the right molecular mass of the fusion protein, a SDS gel was run as well (Figure 3).<br />
<br />
<div><br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=3) SDS-PAGE of K1319020 expression| file=AachenCo3.png}}<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=2) Expression of Galectin-3| file=AachenCo2.png}}<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=1) Expression of Galectin-3| file=AachenCo1.jpg}}<br />
</div><br />
<br />
More information about their project can be found on Aachens [https://2014.igem.org/Team:Aachen/Project/Gal3 Galectin-3 site].<br />
<br />
In exchange they offered to characterise these expression vectors with their own designed analyser by measuring the amount of produced fluorescent proteins. Unfortunately, transporting <i>E.colis</i> to another city wasn't as easy as expected, not all Bacteria were able to grow afterwards and we could only obtain sufficient data from pSBX4C5. <br />
<br />
<br/><br />
<br />
=iGEM Team Tuebingen=<br />
The project of iGEM Team Tuebingen is about blood types and the difficulties that arises during blood donation of the wrong blood type. They aim to develop an easy system to convert blood types A, B, AB to the rares but also most applicable blood type 0. They have applied the method of intein targeting, a method which can also be found in our intein toolbox, to the N-Acetyl-Galactosaminidase protein. More about their project can be read in the very nice [http://igem14-heidelberg.tumblr.com/post/98138990630/igem-team-tubingen article] they have wrote for our tumblr blog or on their own [[Team:Tuebingen|wiki]].<br />
<br />
In this collaboration, Tuebingen conducted a mass spectrometry analysis of our Lambda lysozyme to proof circularization. The data still has to be evaluated, but we will have them ready until the jamboree. There is a high demand for our expression vectors. Tuebingen had problems with expressing their constructs, therefore we send them some of our plasmids pSBX1K30 ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362093 BBa_K1362093]) and pSBX4K50 ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362097 BBa_K1362097]), as they showed to work really nicely even with big proteins like the [[Team:Heidelberg/Project/PCR2.0|Dnmt1]] with 105kDa.<br />
<br />
<br/><br />
<br />
=iGEM Team Freiburg=<br />
<br />
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<br />
Three of our team members, Anna, Caro, and Charlotte visited the iGEM team Freiburg. We met the team right after a big team meeting, therefore we were able to hear their latest project developments. We spend the evening talking about experiences in the lab and about iGEM. It was relieving to hear that they faced similar difficulties in the lab. <br />
The morning after we presented our project to the iGEM team Freiburg. Freiburg was very interested in our program iGEM@home as we offered them to promote their own human practice projects. You can also find an [http://igem14-heidelberg.tumblr.com/post/99936805780/optogenetics-the-lock-and-key-for-daily-life article] about optogenetics on our tumblr blog.<br />
Both our teams are working with light inducible systems, manly the LOV domain. Although working in different organisms, the constructs were compatible, therefore we could interchange protocols for induction and preliminary results. Furthermore we gazed at their professional light induction box - we can definitely improved ours. <br />
<br />
After cooperation with the iGEM Team Freiburg for two years in a row we are hoping to keep Freiburg as a reliable partner in the next years as well.<br />
<br />
<br/><br />
<br />
=iGEM Team Marburg=<br />
<br />
<br />
The screensaver of [[Team:Heidelberg/Human_Practice/igemathome|iGEM@home]] is a great possibility to spread news and information about Synthetic Biology around the world. Lately, even other iGEM team have used this new option and filled our screensaver with slides about their own project. Marburg was able to promote their quiz app about Synthetic Biology.</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/CollaborationsTeam:Heidelberg/pages/Collaborations2014-10-17T22:25:27Z<p>Huhny: </p>
<hr />
<div><br/><br />
<br />
Besides spending a whole summer in the lab and developing an own project, iGEM is about meeting new people, people with the same interests, the same motivation, the same dedication. Collaborations between team ensure the exchange of ideas, therefore helping the field of Synthetic Biology to be interdisciplinary and creative - the best conditions to give rise to revolutionary projects. We are very happy to met some of the team members from Freiburg, Aachen, Tuebingen, Marburg and London. <br />
<br />
=iGEM Team Aachen=<br />
<br />
<br/><br />
<br />
Michael from the iGEM team Aachen visited us in Heidelberg. Their team worked on the development of a real-time pathogen detection technique for what they have build their own fluorescence measurement camera. We provided our expression vectors to the iGEM team Aachen, which they used to express their part [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1319003 E1010-K1319003], the human galectin-3 protein fused to mRFP. Nicely seen in Figure 1 and 2 the the intensity of the red color is significantly higher for the induced sample with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362091 pSBX1A30] compared to the uninduced ones. To ensure the right molecular mass of the fusion protein, a SDS gel was run as well (Figure 3).<br />
<br />
<div><br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=3) SDS-PAGE of K1319020 expression| file=AachenCo3.png}}<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=2) Expression of Galectin-3| file=AachenCo2.png}}<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=1) Expression of Galectin-3| file=AachenCo1.jpg}}<br />
</div><br />
<br />
More information about their project can be found on Aachens [https://2014.igem.org/Team:Aachen/Project/Gal3 Galectin-3 site].<br />
<br />
In exchange they offered to characterise these expression vectors with their own designed analyser by measuring the amount of produced fluorescent proteins. Unfortunately, transporting <i>E.colis</i> to another city wasn't as easy as expected, not all Bacteria were able to grow afterwards and we could only obtain sufficient data from pSBX4C5. <br />
<br />
<br/><br />
<br />
=iGEM Team Tuebingen=<br />
The project of iGEM Team Tuebingen is about blood types and the difficulties that arises during blood donation of the wrong blood type. They aim to develop an easy system to convert blood types A, B, AB to the rares but also most applicable blood type 0. They have applied the method of intein targeting, a method which can also be found in our intein toolbox, to the N-Acetyl-Galactosaminidase protein. More about their project can be read in the very nice [http://igem14-heidelberg.tumblr.com/post/98138990630/igem-team-tubingen article] they have wrote for our tumblr blog or on their own [[Team:Tuebingen|wiki]].<br />
<br />
In this collaboration, Tuebingen conducted a mass spectrometry analysis of our Lambda lysozyme to proof circularization. The data still has to be evaluated, but we will have them ready until the jamboree. There is a high demand for our expression vectors. Tuebingen had problems with expressing their constructs, therefore we send them some of our plasmids pSBX1K30 ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362093 BBa_K1362093]) and pSBX4K50 ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362097 BBa_K1362097]), as they showed to work really nicely even with big proteins like the [[Team:Heidelberg/Project/PCR2.0|Dnmt1]] with 105kDa.<br />
<br />
=iGEM Team Freiburg=<br />
<br />
<br />
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}}<br />
<br />
Three of our team members, Anna, Caro, and Charlotte visited the iGEM team Freiburg. We met the team right after a big team meeting, therefore we were able to hear their latest project developments. We spend the evening talking about experiences in the lab and about iGEM. It was relieving to hear that they faced similar difficulties in the lab. <br />
The morning after we presented our project to the iGEM team Freiburg. Freiburg was very interested in our program iGEM@home as we offered them to promote their own human practice projects. You can also find an [http://igem14-heidelberg.tumblr.com/post/99936805780/optogenetics-the-lock-and-key-for-daily-life article] about optogenetics on our tumblr blog.<br />
Both our teams are working with light inducible systems, manly the LOV domain. Although working in different organisms, the constructs were compatible, therefore we could interchange protocols for induction and preliminary results. Furthermore we gazed at their professional light induction box - we can definitely improved ours. <br />
<br />
After cooperation with the iGEM Team Freiburg for two years in a row we are hoping to keep Freiburg as a reliable partner in the next years as well.<br />
<br />
==iGEM Team Marburg==<br />
<br />
<br />
The screensaver of [[Team:Heidelberg/Human_Practice/igemathome|iGEM@home]] is a great possibility to spread news and information about Synthetic Biology around the world. Lately, even other iGEM team have used this new option and filled our screensaver with slides about their own project. Marburg was able to promote their quiz app about Synthetic Biology.</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/CollaborationsTeam:Heidelberg/pages/Collaborations2014-10-17T22:17:49Z<p>Huhny: /* iGEM Team Tuebingen */</p>
<hr />
<div><br/><br />
<br />
Besides spending a whole summer in the lab and developing an own project, iGEM is about meeting new people, people with the same interests, the same motivation, the same dedication. Collaborations between team ensure the exchange of ideas, therefore helping the field of Synthetic Biology to be interdisciplinary and creative - the best conditions to give rise to revolutionary projects. We are very happy to met some of the team members from Freiburg, Aachen, Tuebingen, Marburg and London. <br />
<br />
=iGEM Team Aachen=<br />
<br />
<br/><br />
<br />
Michael from the iGEM team Aachen visited us in Heidelberg. Their team worked on the development of a real-time pathogen detection technique for what they have build their own fluorescence measurement camera. We provided our expression vectors to the iGEM team Aachen, which they used to express their part [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1319003 E1010-K1319003], the human galectin-3 protein fused to mRFP. Nicely seen in Figure 1 and 2 the the intensity of the red color is significantly higher for the induced sample with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362091 pSBX1A30] compared to the uninduced ones. To ensure the right molecular mass of the fusion protein, a SDS gel was run as well (Figure 3).<br />
<br />
<div><br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=3) SDS-PAGE of K1319020 expression| file=AachenCo3.png}}<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=2) Expression of Galectin-3| file=AachenCo2.png}}<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=1) Expression of Galectin-3| file=AachenCo1.jpg}}<br />
</div><br />
<br />
More information about their project can be found on Aachens [https://2014.igem.org/Team:Aachen/Project/Gal3 Galectin-3 site].<br />
<br />
In exchange they offered to characterise these expression vectors with their own designed analyser by measuring the amount of produced fluorescent proteins. Unfortunately, transporting <i>E.colis</i> to another city wasn't as easy as expected, not all Bacteria were able to grow afterwards and we could only obtain sufficient data from pSBX4C5. <br />
<br />
<br/><br />
<br />
=iGEM Team Tuebingen=<br />
The project of iGEM Team Tuebingen is about blood types and the difficulties that arises during blood donation of the wrong blood type. They aim to develop an easy system to convert blood types A, B, AB to the rares but also most applicable blood type 0. They have applied the method of intein targeting, a method which can also be found in our intein toolbox, to the N-Acetyl-Galactosaminidase protein. More about their project can be read in the very nice [http://igem14-heidelberg.tumblr.com/post/98138990630/igem-team-tubingen article] they have wrote for our tumblr blog or on their own [[Team:Tuebingen|wiki]].<br />
<br />
In this collaboration, Tuebingen conducted a mass spectrometry analysis of our Lambda lysozyme to proof circularization. The data still has to be evaluated, but we will have them ready until the jamboree. There is a high demand for our expression vectors. Tuebingen had problems with expressing their constructs, therefore we send them some of our plasmids pSBX1K30 ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362093 BBa_K1362093]) and pSBX4K50 ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362097 BBa_K1362097]), as they showed to work really nicely even with big proteins like the [[Team:Heidelberg/Project/PCR2.0|Dnmt1]] with 105kDa.<br />
<br />
=iGEM Team Freiburg=<br />
<br />
<br />
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}}<br />
<br />
Three of our team members, Anna, Caro, and Charlotte visited the iGEM team Freiburg. We met the team right after a big team meeting, therefore we were able to hear their latest project developments. We spend the evening talking about experiences in the lab and about iGEM. It was relieving to hear that they faced similar difficulties in the lab. <br />
The morning after we presented our project to the iGEM team Freiburg. Freiburg was very interested in our program iGEM@home as we offered them to promote their own human practice projects. You can also find an [http://igem14-heidelberg.tumblr.com/post/99936805780/optogenetics-the-lock-and-key-for-daily-life article] about optogenetics on our tumblr blog.<br />
Both our teams are working with light inducible systems, manly the LOV domain. Although working in different organisms, the constructs were compatible, therefore we could interchange protocols for induction and preliminary results. Furthermore we gazed at their professional light induction box - we can definitely improved ours. <br />
<br />
After cooperation with the iGEM Team Freiburg for two years in a row we are hoping to keep Freiburg as a reliable partner in the next years as well.</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/CollaborationsTeam:Heidelberg/pages/Collaborations2014-10-17T22:17:14Z<p>Huhny: /* iGEM Team Tuebingen */</p>
<hr />
<div><br/><br />
<br />
Besides spending a whole summer in the lab and developing an own project, iGEM is about meeting new people, people with the same interests, the same motivation, the same dedication. Collaborations between team ensure the exchange of ideas, therefore helping the field of Synthetic Biology to be interdisciplinary and creative - the best conditions to give rise to revolutionary projects. We are very happy to met some of the team members from Freiburg, Aachen, Tuebingen, Marburg and London. <br />
<br />
=iGEM Team Aachen=<br />
<br />
<br/><br />
<br />
Michael from the iGEM team Aachen visited us in Heidelberg. Their team worked on the development of a real-time pathogen detection technique for what they have build their own fluorescence measurement camera. We provided our expression vectors to the iGEM team Aachen, which they used to express their part [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1319003 E1010-K1319003], the human galectin-3 protein fused to mRFP. Nicely seen in Figure 1 and 2 the the intensity of the red color is significantly higher for the induced sample with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362091 pSBX1A30] compared to the uninduced ones. To ensure the right molecular mass of the fusion protein, a SDS gel was run as well (Figure 3).<br />
<br />
<div><br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=3) SDS-PAGE of K1319020 expression| file=AachenCo3.png}}<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=2) Expression of Galectin-3| file=AachenCo2.png}}<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=1) Expression of Galectin-3| file=AachenCo1.jpg}}<br />
</div><br />
<br />
More information about their project can be found on Aachens [https://2014.igem.org/Team:Aachen/Project/Gal3 Galectin-3 site].<br />
<br />
In exchange they offered to characterise these expression vectors with their own designed analyser by measuring the amount of produced fluorescent proteins. Unfortunately, transporting <i>E.colis</i> to another city wasn't as easy as expected, not all Bacteria were able to grow afterwards and we could only obtain sufficient data from pSBX4C5. <br />
<br />
<br/><br />
<br />
=iGEM Team Tuebingen=<br />
The project of iGEM Team Tuebingen is about blood types and the difficulties that arises during blood donation of the wrong blood type. They aim to develop an easy system to convert blood types A, B, AB to the rares but also most applicable blood type 0. They have applied the method of intein targeting, a method which can also be found in our intein toolbox, to the N-Acetyl-Galactosaminidase protein. More about their project can be read in the very nice [http://igem14-heidelberg.tumblr.com/post/98138990630/igem-team-tubingen article] they have wrote for our tumblr blog or on their own [[Team:Tuebingen|wiki]].<br />
<br />
In this collaboration, Tuebingen conducted a mass spectrometry analysis of our Lambda lysozyme to proof circularization. The data still has to be evaluated, but we will have them ready until the jamboree. There is a high demand for our expression vectors. Tuebingen had problems with expressing their constructs, therefore we send them some of our plasmids pSBX1K30 ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362093 BBa_K1362093]) and pSBX4K50 ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362097 BBa_K1362097]), as they showed to work really nicely even with big proteins like the [Team:Heidelberg/Project/PCR2.0 Dnmt1] with 105kDa.<br />
<br />
=iGEM Team Freiburg=<br />
<br />
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Three of our team members, Anna, Caro, and Charlotte visited the iGEM team Freiburg. We met the team right after a big team meeting, therefore we were able to hear their latest project developments. We spend the evening talking about experiences in the lab and about iGEM. It was relieving to hear that they faced similar difficulties in the lab. <br />
The morning after we presented our project to the iGEM team Freiburg. Freiburg was very interested in our program iGEM@home as we offered them to promote their own human practice projects. You can also find an [http://igem14-heidelberg.tumblr.com/post/99936805780/optogenetics-the-lock-and-key-for-daily-life article] about optogenetics on our tumblr blog.<br />
Both our teams are working with light inducible systems, manly the LOV domain. Although working in different organisms, the constructs were compatible, therefore we could interchange protocols for induction and preliminary results. Furthermore we gazed at their professional light induction box - we can definitely improved ours. <br />
<br />
After cooperation with the iGEM Team Freiburg for two years in a row we are hoping to keep Freiburg as a reliable partner in the next years as well.</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/backgroundTeam:Heidelberg/pages/background2014-10-17T22:16:05Z<p>Huhny: /* Use of inteins in molecular biology and biotechnology */</p>
<hr />
<div><br/><br />
In the variety of proteins that exist, inteins are clearly among the most mysterious.<br />
They behave in such a peculiarly way that scientists are still puzzle about their initial role in the host organisms and are even more astonished when discovering their endless seeming abilities.<br />
<br />
Inteins are integrated as extraneous polypeptide sequences into ordinary proteins. They do not contribute to the original protein function but perform an autocatalytic splicing reaction after protein translation. Analog to intron splicing on RNA level, this posttranslational modification was named protein splicing. Consequentially the protein segments were called inteins, derived from “internal protein sequence”, and the flanking protein chains exteins, “external protein sequences”. Inteins excise themselves out of the host protein while reconnecting the remaining N and C exteins via a new peptide bond. Despite this immense invasion the original protein regains its normal structure and function after splicing [[#References|[1]]]. <br />
<br />
<div><br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 1) Trans-splicing mechanism reaction by split inteins. |<br />
descr=The N-Intein is fused to the C’ terminal end of the N-extein. Complementary, C-Intein is located at the N’ end of the C-extein. After assembly of the two intein fragments, a splicing reaction takes place, where the intein removes itself from the precursor protein and simultaneously ligates the exteins via a peptide bond.|<br />
file=InteinSplicingReaction.png}}<br />
</div><br />
<br />
==History==<br />
<br />
Inteins were discovered in 1990 when dissimilarities between the mature protein sequence of the yeast vacuolar ATPase (Vma1) and its corresponding mRNA were investigated. Surprisingly the mature protein had a lower molecular weight than expected from the encoding sequence, indicating the loss of one part of the protein after translation. <br />
Indeed, a region of 454 amino acids was found to be translated and subsequently removed from the Vma1 protein [[#References|[2]]] [[#References|[3]]]. Since then, over 600 different inteins have been reported in all three domains of life as well as in viruses [[#References|[4]]]. <br />
<br />
Dependent on the organism they belong to, the intein’s name consists out of the genus and species abbreviation followed by the host gene [[#References|[5]]]. For example, the "golden standard" split-intein, NpuDnaE has derived from the DNA polymerase III (DnaE) in <i>Nostoc punctiforme</i> PCC73102 (<i>Npu</i>).<br />
<br />
==Evolution: Inteins as parasitic genes==<br />
Despite many years of research the initial role of inteins in their host organism remains still unclear.<br />
Many natural inteins contain a homing endonuclease (HEN) domain, an enzyme that cleaves DNA and introduces new sequences at homing sites via homologous recombination or reverse transcription. This characteristic led to the perception of the intein as selfish DNA sequence - a gene generating copies of itself without an advantage for the host organism [[#References|[6]]]. This allows horizontal gene transfer of inteins. [[#References|[7]]]. Data showing that 27% of the intein's host proteins are related to DNA metabolism and involved in DNA replication or repair is further supporting the "selfish gene" hypothesis. This ensures expression of the inteins and the HEN domain during DNA replication, where they can take advantage of the DNA replication and repair system for introducing changes in the DNA [[#References|[8]]].<br />
<br />
However recent work on conditional protein splicing has shown sensitivity of some inteins to redox state, temperature and small molecules (reviewed in [[#References|[9]]], [[#References|[10]]]). <br />
This might be a sign for inteins having a role in post-translational protein regulation upon environmental signals. Inserted close to the catalytic core of an protein the intein deactivates the protein function until the splicing reaction has taken place. Evolutionary said, selfish inteins might have adapted due to positive selection pressure to provide a beneficial mechanism for the host cell, thereby becoming selfless [[#References|[8]]].<br />
<br />
==Structure and Classification==<br />
<br />
The structure of inteins contains several conserved motifs. The splicing domains are located at the N and C terminal ends. Before splicing takes place the intein rearranges itself from its initial linear structure into to a horseshoe like structure where the termini are brought in close proximity making up the catalytic core [[#References|[14]]]. <br />
Inteins are divided into three groups: bifunctional (large) inteins, mini inteins and split inteins.<br />
<br />
Large inteins carry both a splicing domain and an endonuclease (HEN) domain whereas mini inteins lack the HEN domain [[#References|[11]]]. <br />
The most promising inteins for biotechnology are split inteins, which are basically mini-inteins, just divided in two fragments and expressed separately connected different proteins. After translation, they assemble with high affinity to become catalytic active and perform a splicing reaction. Split enzymes occure naturally [[#References|[12]]] but can also be engineered artificially [[#References|[13]]]. <br />
<br />
In our project we focused on split inteins, as they present a powerful tool to insert posttranslational modifications, offering a plethora of applications in biotechnology. We have characterized the most promising inteins in our [[Team:Heidelberg/Parts#Intein_Library|Intein Library]].<br />
<br />
==A detailed description of the trans-splicing reaction==<br />
<br />
In contrast to the mini and large inteins that mediate cis-splicing reactions, split inteins are responsible for trans-splicing and fusion of protein parts. The trans-splicing reaction can be divided into the following steps:<br />
<br />
# N-Intein and C-Intein first assemble together to form a dimer like structure with a newly formed catalytic core next to the exteins.<br />
# The tertiary structure of the intein, once correctly formed, facilitates an ''N&rarr;O/S'' acyl rearrangement at its N-terminal serine or cysteine residue. The result is an ester or thioester bond, respectively between the side-chain and the peptide backbone of the N-extein.<br />
# The two exteins are then linked by trans(thio)esterification involving the N-terminal serine or cysteine residue of the C-extein. The C-terminus of the N-extein is now covalently bound to the N-terminal side-chain of the C-extein, while its backbone still retains its normal peptide bond to the intein.<br />
# The C-terminal asparagine of the intein undergoes self-cyclisation to form a succinimidyl moiety. The peptide bond between the intein and the extein is thereby broken, resolving the branched intermediate.<br />
# In a final reaction, an ''O/S&rarr;N'' acyl shift results in the two exteins now being linked by an amide bond, indistinguishable from a ribosome-assembled fusion protein.<br />
(as reviewed in [[#References|[15]]]) <br />
<br />
{{:Team:Heidelberg/templates/image-full|<br />
align=right|<br />
caption=Figure 2) Trans-splicing mechanism reaction by split inteins. |<br />
descr=Picture taken from [[#References|[16]]]|<br />
file=Trans_splicing_OCreaction.PNG}}<br />
<br />
For intein efficiency not only the structure of the intein domain itself is important but also the nature of the flanking extein residues, as they are heavily involved in the splicing reaction. It has been shown, that the first residue has to be a nucleophilic amino acid, preferable Cysteine, Serine or Threonine. Effectiveness of the intein can be mold by changing the these amino acids [[#References|[17]]].<br />
<br />
==Use of inteins in molecular biology and biotechnology==<br />
<br />
Due to their distinct characteristics intein are a powerful tool for molecular biology and biotechnology. Performing an autocatalytic reaction, the inteins are neither dependent on their host protein nor on any other additional substrate. This makes it them broadly applicable for in vivo and in vitro applications. [[#References|[16]]].<br />
<br />
Inteins have been used for protein purification, achieving a higher yield of protein due to more specific targeting. The production of semi-synthetic proteins and the attachment of synthetic groups is of huge interest for recombinant proteins. [[#References|[16]]]. Split Intein-mediated Circular Ligation Of Peptides a ProteinS (SICLOPPS) is a method to produce circular peptides of eight amino acids length, which could be potent therapeutic drugs [[#References|[18]]]. Inteins, fused to split fluorescent proteins as reporter and to the protein of investigation have been used to detect protein-protein interactions in vivo (reviewed in [[#References|[10]]]).<br />
Nevertheless most of this applications are performed in vitro and do not exploit the full potential of inteins as regulatory element for post-translational modification. The iGEM Team Heidelberg employs this excellent mechanism of protein splicing to specifically change whole amino-acid sequences and thereby regulate proteins via (dis-) assembly, protein cleavage or circularization of enzymes in vivo. All this and much more was embedded into our versatile intein toolbox. With this universal toolkit we provide a foundational advance for protein control - introducing the full potential of post-translational modification and therefore a new dimension of genetic engineering to Synthetic Biology. There is much more to [[Team:Heidelberg/Project|explore]]!<br />
<br />
=References=<br />
<br />
[1] Perler FB, Davis EO, Dean GE, Gimble FS, Jack WE, et al. (1994) Protein splicing elements: inteins and exteins–a definition of terms and recommended nomenclature. Nucleic Acids Res 22: 1125–1127<br />
<br />
[2] Kane P.M., et al. Protein Splicing Converts the Yeast TEPI Gene Product to the 69-kD Subunit of the Vacuolar. 13253, (1990)<br />
<br />
[3] Hirata, R. et al. Molecular structure of a gene, VMA1, encoding the catalytic subunit of H(+)-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J. Biol. Chem. 265 , 6726–6733 (1990)<br />
<br />
[4] Perler, F. B. InBase : the Intein Database. 30, 383–384 (2002)<br />
<br />
[5] Perler, F. B. et al. Protein splicing elements : inteins and exteins — a definition of terms and recommended nomenclature. 22, 1125–1127 (1994)<br />
<br />
[6] Barzel, A., Naor, A., Privman, E., Kupiec, M. & Gophna, U. Homing endonucleases residing within inteins: evolutionary puzzles awaiting genetic solutions. Biochem. Soc. Trans. 39, 169–73 (2011)<br />
<br />
[7] Pietrokovski, S. Intein spread and extinction in evolution. Trends Genet. 17, 465-472 (2001)<br />
<br />
[8] Novikova, O., Topilina, N. & Belfort, M. Enigmatic distribution, evolution and function of inteins. J. Biol. Chem. (2014)<br />
<br />
[9] Shah, N. H., and Muir, T. W. Inteins: nature's gift to protein chemists. Chem. Sci. 5, 446- 461 (2014) <br />
<br />
[10] Topilina, N. I. & Mills, K. V. Recent advances in in vivo applications of intein-mediated protein splicing. Mob. DNA 5, 5 (2014)<br />
<br />
[11] Eryilmaz, E., Shah, N., Muir, T. & Cowburn, D. Structural and Dynamical Features of Inteins and Implications on Protein Splicing. J. Biol. Chem. (2014)<br />
<br />
[12] Carvajal-Vallejos, P., Pallissé, R., Mootz, H. D. & Schmidt, S. R. Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. J. Biol. Chem. 287, 28686–96 (2012) <br />
<br />
[13] Lin, Y. et al. Protein trans-splicing of multiple atypical split inteins engineered from natural inteins. PLoS One 8, e59516 (2013). <br />
<br />
[14] Eryilmaz, E., Shah, N., Muir, T. & Cowburn, D. Structural and Dynamical Features of Inteins and Implications on Protein Splicing. J. Biol. Chem. (2014)<br />
<br />
[15] Mills, K. V, Johnson, M. a & Perler, F. B. Protein Splicing: How Inteins Escape from Precursor Proteins. J. Biol. Chem. (2014)<br />
<br />
[16] Mootz, H. D. Split inteins as versatile tools for protein semisynthesis. Chembiochem 10, 2579–89 (2009).<br />
<br />
[17] Amitai, G., Callahan, B. P., Stanger, M. J., Belfort, G. & Belfort, M. Modulation of intein activity by its neighboring extein substrates. Proc. Natl. Acad. Sci. U. S. A. 106, 11005–10 (2009).<br />
<br />
[18] Tavassoli, A. & Benkovic, S. J. Split-intein mediated circular ligation used in the synthesis of cyclic peptide libraries in E. coli. Nat. Protoc. 2, 1126–1133 (2007).</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/backgroundTeam:Heidelberg/pages/background2014-10-17T22:15:47Z<p>Huhny: /* Structure and Classification */</p>
<hr />
<div><br/><br />
In the variety of proteins that exist, inteins are clearly among the most mysterious.<br />
They behave in such a peculiarly way that scientists are still puzzle about their initial role in the host organisms and are even more astonished when discovering their endless seeming abilities.<br />
<br />
Inteins are integrated as extraneous polypeptide sequences into ordinary proteins. They do not contribute to the original protein function but perform an autocatalytic splicing reaction after protein translation. Analog to intron splicing on RNA level, this posttranslational modification was named protein splicing. Consequentially the protein segments were called inteins, derived from “internal protein sequence”, and the flanking protein chains exteins, “external protein sequences”. Inteins excise themselves out of the host protein while reconnecting the remaining N and C exteins via a new peptide bond. Despite this immense invasion the original protein regains its normal structure and function after splicing [[#References|[1]]]. <br />
<br />
<div><br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 1) Trans-splicing mechanism reaction by split inteins. |<br />
descr=The N-Intein is fused to the C’ terminal end of the N-extein. Complementary, C-Intein is located at the N’ end of the C-extein. After assembly of the two intein fragments, a splicing reaction takes place, where the intein removes itself from the precursor protein and simultaneously ligates the exteins via a peptide bond.|<br />
file=InteinSplicingReaction.png}}<br />
</div><br />
<br />
==History==<br />
<br />
Inteins were discovered in 1990 when dissimilarities between the mature protein sequence of the yeast vacuolar ATPase (Vma1) and its corresponding mRNA were investigated. Surprisingly the mature protein had a lower molecular weight than expected from the encoding sequence, indicating the loss of one part of the protein after translation. <br />
Indeed, a region of 454 amino acids was found to be translated and subsequently removed from the Vma1 protein [[#References|[2]]] [[#References|[3]]]. Since then, over 600 different inteins have been reported in all three domains of life as well as in viruses [[#References|[4]]]. <br />
<br />
Dependent on the organism they belong to, the intein’s name consists out of the genus and species abbreviation followed by the host gene [[#References|[5]]]. For example, the "golden standard" split-intein, NpuDnaE has derived from the DNA polymerase III (DnaE) in <i>Nostoc punctiforme</i> PCC73102 (<i>Npu</i>).<br />
<br />
==Evolution: Inteins as parasitic genes==<br />
Despite many years of research the initial role of inteins in their host organism remains still unclear.<br />
Many natural inteins contain a homing endonuclease (HEN) domain, an enzyme that cleaves DNA and introduces new sequences at homing sites via homologous recombination or reverse transcription. This characteristic led to the perception of the intein as selfish DNA sequence - a gene generating copies of itself without an advantage for the host organism [[#References|[6]]]. This allows horizontal gene transfer of inteins. [[#References|[7]]]. Data showing that 27% of the intein's host proteins are related to DNA metabolism and involved in DNA replication or repair is further supporting the "selfish gene" hypothesis. This ensures expression of the inteins and the HEN domain during DNA replication, where they can take advantage of the DNA replication and repair system for introducing changes in the DNA [[#References|[8]]].<br />
<br />
However recent work on conditional protein splicing has shown sensitivity of some inteins to redox state, temperature and small molecules (reviewed in [[#References|[9]]], [[#References|[10]]]). <br />
This might be a sign for inteins having a role in post-translational protein regulation upon environmental signals. Inserted close to the catalytic core of an protein the intein deactivates the protein function until the splicing reaction has taken place. Evolutionary said, selfish inteins might have adapted due to positive selection pressure to provide a beneficial mechanism for the host cell, thereby becoming selfless [[#References|[8]]].<br />
<br />
==Structure and Classification==<br />
<br />
The structure of inteins contains several conserved motifs. The splicing domains are located at the N and C terminal ends. Before splicing takes place the intein rearranges itself from its initial linear structure into to a horseshoe like structure where the termini are brought in close proximity making up the catalytic core [[#References|[14]]]. <br />
Inteins are divided into three groups: bifunctional (large) inteins, mini inteins and split inteins.<br />
<br />
Large inteins carry both a splicing domain and an endonuclease (HEN) domain whereas mini inteins lack the HEN domain [[#References|[11]]]. <br />
The most promising inteins for biotechnology are split inteins, which are basically mini-inteins, just divided in two fragments and expressed separately connected different proteins. After translation, they assemble with high affinity to become catalytic active and perform a splicing reaction. Split enzymes occure naturally [[#References|[12]]] but can also be engineered artificially [[#References|[13]]]. <br />
<br />
In our project we focused on split inteins, as they present a powerful tool to insert posttranslational modifications, offering a plethora of applications in biotechnology. We have characterized the most promising inteins in our [[Team:Heidelberg/Parts#Intein_Library|Intein Library]].<br />
<br />
==A detailed description of the trans-splicing reaction==<br />
<br />
In contrast to the mini and large inteins that mediate cis-splicing reactions, split inteins are responsible for trans-splicing and fusion of protein parts. The trans-splicing reaction can be divided into the following steps:<br />
<br />
# N-Intein and C-Intein first assemble together to form a dimer like structure with a newly formed catalytic core next to the exteins.<br />
# The tertiary structure of the intein, once correctly formed, facilitates an ''N&rarr;O/S'' acyl rearrangement at its N-terminal serine or cysteine residue. The result is an ester or thioester bond, respectively between the side-chain and the peptide backbone of the N-extein.<br />
# The two exteins are then linked by trans(thio)esterification involving the N-terminal serine or cysteine residue of the C-extein. The C-terminus of the N-extein is now covalently bound to the N-terminal side-chain of the C-extein, while its backbone still retains its normal peptide bond to the intein.<br />
# The C-terminal asparagine of the intein undergoes self-cyclisation to form a succinimidyl moiety. The peptide bond between the intein and the extein is thereby broken, resolving the branched intermediate.<br />
# In a final reaction, an ''O/S&rarr;N'' acyl shift results in the two exteins now being linked by an amide bond, indistinguishable from a ribosome-assembled fusion protein.<br />
(as reviewed in [[#References|[15]]]) <br />
<br />
{{:Team:Heidelberg/templates/image-full|<br />
align=right|<br />
caption=Figure 2) Trans-splicing mechanism reaction by split inteins. |<br />
descr=Picture taken from [[#References|[16]]]|<br />
file=Trans_splicing_OCreaction.PNG}}<br />
<br />
For intein efficiency not only the structure of the intein domain itself is important but also the nature of the flanking extein residues, as they are heavily involved in the splicing reaction. It has been shown, that the first residue has to be a nucleophilic amino acid, preferable Cysteine, Serine or Threonine. Effectiveness of the intein can be mold by changing the these amino acids [[#References|[17]]].<br />
<br />
==Use of inteins in molecular biology and biotechnology==<br />
<br />
Due to their distinct characteristics intein are a powerful tool for molecular biology and biotechnology. Performing an autocatalytic reaction, the inteins are neither dependent on their host protein nor on any other additional substrate. This makes it them broadly applicable for in vivo and in vitro applications. [[#References|[16]]].<br />
<br />
Inteins have been used for protein purification, achieving a higher yield of protein due to more specific targeting. The production of semi-synthetic proteins and the attachment of synthetic groups is of huge interest for recombinant proteins. [[#References|[16]]]. Split Intein-mediated Circular Ligation Of Peptides a ProteinS (SICLOPPS) is a method to produce circular peptides of eight amino acids length, which could be potent therapeutic drugs [[#References|[18]]]. Inteins, fused to split fluorescent proteins as reporter and to the protein of investigation have been used to detect protein-protein interactions in vivo (reviewed in [[#References|[10]]]).<br />
Nevertheless most of this applications are performed in vitro and do not exploit the full potential of inteins as regulatory element for post-translational modification. The iGEM Team Heidelberg employs this excellent mechanism of protein splicing to specifically change whole amino-acid sequences and thereby regulate proteins via (dis-) assembly, protein cleavage or circularization of enzymes in vivo. All this and much more was embedded into our versatile intein toolbox. With this universal toolkit we provide a foundational advance for protein control - introducing the full potential of post-translational modification and therefore a new dimension of genetic engineering to Synthetic Biology. There is much more to [[Team:Heidelberg/Project explore]]!<br />
<br />
=References=<br />
<br />
[1] Perler FB, Davis EO, Dean GE, Gimble FS, Jack WE, et al. (1994) Protein splicing elements: inteins and exteins–a definition of terms and recommended nomenclature. Nucleic Acids Res 22: 1125–1127<br />
<br />
[2] Kane P.M., et al. Protein Splicing Converts the Yeast TEPI Gene Product to the 69-kD Subunit of the Vacuolar. 13253, (1990)<br />
<br />
[3] Hirata, R. et al. Molecular structure of a gene, VMA1, encoding the catalytic subunit of H(+)-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J. Biol. Chem. 265 , 6726–6733 (1990)<br />
<br />
[4] Perler, F. B. InBase : the Intein Database. 30, 383–384 (2002)<br />
<br />
[5] Perler, F. B. et al. Protein splicing elements : inteins and exteins — a definition of terms and recommended nomenclature. 22, 1125–1127 (1994)<br />
<br />
[6] Barzel, A., Naor, A., Privman, E., Kupiec, M. & Gophna, U. Homing endonucleases residing within inteins: evolutionary puzzles awaiting genetic solutions. Biochem. Soc. Trans. 39, 169–73 (2011)<br />
<br />
[7] Pietrokovski, S. Intein spread and extinction in evolution. Trends Genet. 17, 465-472 (2001)<br />
<br />
[8] Novikova, O., Topilina, N. & Belfort, M. Enigmatic distribution, evolution and function of inteins. J. Biol. Chem. (2014)<br />
<br />
[9] Shah, N. H., and Muir, T. W. Inteins: nature's gift to protein chemists. Chem. Sci. 5, 446- 461 (2014) <br />
<br />
[10] Topilina, N. I. & Mills, K. V. Recent advances in in vivo applications of intein-mediated protein splicing. Mob. DNA 5, 5 (2014)<br />
<br />
[11] Eryilmaz, E., Shah, N., Muir, T. & Cowburn, D. Structural and Dynamical Features of Inteins and Implications on Protein Splicing. J. Biol. Chem. (2014)<br />
<br />
[12] Carvajal-Vallejos, P., Pallissé, R., Mootz, H. D. & Schmidt, S. R. Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. J. Biol. Chem. 287, 28686–96 (2012) <br />
<br />
[13] Lin, Y. et al. Protein trans-splicing of multiple atypical split inteins engineered from natural inteins. PLoS One 8, e59516 (2013). <br />
<br />
[14] Eryilmaz, E., Shah, N., Muir, T. & Cowburn, D. Structural and Dynamical Features of Inteins and Implications on Protein Splicing. J. Biol. Chem. (2014)<br />
<br />
[15] Mills, K. V, Johnson, M. a & Perler, F. B. Protein Splicing: How Inteins Escape from Precursor Proteins. J. Biol. Chem. (2014)<br />
<br />
[16] Mootz, H. D. Split inteins as versatile tools for protein semisynthesis. Chembiochem 10, 2579–89 (2009).<br />
<br />
[17] Amitai, G., Callahan, B. P., Stanger, M. J., Belfort, G. & Belfort, M. Modulation of intein activity by its neighboring extein substrates. Proc. Natl. Acad. Sci. U. S. A. 106, 11005–10 (2009).<br />
<br />
[18] Tavassoli, A. & Benkovic, S. J. Split-intein mediated circular ligation used in the synthesis of cyclic peptide libraries in E. coli. Nat. Protoc. 2, 1126–1133 (2007).</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/backgroundTeam:Heidelberg/pages/background2014-10-17T22:13:12Z<p>Huhny: </p>
<hr />
<div><br/><br />
In the variety of proteins that exist, inteins are clearly among the most mysterious.<br />
They behave in such a peculiarly way that scientists are still puzzle about their initial role in the host organisms and are even more astonished when discovering their endless seeming abilities.<br />
<br />
Inteins are integrated as extraneous polypeptide sequences into ordinary proteins. They do not contribute to the original protein function but perform an autocatalytic splicing reaction after protein translation. Analog to intron splicing on RNA level, this posttranslational modification was named protein splicing. Consequentially the protein segments were called inteins, derived from “internal protein sequence”, and the flanking protein chains exteins, “external protein sequences”. Inteins excise themselves out of the host protein while reconnecting the remaining N and C exteins via a new peptide bond. Despite this immense invasion the original protein regains its normal structure and function after splicing [[#References|[1]]]. <br />
<br />
<div><br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 1) Trans-splicing mechanism reaction by split inteins. |<br />
descr=The N-Intein is fused to the C’ terminal end of the N-extein. Complementary, C-Intein is located at the N’ end of the C-extein. After assembly of the two intein fragments, a splicing reaction takes place, where the intein removes itself from the precursor protein and simultaneously ligates the exteins via a peptide bond.|<br />
file=InteinSplicingReaction.png}}<br />
</div><br />
<br />
==History==<br />
<br />
Inteins were discovered in 1990 when dissimilarities between the mature protein sequence of the yeast vacuolar ATPase (Vma1) and its corresponding mRNA were investigated. Surprisingly the mature protein had a lower molecular weight than expected from the encoding sequence, indicating the loss of one part of the protein after translation. <br />
Indeed, a region of 454 amino acids was found to be translated and subsequently removed from the Vma1 protein [[#References|[2]]] [[#References|[3]]]. Since then, over 600 different inteins have been reported in all three domains of life as well as in viruses [[#References|[4]]]. <br />
<br />
Dependent on the organism they belong to, the intein’s name consists out of the genus and species abbreviation followed by the host gene [[#References|[5]]]. For example, the "golden standard" split-intein, NpuDnaE has derived from the DNA polymerase III (DnaE) in <i>Nostoc punctiforme</i> PCC73102 (<i>Npu</i>).<br />
<br />
==Evolution: Inteins as parasitic genes==<br />
Despite many years of research the initial role of inteins in their host organism remains still unclear.<br />
Many natural inteins contain a homing endonuclease (HEN) domain, an enzyme that cleaves DNA and introduces new sequences at homing sites via homologous recombination or reverse transcription. This characteristic led to the perception of the intein as selfish DNA sequence - a gene generating copies of itself without an advantage for the host organism [[#References|[6]]]. This allows horizontal gene transfer of inteins. [[#References|[7]]]. Data showing that 27% of the intein's host proteins are related to DNA metabolism and involved in DNA replication or repair is further supporting the "selfish gene" hypothesis. This ensures expression of the inteins and the HEN domain during DNA replication, where they can take advantage of the DNA replication and repair system for introducing changes in the DNA [[#References|[8]]].<br />
<br />
However recent work on conditional protein splicing has shown sensitivity of some inteins to redox state, temperature and small molecules (reviewed in [[#References|[9]]], [[#References|[10]]]). <br />
This might be a sign for inteins having a role in post-translational protein regulation upon environmental signals. Inserted close to the catalytic core of an protein the intein deactivates the protein function until the splicing reaction has taken place. Evolutionary said, selfish inteins might have adapted due to positive selection pressure to provide a beneficial mechanism for the host cell, thereby becoming selfless [[#References|[8]]].<br />
<br />
==Structure and Classification==<br />
<br />
The structure of inteins contains several conserved motifs. The splicing domains are located at the N and C terminal ends. Before splicing takes place the intein rearranges itself from its initial linear structure into to a horseshoe like structure where the termini are brought in close proximity making up the catalytic core [[#References|[14]]]. <br />
Inteins are divided into three groups: bifunctional (large) inteins, mini inteins and split inteins.<br />
<br />
Large inteins carry both a splicing domain and an endonuclease (HEN) domain whereas mini inteins lack the HEN domain [[#References|[11]]]. <br />
The most promising inteins for biotechnology are split inteins, which are basically mini-inteins, just divided in two fragments and expressed separately connected different proteins. After translation, they assemble with high affinity to become catalytic active and perform a splicing reaction. Split enzymes occure naturally [[#References|[12]]] but can also be engineered artificially [[#References|[13]]]. <br />
<br />
In our project we focused on split inteins, as they present a powerful tool to insert posttranslational modifications, offering a plethora of applications in biotechnology. We have characterized the most promising inteins in our [[Team:Heidelberg/Parts#Intein Library Intein Library]].<br />
<br />
<br />
<br />
==A detailed description of the trans-splicing reaction==<br />
<br />
In contrast to the mini and large inteins that mediate cis-splicing reactions, split inteins are responsible for trans-splicing and fusion of protein parts. The trans-splicing reaction can be divided into the following steps:<br />
<br />
# N-Intein and C-Intein first assemble together to form a dimer like structure with a newly formed catalytic core next to the exteins.<br />
# The tertiary structure of the intein, once correctly formed, facilitates an ''N&rarr;O/S'' acyl rearrangement at its N-terminal serine or cysteine residue. The result is an ester or thioester bond, respectively between the side-chain and the peptide backbone of the N-extein.<br />
# The two exteins are then linked by trans(thio)esterification involving the N-terminal serine or cysteine residue of the C-extein. The C-terminus of the N-extein is now covalently bound to the N-terminal side-chain of the C-extein, while its backbone still retains its normal peptide bond to the intein.<br />
# The C-terminal asparagine of the intein undergoes self-cyclisation to form a succinimidyl moiety. The peptide bond between the intein and the extein is thereby broken, resolving the branched intermediate.<br />
# In a final reaction, an ''O/S&rarr;N'' acyl shift results in the two exteins now being linked by an amide bond, indistinguishable from a ribosome-assembled fusion protein.<br />
(as reviewed in [[#References|[15]]]) <br />
<br />
{{:Team:Heidelberg/templates/image-full|<br />
align=right|<br />
caption=Figure 2) Trans-splicing mechanism reaction by split inteins. |<br />
descr=Picture taken from [[#References|[16]]]|<br />
file=Trans_splicing_OCreaction.PNG}}<br />
<br />
For intein efficiency not only the structure of the intein domain itself is important but also the nature of the flanking extein residues, as they are heavily involved in the splicing reaction. It has been shown, that the first residue has to be a nucleophilic amino acid, preferable Cysteine, Serine or Threonine. Effectiveness of the intein can be mold by changing the these amino acids [[#References|[17]]].<br />
<br />
==Use of inteins in molecular biology and biotechnology==<br />
<br />
Due to their distinct characteristics intein are a powerful tool for molecular biology and biotechnology. Performing an autocatalytic reaction, the inteins are neither dependent on their host protein nor on any other additional substrate. This makes it them broadly applicable for in vivo and in vitro applications. [[#References|[16]]].<br />
<br />
Inteins have been used for protein purification, achieving a higher yield of protein due to more specific targeting. The production of semi-synthetic proteins and the attachment of synthetic groups is of huge interest for recombinant proteins. [[#References|[16]]]. Split Intein-mediated Circular Ligation Of Peptides a ProteinS (SICLOPPS) is a method to produce circular peptides of eight amino acids length, which could be potent therapeutic drugs [[#References|[18]]]. Inteins, fused to split fluorescent proteins as reporter and to the protein of investigation have been used to detect protein-protein interactions in vivo (reviewed in [[#References|[10]]]).<br />
Nevertheless most of this applications are performed in vitro and do not exploit the full potential of inteins as regulatory element for post-translational modification. The iGEM Team Heidelberg employs this excellent mechanism of protein splicing to specifically change whole amino-acid sequences and thereby regulate proteins via (dis-) assembly, protein cleavage or circularization of enzymes in vivo. All this and much more was embedded into our versatile intein toolbox. With this universal toolkit we provide a foundational advance for protein control - introducing the full potential of post-translational modification and therefore a new dimension of genetic engineering to Synthetic Biology. There is much more to [[Team:Heidelberg/Project explore]]!<br />
<br />
=References=<br />
<br />
[1] Perler FB, Davis EO, Dean GE, Gimble FS, Jack WE, et al. (1994) Protein splicing elements: inteins and exteins–a definition of terms and recommended nomenclature. Nucleic Acids Res 22: 1125–1127<br />
<br />
[2] Kane P.M., et al. Protein Splicing Converts the Yeast TEPI Gene Product to the 69-kD Subunit of the Vacuolar. 13253, (1990)<br />
<br />
[3] Hirata, R. et al. Molecular structure of a gene, VMA1, encoding the catalytic subunit of H(+)-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J. Biol. Chem. 265 , 6726–6733 (1990)<br />
<br />
[4] Perler, F. B. InBase : the Intein Database. 30, 383–384 (2002)<br />
<br />
[5] Perler, F. B. et al. Protein splicing elements : inteins and exteins — a definition of terms and recommended nomenclature. 22, 1125–1127 (1994)<br />
<br />
[6] Barzel, A., Naor, A., Privman, E., Kupiec, M. & Gophna, U. Homing endonucleases residing within inteins: evolutionary puzzles awaiting genetic solutions. Biochem. Soc. Trans. 39, 169–73 (2011)<br />
<br />
[7] Pietrokovski, S. Intein spread and extinction in evolution. Trends Genet. 17, 465-472 (2001)<br />
<br />
[8] Novikova, O., Topilina, N. & Belfort, M. Enigmatic distribution, evolution and function of inteins. J. Biol. Chem. (2014)<br />
<br />
[9] Shah, N. H., and Muir, T. W. Inteins: nature's gift to protein chemists. Chem. Sci. 5, 446- 461 (2014) <br />
<br />
[10] Topilina, N. I. & Mills, K. V. Recent advances in in vivo applications of intein-mediated protein splicing. Mob. DNA 5, 5 (2014)<br />
<br />
[11] Eryilmaz, E., Shah, N., Muir, T. & Cowburn, D. Structural and Dynamical Features of Inteins and Implications on Protein Splicing. J. Biol. Chem. (2014)<br />
<br />
[12] Carvajal-Vallejos, P., Pallissé, R., Mootz, H. D. & Schmidt, S. R. Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. J. Biol. Chem. 287, 28686–96 (2012) <br />
<br />
[13] Lin, Y. et al. Protein trans-splicing of multiple atypical split inteins engineered from natural inteins. PLoS One 8, e59516 (2013). <br />
<br />
[14] Eryilmaz, E., Shah, N., Muir, T. & Cowburn, D. Structural and Dynamical Features of Inteins and Implications on Protein Splicing. J. Biol. Chem. (2014)<br />
<br />
[15] Mills, K. V, Johnson, M. a & Perler, F. B. Protein Splicing: How Inteins Escape from Precursor Proteins. J. Biol. Chem. (2014)<br />
<br />
[16] Mootz, H. D. Split inteins as versatile tools for protein semisynthesis. Chembiochem 10, 2579–89 (2009).<br />
<br />
[17] Amitai, G., Callahan, B. P., Stanger, M. J., Belfort, G. & Belfort, M. Modulation of intein activity by its neighboring extein substrates. Proc. Natl. Acad. Sci. U. S. A. 106, 11005–10 (2009).<br />
<br />
[18] Tavassoli, A. & Benkovic, S. J. Split-intein mediated circular ligation used in the synthesis of cyclic peptide libraries in E. coli. Nat. Protoc. 2, 1126–1133 (2007).</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/backgroundTeam:Heidelberg/pages/background2014-10-17T22:10:31Z<p>Huhny: </p>
<hr />
<div><br/><br />
In the variety of proteins that exist, inteins are clearly among the most mysterious.<br />
They behave in such a peculiarly way that scientists are still puzzle about their initial role in the host organisms and are even more astonished when discovering their endless seeming abilities.<br />
<br />
Inteins are integrated as extraneous polypeptide sequences into ordinary proteins. They do not contribute to the original protein function but perform an autocatalytic splicing reaction after protein translation. Analog to intron splicing on RNA level, this posttranslational modification was named protein splicing. Consequentially the protein segments were called inteins, derived from “internal protein sequence”, and the flanking protein chains exteins, “external protein sequences”. Inteins excise themselves out of the host protein while reconnecting the remaining N and C exteins via a new peptide bond. Despite this immense invasion the original protein regains its normal structure and function after splicing [[#References|[1]]]. <br />
<br />
<div><br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 1) Trans-splicing mechanism reaction by split inteins. |<br />
descr=The N-Intein is fused to the C’ terminal end of the N-extein. Complementary, C-Intein is located at the N’ end of the C-extein. After assembly of the two intein fragments, a splicing reaction takes place, where the intein removes itself from the precursor protein and simultaneously ligates the exteins via a peptide bond.|<br />
file=InteinSplicingReaction.png}}<br />
</div><br />
<br />
==History==<br />
<br />
Inteins were discovered in 1990 when dissimilarities between the mature protein sequence of the yeast vacuolar ATPase (Vma1) and its corresponding mRNA were investigated. Surprisingly the mature protein had a lower molecular weight than expected from the encoding sequence, indicating the loss of one part of the protein after translation. <br />
Indeed, a region of 454 amino acids was found to be translated and subsequently removed from the Vma1 protein [[#References|[2]]] [[#References|[3]]]. Since then, over 600 different inteins have been reported in all three domains of life as well as in viruses [[#References|[4]]]. <br />
<br />
Dependent on the organism they belong to, the intein’s name consists out of the genus and species abbreviation followed by the host gene [[#References|[5]]]. For example, the "golden standard" split-intein, NpuDnaE has derived from the DNA polymerase III (DnaE) in <i>Nostoc punctiforme</i> PCC73102 (<i>Npu</i>).<br />
<br />
==Evolution: Inteins as parasitic genes==<br />
Despite many years of research the initial role of inteins in their host organism remains still unclear.<br />
Many natural inteins contain a homing endonuclease (HEN) domain, an enzyme that cleaves DNA and introduces new sequences at homing sites via homologous recombination or reverse transcription. This characteristic led to the perception of the intein as selfish DNA sequence - a gene generating copies of itself without an advantage for the host organism [[#References|[6]]]. This allows horizontal gene transfer of inteins. [[#References|[7]]]. Data showing that 27% of the intein's host proteins are related to DNA metabolism and involved in DNA replication or repair is further supporting the "selfish gene" hypothesis. This ensures expression of the inteins and the HEN domain during DNA replication, where they can take advantage of the DNA replication and repair system for introducing changes in the DNA [[#References|[8]]].<br />
<br />
However recent work on conditional protein splicing has shown sensitivity of some inteins to redox state, temperature and small molecules (reviewed in [[#References|[9]]], [[#References|[10]]]). <br />
This might be a sign for inteins having a role in post-translational protein regulation upon environmental signals. Inserted close to the catalytic core of an protein the intein deactivates the protein function until the splicing reaction has taken place. Evolutionary said, selfish inteins might have adapted due to positive selection pressure to provide a beneficial mechanism for the host cell, thereby becoming selfless [[#References|[8]]].<br />
<br />
==Structure and Classification==<br />
<br />
The structure of inteins contains several conserved motifs. The splicing domains are located at the N and C terminal ends. Before splicing takes place the intein rearranges itself from its initial linear structure into to a horseshoe like structure where the termini are brought in close proximity making up the catalytic core [[#References|[14]]]. <br />
Inteins are divided into three groups: bifunctional (large) inteins, mini inteins and split inteins.<br />
<br />
Large inteins carry both a splicing domain and an endonuclease (HEN) domain whereas mini inteins lack the HEN domain [[#References|[11]]]. <br />
The most promising inteins for biotechnology are split inteins, which are basically mini-inteins, just divided in two fragments and expressed separately connected different proteins. After translation, they assemble with high affinity to become catalytic active and perform a splicing reaction. Split enzymes occure naturally [[#References|[12]]] but can also be engineered artificially [[#References|[13]]]. <br />
<br />
In our project we focused on split inteins, as they present a powerful tool to insert posttranslational modifications, offering a plethora of applications in biotechnology. We have characterized the most promising inteins in our [Team:Heidelberg/Parts#Intein Library Intein Library].<br />
<br />
<br />
<br />
==A detailed description of trans-splicing reaction==<br />
<br />
In contrast to the mini and large inteins that mediate cis-splicing reactions, split inteins are responsible for trans-splicing and fusion of protein parts. The trans-splicing reaction can be divided into the following steps:<br />
<br />
# N-Intein and C-Intein first assemble together to form a dimer like structure with a newly formed catalytic core next to the exteins.<br />
# The tertiary structure of the intein, once correctly formed, facilitates an ''N&rarr;O/S'' acyl rearrangement at its N-terminal serine or cysteine residue. The result is an ester or thioester bond, respectively between the side-chain and the peptide backbone of the N-extein.<br />
# The two exteins are then linked by trans(thio)esterification involving the N-terminal serine or cysteine residue of the C-extein. The C-terminus of the N-extein is now covalently bound to the N-terminal side-chain of the C-extein, while its backbone still retains its normal peptide bond to the intein.<br />
# The C-terminal asparagine of the intein undergoes self-cyclisation to form a succinimidyl moiety. The peptide bond between the intein and the extein is thereby broken, resolving the branched intermediate.<br />
# In a final reaction, an ''O/S&rarr;N'' acyl shift results in the two exteins now being linked by an amide bond, indistinguishable from a ribosome-assembled fusion protein.<br />
(as reviewed in [[#References|[15]]]) <br />
<br />
{{:Team:Heidelberg/templates/image-full|<br />
align=right|<br />
caption=Figure 2) Trans-splicing mechanism reaction by split inteins. |<br />
descr=Picture taken from [[#References|[16]]]|<br />
file=Trans_splicing_OCreaction}}<br />
<br />
For intein efficiency not only the structure of the intein domain itself is important but also the nature of the flanking extein residues, as they are heavily involved in the splicing reaction. It has been shown, that the first residue has to be a nucleophilic amino acid, preferable Cysteine, Serine or Threonine. Effectiveness of the intein can be mold by changing the these amino acids [[#References|[17]]].<br />
<br />
==Use of inteins in molecular biology and biotechnology==<br />
<br />
Due to their distinct characteristics intein are a powerful tool for molecular biology and biotechnology. Performing an autocatalytic reaction, the inteins are neither dependent on their host protein nor on any other additional substrate. This makes it them broadly applicable for in vivo and in vitro applications. [[#References|[16]]].<br />
<br />
Inteins have been used for protein purification, achieving a higher yield of protein due to more specific targeting. The production of semi-synthetic proteins and the attachment of synthetic groups is of huge interest for recombinant proteins. [[#References|[16]]]. Split Intein-mediated Circular Ligation Of Peptides a ProteinS (SICLOPPS) is a method to produce circular peptides of eight amino acids length, which could be potent therapeutic drugs [[#References|[18]]]. Inteins, fused to split fluorescent proteins as reporter and to the protein of investigation have been used to detect protein-protein interactions in vivo (reviewed in [[#References|[10]]]).<br />
Nevertheless most of this applications are performed in vitro and do not exploit the full potential of inteins as regulatory element for post-translational modification. The iGEM Team Heidelberg employs this excellent mechanism of protein splicing to specifically change whole amino-acid sequences and thereby regulate proteins via (dis-) assembly, protein cleavage or circularization of enzymes in vivo. All this and much more was embedded into our versatile intein toolbox. With this universal toolkit we provide a foundational advance for protein control - introducing the full potential of post-translational modification and therefore a new dimension of genetic engineering to Synthetic Biology. There is much more to [Team:Heidelberg/Project explore]!<br />
<br />
=References=<br />
<br />
[1] Perler FB, Davis EO, Dean GE, Gimble FS, Jack WE, et al. (1994) Protein splicing elements: inteins and exteins–a definition of terms and recommended nomenclature. Nucleic Acids Res 22: 1125–1127<br />
<br />
[2] Kane P.M., et al. Protein Splicing Converts the Yeast TEPI Gene Product to the 69-kD Subunit of the Vacuolar. 13253, (1990)<br />
<br />
[3] Hirata, R. et al. Molecular structure of a gene, VMA1, encoding the catalytic subunit of H(+)-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J. Biol. Chem. 265 , 6726–6733 (1990)<br />
<br />
[4] Perler, F. B. InBase : the Intein Database. 30, 383–384 (2002)<br />
<br />
[5] Perler, F. B. et al. Protein splicing elements : inteins and exteins — a definition of terms and recommended nomenclature. 22, 1125–1127 (1994)<br />
<br />
[6] Barzel, A., Naor, A., Privman, E., Kupiec, M. & Gophna, U. Homing endonucleases residing within inteins: evolutionary puzzles awaiting genetic solutions. Biochem. Soc. Trans. 39, 169–73 (2011)<br />
<br />
[7] Pietrokovski, S. Intein spread and extinction in evolution. Trends Genet. 17, 465-472 (2001)<br />
<br />
[8] Novikova, O., Topilina, N. & Belfort, M. Enigmatic distribution, evolution and function of inteins. J. Biol. Chem. (2014)<br />
<br />
[9] Shah, N. H., and Muir, T. W. Inteins: nature's gift to protein chemists. Chem. Sci. 5, 446- 461 (2014) <br />
<br />
[10] Topilina, N. I. & Mills, K. V. Recent advances in in vivo applications of intein-mediated protein splicing. Mob. DNA 5, 5 (2014)<br />
<br />
[11] Eryilmaz, E., Shah, N., Muir, T. & Cowburn, D. Structural and Dynamical Features of Inteins and Implications on Protein Splicing. J. Biol. Chem. (2014)<br />
<br />
[12] Carvajal-Vallejos, P., Pallissé, R., Mootz, H. D. & Schmidt, S. R. Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. J. Biol. Chem. 287, 28686–96 (2012) <br />
<br />
[13] Lin, Y. et al. Protein trans-splicing of multiple atypical split inteins engineered from natural inteins. PLoS One 8, e59516 (2013). <br />
<br />
[14] Eryilmaz, E., Shah, N., Muir, T. & Cowburn, D. Structural and Dynamical Features of Inteins and Implications on Protein Splicing. J. Biol. Chem. (2014)<br />
<br />
[15] Mills, K. V, Johnson, M. a & Perler, F. B. Protein Splicing: How Inteins Escape from Precursor Proteins. J. Biol. Chem. (2014)<br />
<br />
[16] Mootz, H. D. Split inteins as versatile tools for protein semisynthesis. Chembiochem 10, 2579–89 (2009).<br />
<br />
[17] Amitai, G., Callahan, B. P., Stanger, M. J., Belfort, G. & Belfort, M. Modulation of intein activity by its neighboring extein substrates. Proc. Natl. Acad. Sci. U. S. A. 106, 11005–10 (2009).<br />
<br />
[18] Tavassoli, A. & Benkovic, S. J. Split-intein mediated circular ligation used in the synthesis of cyclic peptide libraries in E. coli. Nat. Protoc. 2, 1126–1133 (2007).</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/CollaborationsTeam:Heidelberg/pages/Collaborations2014-10-17T21:38:28Z<p>Huhny: /* iGEM Team Freiburg */</p>
<hr />
<div><br/><br />
<br />
Besides spending a whole summer in the lab and developing an own project, iGEM is about meeting new people, people with the same interests, the same motivation, the same dedication. Collaborations between team ensure the exchange of ideas, therefore helping the field of Synthetic Biology to be interdisciplinary and creative - the best conditions to give rise to revolutionary projects. We are very happy to met some of the team members from Freiburg, Aachen, Tuebingen, Marburg and London. <br />
<br />
=iGEM Team Aachen=<br />
<br />
<br/><br />
<br />
Michael from the iGEM team Aachen visited us in Heidelberg. Their team worked on the development of a real-time pathogen detection technique for what they have build their own fluorescence measurement camera. We provided our expression vectors to the iGEM team Aachen, which they used to express their part [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1319003 E1010-K1319003], the human galectin-3 protein fused to mRFP. Nicely seen in Figure 1 and 2 the the intensity of the red color is significantly higher for the induced sample with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362091 pSBX1A30] compared to the uninduced ones. To ensure the right molecular mass of the fusion protein, a SDS gel was run as well (Figure 3).<br />
<br />
<div><br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=3) SDS-PAGE of K1319020 expression| file=AachenCo3.png}}<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=2) Expression of Galectin-3| file=AachenCo2.png}}<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=1) Expression of Galectin-3| file=AachenCo1.jpg}}<br />
</div><br />
<br />
More information about their project can be found on Aachens [https://2014.igem.org/Team:Aachen/Project/Gal3 Galectin-3 site].<br />
<br />
In exchange they offered to characterise these expression vectors with their own designed analyser by measuring the amount of produced fluorescent proteins. Unfortunately, transporting <i>E.colis</i> to another city wasn't as easy as expected, not all Bacteria were able to grow afterwards and we could only obtain sufficient data from pSBX4C5. <br />
<br />
<br/><br />
<br />
=iGEM Team Tuebingen=<br />
The project of iGEM Team Tuebingen is about blood types and the difficulties that arises during blood donation of the wrong blood type. They aim to develop an easy system to convert blood types A, B, AB to the rares but also most applicable blood type 0. They have applied the method of intein targeting, a method which can also be found in our intein toolbox, to the N-Acetyl-Galactosaminidase protein. More about their project can be read in the very nice [http://igem14-heidelberg.tumblr.com/post/98138990630/igem-team-tubingen article] they have wrote for our tumblr blog or on their own [Team:Tuebingen wiki].<br />
<br />
In this collaboration, Tuebingen conducted a mass spectrometry analysis of our Lambda lysozyme to proof circularisation. The data still has to be evaluated, but we will have them ready until the jamboree. There is a high demand for our expression vectors. Tuebingen had problems with expressing their constructs, therefore we send them some of our plasmids pSBX1K30 ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362093 BBa_K1362093]) and pSBX4K50 ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362097 BBa_K1362097]), as they showed to work really nicely even with big proteins like the [Team:Heidelberg/Project/PCR2.0 Dnmt1] with 105kDa.<br />
<br />
=iGEM Team Freiburg=<br />
<br />
<br />
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<br />
Three of our team members, Anna, Caro, and Charlotte visited the iGEM team Freiburg. We met the team right after a big team meeting, therefore we were able to hear their latest project developments. We spend the evening talking about experiences in the lab and about iGEM. It was relieving to hear that they faced similar difficulties in the lab. <br />
The morning after we presented our project to the iGEM team Freiburg. Freiburg was very interested in our program iGEM@home as we offered them to promote their own human practice projects. You can also find an [http://igem14-heidelberg.tumblr.com/post/99936805780/optogenetics-the-lock-and-key-for-daily-life article] about optogenetics on our tumblr blog.<br />
Both our teams are working with light inducible systems, manly the LOV domain. Although working in different organisms, the constructs were compatible, therefore we could interchange protocols for induction and preliminary results. Furthermore we gazed at their professional light induction box - we can definitely improved ours. <br />
<br />
After cooperation with the iGEM Team Freiburg for two years in a row we are hoping to keep Freiburg as a reliable partner in the next years as well.</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/CollaborationsTeam:Heidelberg/pages/Collaborations2014-10-17T21:36:20Z<p>Huhny: </p>
<hr />
<div><br/><br />
<br />
Besides spending a whole summer in the lab and developing an own project, iGEM is about meeting new people, people with the same interests, the same motivation, the same dedication. Collaborations between team ensure the exchange of ideas, therefore helping the field of Synthetic Biology to be interdisciplinary and creative - the best conditions to give rise to revolutionary projects. We are very happy to met some of the team members from Freiburg, Aachen, Tuebingen, Marburg and London. <br />
<br />
=iGEM Team Aachen=<br />
<br />
<br/><br />
<br />
Michael from the iGEM team Aachen visited us in Heidelberg. Their team worked on the development of a real-time pathogen detection technique for what they have build their own fluorescence measurement camera. We provided our expression vectors to the iGEM team Aachen, which they used to express their part [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1319003 E1010-K1319003], the human galectin-3 protein fused to mRFP. Nicely seen in Figure 1 and 2 the the intensity of the red color is significantly higher for the induced sample with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362091 pSBX1A30] compared to the uninduced ones. To ensure the right molecular mass of the fusion protein, a SDS gel was run as well (Figure 3).<br />
<br />
<div><br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=3) SDS-PAGE of K1319020 expression| file=AachenCo3.png}}<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=2) Expression of Galectin-3| file=AachenCo2.png}}<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=1) Expression of Galectin-3| file=AachenCo1.jpg}}<br />
</div><br />
<br />
More information about their project can be found on Aachens [https://2014.igem.org/Team:Aachen/Project/Gal3 Galectin-3 site].<br />
<br />
In exchange they offered to characterise these expression vectors with their own designed analyser by measuring the amount of produced fluorescent proteins. Unfortunately, transporting <i>E.colis</i> to another city wasn't as easy as expected, not all Bacteria were able to grow afterwards and we could only obtain sufficient data from pSBX4C5. <br />
<br />
<br/><br />
<br />
=iGEM Team Tuebingen=<br />
The project of iGEM Team Tuebingen is about blood types and the difficulties that arises during blood donation of the wrong blood type. They aim to develop an easy system to convert blood types A, B, AB to the rares but also most applicable blood type 0. They have applied the method of intein targeting, a method which can also be found in our intein toolbox, to the N-Acetyl-Galactosaminidase protein. More about their project can be read in the very nice [http://igem14-heidelberg.tumblr.com/post/98138990630/igem-team-tubingen article] they have wrote for our tumblr blog or on their own [Team:Tuebingen wiki].<br />
<br />
In this collaboration, Tuebingen conducted a mass spectrometry analysis of our Lambda lysozyme to proof circularisation. The data still has to be evaluated, but we will have them ready until the jamboree. There is a high demand for our expression vectors. Tuebingen had problems with expressing their constructs, therefore we send them some of our plasmids pSBX1K30 ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362093 BBa_K1362093]) and pSBX4K50 ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362097 BBa_K1362097]), as they showed to work really nicely even with big proteins like the [Team:Heidelberg/Project/PCR2.0 Dnmt1] with 105kDa.<br />
<br />
=iGEM Team Freiburg=<br />
<br />
<br />
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<br />
Three of our team members, Anna, Caro, and Charlotte visited the iGEM team Freiburg. We met the team right after a big team meeting, therefore we were able to hear their latest project developments. We spend the evening talking about experiences in the lab and about iGEM. It was relieving to hear that they faced similar difficulties in the lab. <br />
The morning after we presented our project to the iGEM team Freiburg. Freiburg was very interested in our program iGEM@home as we offered them to promote their own human practice projects. You can also find an [http://igem14-heidelberg.tumblr.com/post/99936805780/optogenetics-the-lock-and-key-for-daily-life article] about optogenetics on our tumblr blog.<br />
Both our teams are working with light inducible systems, manly the LOV domain. Although working in different organisms, the constructs were compatible, therefore we could interchange protocols for induction and preliminary results. Furthermore we gazed at their professional light induction box - we can definitely improved ours. <br />
<br />
After cooperation with the iGEM Team Freiburg for two years in a row we are hoping to keep Freiburg as a reliable partner in the next years as well.</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/CollaborationsTeam:Heidelberg/pages/Collaborations2014-10-17T20:50:06Z<p>Huhny: </p>
<hr />
<div>Beside spending a whole summer in the lab, iGEM is about meeting new people, people with the same interests, the same motivation, the same dedication. Collaborations between team ensure the exchange of ideas, therefore helping the field of Synthetic Biology to be interdisciplinary and creative - the best conditions to give rise to revolutionary projects. We are very happy to met some of the team members from Freiburg, Aachen, Tuebingen, Marburg and London. <br />
<br />
=iGEM Team Aachen=<br />
<br />
<br/><br />
<br />
Michael from the iGEM team Aachen visited us in Heidelberg. Their team worked on the development of a real-time pathogen detection technique for what they have build their own fluorescence measurement camera. We provided our expression vectors to the iGEM team Aachen, which they used to express their part [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1319003 E1010-K1319003], the human galectin-3 protein fused to mRFP. Nicely seen in Figure 1 and 2 the the intensity of the red color is significantly higher for the induced sample with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362091 pSBX1A30] compared to the uninduced ones. To ensure the right molecular mass of the fusion protein, a SDS gel was run as well (Figure 3).<br />
<br />
<div><br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=3) SDS-PAGE of K1319020 expression| file=AachenCo3.png}}<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=2) Expression of Galectin-3| file=AachenCo2.png}}<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=1) Expression of Galectin-3| file=AachenCo1.jpg}}<br />
</div><br />
<br />
More information about their project can be found on Aachens [https://2014.igem.org/Team:Aachen/Project/Gal3 Galectin-3 site].<br />
<br />
In exchange they offered to characterise these expression vectors with their own designed analyser by measuring the amount of produced fluorescent proteins. Unfortunately, transporting <i>E.colis</i> to another city wasn't as easy as expected, not all Bacteria were able to grow afterwards and we could only obtain sufficient data from pSBX4C5. <br />
<br />
<br/><br />
<br />
=iGEM Team Tuebingen=<br />
<br />
<br />
=iGEM Team Freiburg=<br />
<br />
<br />
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<br />
Three of our team members, Anna, Caro, and Charlotte visited the iGEM team Freiburg. We met the team right after a big team meeting, therefore we were able to hear their latest project developments. We spend the evening talking about experiences in the lab and about iGEM. It was relieving to hear that they faced similar difficulties in the lab. <br />
The morning after we presented our project to the iGEM team Freiburg. Freiburg was very interested in our program iGEM@home as we offered them to promote their own human practise projects.<br />
Both our teams are working with light inducible systems, manly the LOV domain. Although working in different organisms, the constructs were compatible, therefore we could interchange protocols for induction and preliminary results. Furthermore we gazed at their professional light induction box - we can definitely improved ours. <br />
<br />
Gibson vs. Cpec?<br />
<br />
After cooperation with the iGEM Team Freiburg for two years in a row we are hoping to keep Freiburg as a reliable partner in the next years as well.<br />
<br />
<br />
=iGEM Team Imperial College London=</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/CollaborationsTeam:Heidelberg/pages/Collaborations2014-10-17T20:40:55Z<p>Huhny: </p>
<hr />
<div>Kurze Einleitung<br />
<br />
=iGEM Team Aachen=<br />
<br />
<br/><br />
<br />
Michael from the iGEM team Aachen visited us in Heidelberg. Their team worked on the development of a real-time pathogen detection technique for what they have build their own fluorescence measurement camera. We provided our expression vectors to the iGEM team Aachen, which they used to express their part [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1319003 E1010-K1319003], the human galectin-3 protein fused to mRFP. Nicely seen in Figure 1 and 2 the the intensity of the red color is significantly higher for the induced sample with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362091 pSBX1A30] compared to the uninduced ones. To ensure the right molecular mass of the fusion protein, a SDS gel was run as well (Figure 3).<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=3) SDS-PAGE of K1319020 expression| file=AachenCo3.png}}<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=2) Expression of Galectin-3| file=AachenCo2.png}}<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=1) Expression of Galectin-3| file=AachenCo1.jpg}}<br />
<br />
More information about their project can be found on Aachens [https://2014.igem.org/Team:Aachen/Project/Gal3 Galectin-3 site].<br />
<br />
In exchange they offered to characterise these expression vectors with their own designed analyser by measuring the amount of produced fluorescent proteins. Unfortunately, transporting <i>E.colis</i> to another city wasn't as easy as expected, not all Bacteria were able to grow afterwards and we could only obtain sufficient data from pSBX4C5. <br />
<br />
=iGEM Team Tuebingen=<br />
<br />
<br />
=iGEM Team Freiburg=<br />
<br />
<br />
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<br />
Three of our team members, Anna, Caro, and Charlotte visited the iGEM team Freiburg. We met the team right after a big team meeting, therefore we were able to hear their latest project developments. We spend the evening talking about experiences in the lab and about iGEM. It was relieving to hear that they faced similar difficulties in the lab. <br />
The morning after we presented our project to the iGEM team Freiburg. Freiburg was very interested in our program iGEM@home as we offered them to promote their own human practise projects.<br />
Both our teams are working with light inducible systems, manly the LOV domain. Although working in different organisms, the constructs were compatible, therefore we could interchange protocols for induction and preliminary results. Furthermore we gazed at their professional light induction box - we can definitely improved ours. <br />
<br />
Gibson vs. Cpec?<br />
<br />
After cooperation with the iGEM Team Freiburg for two years in a row we are hoping to keep Freiburg as a reliable partner in the next years as well.<br />
<br />
<br />
=iGEM Team Imperial College London=</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/CollaborationsTeam:Heidelberg/pages/Collaborations2014-10-17T20:36:54Z<p>Huhny: </p>
<hr />
<div>Kurze Einleitung<br />
<br />
=iGEM Team Aachen=<br />
<br />
Michael from the iGEM team Aachen visited us in Heidelberg. Their team worked on the development of a real-time pathogen detection technique for what they have build their own fluorescence measurement camera. We provided our expression vectors to the iGEM team Aachen, which they used to express their part [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1319003 E1010-K1319003], the human galectin-3 protein fused to mRFP. Nicely seen in Figure 1 and 2 the the intensity of the red color is significantly higher for the induced sample with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362091 pSBX1A30] compared to the uninduced ones. To ensure the right molecular mass of the fusion protein, a SDS gel was run as well (Figure 3).<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=3) SDS-PAGE of K1319020 expression| file=AachenCo3.png}}<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=2) Expression of Galectin-3| file=AachenCo2.png}}<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=1) Expression of Galectin-3| file=AachenCo1.jpg}}<br />
<br />
More information about their project can be found on Aachens [https://2014.igem.org/Team:Aachen/Project/Gal3 Galectin-3 site].<br />
In exchange they offered to characterised these expression vectors with their own designed analyser by measuring the amount of produced fluorescent proteins. Unfortunately, transporting <i>E.colis</i> to another city wasn't as easy as expected, not all Bacteria were able to grow afterwards and we could only obtain sufficient data from pSBX4C5. <br />
<br />
=iGEM Team Tuebingen=<br />
<br />
<br />
=iGEM Team Freiburg=<br />
<br />
<br />
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<br />
Three of our team members, Anna, Caro, and Charlotte visited the iGEM team Freiburg. We met the team right after a big team meeting, therefore we were able to hear their latest project developments. We spend the evening talking about experiences in the lab and about iGEM. It was relieving to hear that they faced similar difficulties in the lab. <br />
The morning after we presented our project to the iGEM team Freiburg. Freiburg was very interested in our program iGEM@home as we offered them to promote their own human practise projects.<br />
Both our teams are working with light inducible systems, manly the LOV domain. Although working in different organisms, the constructs were compatible, therefore we could interchange protocols for induction and preliminary results. Furthermore we gazed at their professional light induction box - we can definitely improved ours. <br />
<br />
Gibson vs. Cpec?<br />
<br />
After cooperation with the iGEM Team Freiburg for two years in a row we are hoping to keep Freiburg as a reliable partner in the next years as well.<br />
<br />
<br />
=iGEM Team Imperial College London=</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/CollaborationsTeam:Heidelberg/pages/Collaborations2014-10-17T20:34:50Z<p>Huhny: </p>
<hr />
<div>Kurze Einleitung<br />
<br />
=iGEM Team Aachen=<br />
<br />
Michael from the iGEM team Aachen visited us in Heidelberg. Their team worked on the development of a real-time pathogen detection technique for what they have build their own fluorescence measurement camera. We provided our expression vectors to the iGEM team Aachen, which they used to express their part [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1319003 E1010-K1319003], the human galectin-3 protein fused to mRFP. Nicely seen in Figure 1 and 2 the the intensity of the red color is significantly higher for the induced sample with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362091 pSBX1A30] compared to the uninduced ones. To ensure the right molecular mass of the fusion protein, a SDS gel was run as well (Figure 3).<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=Expression of Galectin-3| file=AachenCo1.png}}<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=Expression of Galectin-3| file=AachenCo2.png}}<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=SDS-PAGE of K1319020 expression| file=AachenCo3.png}}<br />
<br />
More information about their project can be found on Aachens [https://2014.igem.org/Team:Aachen/Project/Gal3 Galectin-3 site].<br />
In exchange they offered to characterised these expression vectors with their own designed analyser by measuring the amount of produced fluorescent proteins. Unfortunately, transporting <i>E.colis</i> to another city wasn't as easy as expected, not all Bacteria were able to grow afterwards and we could only obtain sufficient data from pSBX4C5. <br />
<br />
=iGEM Team Tuebingen=<br />
<br />
<br />
=iGEM Team Freiburg=<br />
<br />
<br />
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<br />
Three of our team members, Anna, Caro, and Charlotte visited the iGEM team Freiburg. We met the team right after a big team meeting, therefore we were able to hear their latest project developments. We spend the evening talking about experiences in the lab and about iGEM. It was relieving to hear that they faced similar difficulties in the lab. <br />
The morning after we presented our project to the iGEM team Freiburg. Freiburg was very interested in our program iGEM@home as we offered them to promote their own human practise projects.<br />
Both our teams are working with light inducible systems, manly the LOV domain. Although working in different organisms, the constructs were compatible, therefore we could interchange protocols for induction and preliminary results. Furthermore we gazed at their professional light induction box - we can definitely improved ours. <br />
<br />
Gibson vs. Cpec?<br />
<br />
After cooperation with the iGEM Team Freiburg for two years in a row we are hoping to keep Freiburg as a reliable partner in the next years as well.<br />
<br />
<br />
=iGEM Team Imperial College London=</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/PCR_2.0Team:Heidelberg/pages/PCR 2.02014-10-17T20:11:42Z<p>Huhny: </p>
<hr />
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<br />
</div><br />
<div class="col-md-6"><br />
<p style="font-size:25px; font-weight:bold">Abstract</p><br />
<br/><br />
<p>DNA methylation is the most abundant DNA modification and essential for embryonic development, gene regulation and genomic stability. Although several methods for the detection of methylation patterns exist, there is no easy way to amplify methylated DNA for <i>in vitro</i> or <i>in vivo</i> studies.</p><br />
<p>To empower epigenetic research, we envisioned a PCR2.0 which maintains DNA methylation patterns during amplification. The central element of this PCR2.0 is a heat-resistant DNA methyltransferase - DNMT1 (731-1602) - which we created by circularization using our intein toolbox and the CRAUT linker software.</p><br />
<p>So far, our DNMT1 represents the largest circularized protein which highlights the usefulness of our intein toolbox in combination with the CRAUT linker software. Increased heat resistance of our circular DNMT1 which was observed in initial assays smoothens the path for the establishment of a PCR2.0 and illustrates the suitability of intein-mediated circularization for the advancement of heat resistant proteins.</p><br />
<p style="font-size:25px; font-weight:bold">Highlights</p><br />
<ul><br />
<li>Heat stabilization of mDNMT1 (731-1602) as pivotal element of a methylation maintaining PCR2.0.</li><br />
<li>Largest circularized protein so far.</li><br />
<li>Successful application of rational linker design with CRAUT linker software.</li><br />
<li>Efficient expression and purification of active linear/circular mDNMT1 (731-1602) in E.coli.</li><br />
<li>Establishment of a methylation activity assay that allows quantification of activity and specificity.</li><br />
</ul><br />
</div><br />
</div><br />
</div><br />
</div><br />
<br />
</html><br />
<br />
=Introduction=<br />
<br />
==Motivation==<br />
<br />
The invention of the polymerase chain reaction in 1983 by Kary Mullis revolutionized our world by enabling amplification of DNA in an exponential manner. Further improvements including the use of thermo-stable DNA polymerases made the method even more efficient, allowing its widespread use in nearly every field of modern diagnostics and research. However, a major part of information is lost when using conventional PCR as DNA modifications are not copied. The most abundant modification is DNA methylation, which is a prominent regulator of gene expression in all kingdoms of life. Methylation and other modifications that influence the DNA function without changing its actual sequence are studied in the fast-growing field of epigenetics. Even though the detection and mapping of methylation patterns have become feasible in a high-throughput manner by using bisulfite sequencing and array techniques [[#References|[1]]], further functional analysis is still hindered by the small amount of primary material. Up to now, there is no method available to amplify methylated DNA without knowledge of methylation patterns and expensive de-novo synthesis.<br />
<br />
To empower epigenetics, we propose the '''“PCR 2.0”''' as an easy and efficient way to amplify DNA templates in an exponential manner while maintaining their specific methylation pattern. The pivotal element of this PCR reaction is a heat-stable DNA methyltransferase (DNMT). Although comparable enzymes exist in thermophile organisms [[#References|[2]]], until now no suitable protein has been found or synthesized that withstands the harsh conditions of a PCR.<br />
<br />
To achieve this goal of a PCR2.0 that can be used to amplify DNA including its intrinsic methylation pattern, our main aim was the generation of a heat-stable Dnmt1 that can be produced in a larger scale and shows efficient and specific methylation of DNA. It was reported that the insertion of additional intramolecular bonds can increase the stability of proteins [[#References|[3]]][[#References|[4]]], and that specifically joining the C- and N-terminus improves the thermostability of smaller peptides [[#References|[5]]]. This is why our approach to generate a heat-stable Dnmt1 applies:<br />
* protein circularization using our intein toolbox<br />
* creating a potent [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software Software] to design efficient linker peptides connecting C- and N-terminus<br />
<br />
To enable the PCR2.0 we started the circularization of the largest protein so far…<br />
<br />
==Epigenetics - there is more than just A, T, C and G…==<br />
In mammals and other vertebrates DNA methylation occurs at the C5 position of cytosine nucleotides that are followed by guanines (CpG). It is a prominent key regulator of transcription, embryonic development, X chromosome inactivation and many other cellular functions [[#References|[6]]][[#References|[7]]]. The human genome contains about 28 million CpGs of which 60-80% are methylated [[#References|[28]]]. Due to its great prevalence and the heredity of this modification, the 5-methylcytosine (5mC) is also known as the “fifth base” of eukaryotic genomes [[#References|[9]]]. DNA methylation is preferentially occurring at intergenic regions and repetitive sequences, where it is known to silence gene expression [[#References|[10]]][[#References|[11]]][[#References|[12]]]. Inappropriate cytosine methylation and silencing has been reported to play a crucial role in the development of numerous diseases, including cancer, imprinting diseases and repeat-instability based diseases such Huntington's disease [[#References|[13]]].<br />
<br />
==A closer look at DNA Methyltransferases==<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 1) Principle of mDNMT1-mediated maintenance methylation|<br />
descr=The enzyme DNMT1 is of particular interest for methylation maintenance PCR, as it allows exact duplication of the original methylation pattern to every new DNA copy.|<br />
file=methylation_principle.png}}<br />
<br />
<br />
The family of enzymes called DNA methyltransferases (DNMTs) is responsible for the establishment and maintenance of cell-type specific DNA methylation patterns. Whereas the two enzymes DNMT3a and DNMT3b contribute to de novo methylation of DNA during development, DNMT1 is preserving existing methylation patterns throughout cell divisions. To do so, DNMT1 exploits the principle of semi-conservative replication, using the parental strand as a template to create an exact copy of methylation patterns on the daughter strand (Figure 1). After DNA synthesis, the DNA consists of a methylated and an unmethylated strand. DNMT1 recognizes these so called hemi-methylated CpG sites, and transfers a methyl group from the methyl donor S-Adenosyl-methionine (SAM) to the cytosine belonging to the newly synthesized strand [[#References|[15]]][[#References|[19]]].<br />
<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 2) Crystal structure of full-length and truncated (731-1602) mDNMT1|<br />
descr=Structural overview of full length mDNMT1 and truncated mDNMT1(731–1602) (A) Color-coded schematic overview of domain structure and numbering of mDNMT1 sequence. (B) Ribbon presentation of full-length DNMT1 with the CXXC, BAH1, BAH2,and methyltransferase domain colored in light blue, yellow, orange and red, respectively. (C) Crystal structure of truncated DNMT1, missing the CXXC domain and schowing more adjacent protein termini that the full-length version.|<br />
file=dnmt1_structure.png}}<br />
<br />
<br />
<br />
The complex structure of DNMT1 and several truncated versions have been recently solved by X-ray crystallography [[#References|[19]]]. For our experiments we used the smallest truncated version of DNMT1 - mDNMT1 (731-1602) - that has been reported to efficiently mediate methylation maintenance. This truncated version is derived from <i>Mus musculus</i> and comprises the amino acids 731-1602 of the full length protein [[#References|[15]]][[#References|[19]]] (kindly provided by Dr. Bashtrykov after approval from Prof. Patel). Murine DNMT1 (731-1602) contains a C-terminal catalytic methyltransferase domain as well as the bromo-adjacent homology (BAH) domains, which prevent the binding of unmethylated DNA to the catalytic core. The truncated enzyme is missing the CXXC domain, which has a high affinity to hemi-methylated CpG dinucleotides, but was shown to be dispensable for protein function [[#References|[16]]]. In contrast to the full-length DNMT1, mDNMT1 (731-1602) has the great advantage of being efficiently expressed in E.coli. Another benefit of the truncated DNMT1 is that the N- and C- terminus are closer together than in the full-length DNMT1 (Figure 2). Thus circularization might cause less deformation and show lower impact on the overall activity, which makes this truncated mDNMT1 an ideal candidate for our purposes.<br />
<br />
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<br />
==Circularization - The missing link== <br />
<br />
Heat stabilization of mDNMT1 as a pivotal element of our methylation-maintaining PCR2.0 was approached by circularization of the protein. Until now, only smaller proteins have been stabilized using this method. Therefore, to our knowledge, mDNMT1 – even in its shortest truncated version of 871 amino acids and a molecular weight of approximately 100kDa – is the largest protein that has ever been tried to circularize. According to the crystal structure, the distance between the termini of the truncated DNMT1 is 48 Å. We suspected that circularization of the protein by direct fusion of both termini might cause deformation of the protein structure. Therefore we used our own [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software CRAUT Linker Software] to create a linking amino acid sequence, adapted to the structure of DNMT1. The software is able to design protein linkers with the required length to bridge the gap between the protein termini while bypassing the catalytic core of the enzyme.<br />
<br />
Since we could not estimate the impact of the linker introduction, we focused on two different kinds of linkers: a so called rigid linker that has been calculated and optimized during the establishment of our linker software and a flexible peptide connection consisting mostly of glycine and serine. To perform circularization we have used the split NpuDnaE Intein, since it is used as the <i>golden standard</i> for protein splicing and has been efficiently tested by us through circularization of GFP, Lysozyme and Xylanase. Moreover, we tried to implement protein circularization by using sortase A, which is recognizing and cleaving a carboxyterminal sorting signal followed by a transpeptidation reaction that can be exploited for protein circularization. This method has been reported to be very efficient [[#References|[4]]] and was therefore included in our study as possible candidate for large scale productions of circular proteins.<br />
<br />
=Materials and Methods=<br />
<br />
==Constructs==<br />
<br />
We designed constructs in order to characterize the efficiency of sortase A as well as intein mediated circularization of DNMT1. Both approaches are based on the comparison of the circularized protein with a corresponding linear counterpart at different temperatures and incubation times.<br />
<br />
{{:Team:Heidelberg/templates/image-quarter|<br />
align=right|<br />
caption=Construct for circularization of mDNMT1 using inteins.|<br />
descr= |<br />
file=Dnmt1_construct1.png}}<br />
<br />
For circularization of DNMT1 with inteins we fused the obtained truncated version of DNMT1 (731-1602) [[#References|[15]]] with an appropriate linker and split NpuDnaE domains at either site of the construct. For efficient purification of the protein, all constructs contained a hexa-histidine tag. Moreover, we have cloned the ubiquitin-like Smt3 protein from <i> Saccharomyces cerevisiae </i> in front of the DNMT1-intein complex. This attachment is used to increase the yield when expressing large mammalian proteins like DNMT1 in <i>E.coli</i> [[#References|[17]]]. The constructs have been designed in a way that Smt3 is no longer included in the final circular protein, and therefore cannot interfere with its function. <br />
All cloning steps were carried out using our new RFC[i] standard procedure. <br />
<br />
The mDNMT1 (731-1602) constructs that have been designed to allow sortase A mediated cirularization are flanked by the sortase A recognition sequence LPETGG on the C–terminus as well as N-terminal glycines that that can be exposed through previous TEV treatment. Since the transpeptidation via sortase requires an additional <i> in vitro </i> reaction, subsequent purification is necessary. To facilitate this step and to enrich the circular product in the sample, a hexa-histidine tag is present in the initial protein that is lost during circularization. Hence, an additional affinity chromatography via His trap can be used to separate the histidine tag containing sortase and not circularized educt from the circular flow-through. <br />
All cloning steps were carried out using our sortase standard. <br />
<br />
The following linkers that had been optimized along with our [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software CRAUT Linker Software]:<br />
<br />
{| class="table table-hover"<br />
|-<br />
! Definition!! Sequence <br />
|-<br />
|Rigid linker|| GGAEAAAKEAAAKVNLTAAEAAAKEAAAKEAAAKEAAAKEAAAKAVNLTAAEAAAKAHHHHHHSGRGT <br />
|-<br />
|Flexible linker|| CWEGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSHHHHHHSGRGT<br />
|}<br />
<br />
== Expression and purification==<br />
<br />
{{:Team:Heidelberg/templates/image-quarter|<br />
align=right|<br />
caption=Figure 3) Purification of recombinant mDNMT1|<br />
descr= The purification of recombinantly expressed mDNMT1 is a challenging task including large scale induction, efficient cell lysis, reduction of sample volume via ultracentrifugation, immobilized Ni-ion affinity chromatography, dialysis and concentration through centrifugation. |<br />
file=Purification_procedure_new.png}}<br />
<br />
In comparison to the full length murine DNMT1, our modified mDNMT1(731-1602) can be expressed in <i>E.coli (Rosetta DE3)</i> and does not need the establishment of Baculovirus based system and the use of insect cell cultures. First experiments were conducted to determine the optimal conditions for protein induction and expression. We tested several concentrations of IPTG as well as different temperatures to maximize the yield of mDNMT1. Still, even though expression of the constructs can be simplified by using <i>E.coli</i>, purification of the recombinant protein remains a challenging task requiring multiple different steps that had to be adapted and established from published protocols [[#References|[19]]]. <br />
<br />
In a first step, we optimized the process of cell lysis, to be able to extract a maximum amount of active protein from our cultures. Therefore we compared the two commonly used methods sonication and disruption via French press. Subsequently, the sample was cleared from cell debris by ultracentrifugation. The protein of interest containing a hexa-histidine tag was further enriched via immobilized metal ion affinity chromatography (IMAC) using a His trap and eluted with imidazol. In a next step, low molecular weight solutes such as salts and imidazol that would interfere with protein function were removed from the sample by dialysis. Finally, the purified protein was concentrated by size-exclusion centrifugation using filters with an appropriate mass cutoff.<br />
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<br />
==Methylation activity assay==<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 4) Sequence of 40-mer DNA template for methylation activity assay|<br />
descr=Sequence of the 40-mer DNA template. CpG dinucleotides are shaded in gray, Sau3AI and HpaII recognition sites are depicted in red. The first CpG dinucleotide is methylated on the reverse strand.|<br />
file=DNMT1_substrate.png}}<br />
<br />
To measure the efficiency of mDNMT1 maintenance methylation, we used an assay that is based on two methylation-sensitive restriction enzymes Sau3AI and HpaII. It relies on the inhibition of restriction enzymes when methyl groups are attached to the cleavage site. We used a 40-mer DNA template with one hemi-methylated and one unmethylated CpG site which are located within the Sau3AI and HpaII restriction site, respectively (adapted from Bashtrykov et colleagues [[#References|[16]]].<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 4) Principle of the mDNMT1 methylation activity assay|<br />
descr=Principle of mDNMT1 methylation assay using a 40-mer substrate with Sau3AI and HpaII cleavage sites that are blocked upon further CpG methylation. The lack of methylation activity leads to fragmentation of the DNA template with either of the two restriction enzymes. Whereas specific methylation of hemimethylated CpGs hampers only cleavage by Sau3AI, <i>de novo</i> methylation activity of mDNMT1 can be detected when HpaII cleavage is blocked as well.|<br />
file=MethylationAssay_overview.png}}<br />
<br />
The restriction enzyme Sau3AI is capable of cleaving the template despite of the hemimethylated state of the first CpG nucleotide. Similarly, HpaII can attack its unmethylated native site, leading to fragmentation of the template as long as no further methylation occurs. Accordingly, the methylation activity of circular and linear mDNMT1 can be measured by quantifying the cleavage efficiency of the restriction enzymes after incubation with the protein. Impairment of Sau3AI cleavage indicates maintenance methylation activity, whereas a decrease in HpaII activity would result from <i>de novo </i> methylation. Therefore the assay is not only detecting activity of the enzyme but also includes its specificity for hemimethylated DNA over unmethylated DNA. To quantify maintenance and de novo methylation activity, the 40-mer fragments remaining after DNMT1 incubation and restriction digest were separated by gel electrophoresis. The intensities of the detected bands corresponding to different fragments were measured using ImageJ Gel Analyzer and quantified using gaussian fitting after background substraction. <br />
<br />
<br />
<div style="clear:both;"></div><br />
<br />
=Results=<br />
<br />
==Optimizing expression and purification of linear mDNMT1(731-602)==<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 5) Optimisation of induction|<br />
descr=As soon as transformed Rosetta (DE3) cultures reached an OD600 of approximately 0.6, induction of linear mDNMT1(731-1602) was perfomed over night at either 15°C or 25°C, using different concentrations of IPTG. Higest expression of DNMT1 was observed at 15°C using 0.7mM IPTG.|<br />
file=DNMT1-Induction2.png}}<br />
<br />
Before starting circularization of DNMT1, we first tested if we can actually express and purify active linear DNMT1. Since DNMT1 expression in E.coli normally results in low yields (<1mg/liter of culture)[[#References|[15]]], we started with an optimization of the expression conditions. <br />
<br />
In general, low temperatures and low IPTG concentrations are known to prevent the occurrence of insoluble inclusion bodies under conditions of overexpression. Thus, we tested different IPTG concentrations and temperatures during induction in order to maximize yields of active mDNMT1(731-1602). Generally, solubility tags such as the fused Smt3 as well as lower temperatures are known to prevent the occurrence of insoluble inclusion bodies under conditions of overexpression. Accordingly, we observed an optimal induction of our mDNMT1(731-1602) construct after incubation at 15 °C using 0.7mM IPTG (Figure 5).<br />
<br />
Even though the overall yield of recombinant protein is quite low, the presence of mDNMT1(731-1602) after induction with IPTG was detectable through Coomassie staining and specifically verified by Western blot (Figure 6A and 6B). In the western blot, several other peptides with lower molecular weight were detected (Figure 6B). These peptides possibly result from incomplete translation or degradation processes, since the construct used for initial mDNMT1 expression and purification comprised an N-terminal hexa-histidine tag. To minimize the amount of incomplete protein products that would be enriched in the following purification, we optimized the mDNMT1(731-1602) constructs for circularization by shifting the His-tag to the C-terminus.<br />
<br />
<div style="clear:both;"></div><br />
<br />
After successful expression, the next critical step for high yields of recombinant protein after purification is protein extraction. The lysis method has to be efficient but still gentle enough to preserve the proteins function - especially when dealing with sensitive and low expressed proteins like DNMT1. In order to optimize protein extraction we therefore compared cell lysis by sonification with disruption via French press (Figure 6A and 6B). Due to the low overall yield, mDNMT1 does not appear as a prominent band on the Coomassie-stained gel as we expected for an overexpressed recombinant proteins. <br />
Assuming similar amounts of overall protein that should be present in the consecutively generated samples, sonification seems to be less efficient for extraction of recombinant protein compared to usage of the French press. Moreover, the disruption of <i>E.coli</i> using French press has been reported to preserve the biological activity of susceptible proteins more efficiently than sonication [18]. Therefore we used the French press for further extraction of linear as well as circular mDNMT1.<br />
<br />
Further purification of the protein using a His-trap resulted in successful enrichment of mDNMT1f(731-1602) (Figure 6C). Corresponding eluates were pooled and further concentrated for use in functional assays. <br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 6A) Comparison of DNMT1 extraction from bacteria by French press and sonification|<br />
descr=mDNMT1(731-1602) expression in Rosetta (DE3) was performed over night at 15°C. Subsequently, the bacteria were lysed by sonification or French press and ultracentrifuged. After each step, samples were collected for analysis by SDS-PAGE.<br />
(Untransformed negative control (UnTra), intervals of cell disruption (1, 2), supernatant (SN)).|<br />
file=DNMT1-Celllysis.jpg}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 6B)Lysis via French press efficiently etracts mDNMT1(731-1602)|<br />
descr=Western Blot of lysed samples was perfomed with an anti-hexa-histidine antibody for specific detection of mDNMT1. Full length mDNMT1(731-1602) is indicated with an arrow.|<br />
file=DNMT1-Celllysis-WB.jpg}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 6C) Purification of linear mDNMT1(731-1602)|<br />
descr=Coomassie stained SDS-PAGE showing efficient purification of linear mDNMT1(731-1602) via His-column. Arrow indicates peptides corresponding to the molecular weight of mDNMT1(731-1602) (133kDa).|<br />
file=DNMT1-Purification-linear.png}}<br />
<br />
<div style="clear:both;"></div><br />
<br />
==Activity and specificity of linear mDNMT1(731-1602)==<br />
<br />
After successful expression and purification of linear DNMT1, we characterized the proteins activity and specificity for hemi-methylated DNA by performing the methylation activity assay described above. To test the maintenance methylation activity of our linear DNMT1, we performed the methylation assay with different incubation times of DNMT1. We observe an increasing amount of completely methylated template over time (Figure 7), represented by increasing amounts of template that are unsusceptible towards SauAI cleavage. <br />
The truncated DNMT1 (731-1602) showed maximal methylation of the template after approximately 1/2 to 1hour. Therefore, our data agrees with efficiencies, that have been reported in literature for wild type and truncated versions of mDNMT1 [[#References|[15]]] [[#References|[16]]] .Overall, the reaction kinetics of the linear protein seem to be promising for later use in a PCR2.0, assuming that circularization of the protein will not ablate its function. <br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 7) Maintenance Methylation deposition of linear Dnmt1(731-1602) over time|<br />
descr= Purified linear mDNMT1(731-1602) was incubated with 40mer DNA template (see Figure4) for indicated times at 37°C. Subsequently the samples were digested with Sau3AI. Fragments were separated by PAGE and stained with ethidium bromide. (Homo me= homomethylated substrate, control for enzyme inhibition through methylation, Hemi me=hemimethylated substrate positive control for digest).|<br />
file=activityovertime.jpg}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 8) <i>De novo</i> methylation activity of mDNMT1|<br />
descr=Purified linear mDNMT1(731-1602) was incubated with 40mer DNA template (see Figure4) for indicated times at 37°C. Subsequently the samples were digested with HpaII. Fragments were separated using gel-electrophoresis and visualized with ethidiumbromid staining. (Homo me= homomethylated substrate,control for enzyme inhibition through methylation, Hemi me=hemimethylated substrate positive control for digest).|<br />
file=specificityovertime.jpg}}<br />
<br />
In oder to be used in the targeted PCR2.0 mDNMT1 needs to be highly specific for hemi-methylated sites and should not display <i>de novo</i> methylation activity. Therefore, we included HpaII digestion in our assay to determine <i>de novo</i> methylation of CpG sites. The first results indicate that the purified mDNMT1(731-1602) does not exhibit de novo methylation activity (Figure 8). Even with increasing DNMT1 incubation time, HpaII is not repressed which indicates absence of <i>de novo</i> methylation. <br />
However, the analysis of the gel pictures is difficult since restriction efficiency of HpaII was not 100% as it can be seen from the hemimethylated control. Therefore, uncut template which is not resulting from unspecific methylation activity of the enzyme complicates quantitative analysis. Further tests, especially with the circularized protein will have to be performed to confirm these initial data.<br />
<div style="clear:both;"></div><br />
<br />
=Circular mDNMT1(731-1602) - The pivotal element of PCR2.0=<br />
<br />
==Expression and purification of mDNMT1(731-1602) for circularization==<br />
<br />
After we succeeded in purifying active and specific linear mDNMT1, we went on with cloning, expression and purification of the circular DNMTs. <br />
<br />
Expression and purification of mDNMT1(731-1602) for the intein as well as the sortase approach were conducted as they had been established for the linear DNMT1. Elutions from the His-Trap showed an enrichment and concentration of peptides that correlate with the expected molecular weight of the truncated mDNMT1(731-1602) (Figure 9A and 9B). Nevertheless, purification with a His-Trap alone does not seem to exclude a variety of impurities that either result from degraded mDNMT1 that still contains a His-Tag or unspecific binding to the affinity column. Advanced protocols as they were used by Song and collegues [[#References|[19]]] for analysis of the proteins crystal structure are therefore including several more steps. Nevertheless, additional steps of purification increase the risk of reducing the overall protein yield and activity, necessitating greater amounts of starting material. Since the active protein should already be included and efficiently enriched in our sample despite of present impurities, we continued with the functional analysis of the sample.<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 9A) Efficiency of mDNMT1(731-1602) purification for the intein approach|<br />
descr=Induction of Rosetta (DE3) and purification were performed as described above. After each step, samples were collected for analysis by Coomassie stained SDS-PAGE. Samples are numbered according to their chronology of collection. Arrow indicates mDNMT1(731-1602).|<br />
file=DNMT1-intein-Purification3.png}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 9B) Sortase approach - Efficiency of Purification|<br />
descr=Induction of Rosetta (DE3) and purification were performed as described above. After each step, samples were collected for analysis by Coomassie stained SDS-PAGE. Samples are numbered according to their chronology of collection. Arrow indicates mDNMT1(731-1602).|<br />
file=DNMT1-sortase-Purification2.png}}<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 10) Expression and Purification of intein and sortase constructs with different linkers|<br />
descr=Expression and Purification of different constructs in Rosetta (DE3) was performed as described above. Unpurified as well as purified samples were collected for analysis by Coomassie stained SDS-PAGE. Ctrl indicates the untransformed control. D= no linker, R= Rigid linker, F=Flexible linker, L= linear. Arrow indicates peptides with molecular weight corresponding to mDNMT1(731-1602). Asterix indicates possibly spliced Intein constructs.|<br />
file=DNMT1-Purification1.png}}<br />
<br />
Our approach of heat-stabilization includes the testing of different linkers that had been calculated by our [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software CRAUT Linker Software]. We tested a flexible linker, consisting mostly of glycines and serines and a rigid linker, which was introduced to maximize stabilization of the protein through cicularization. Purification of the mDNMT1(731-1602) variants with different linkers for circularization was successful (Figure 10), but the yields were lower compared to different batches of linear mDNMT1(731-1602) that had been produced in a similar way. <br />
Interestingly, a shift between mDNMT1(731-1602) expressed from the intein construct compared to the sortase or linear versions was detected. Since protein circularization via inteins is an autocatalytic process that takes place inside the bacteria, this shift could possibly result from successful splicing activity of the inteins. The resulting product lacks Smt3 and is therefore approximately 30kDa smaller than its precursor (Figure 10, indicated by an asterix). Since intein splicing likely occurred, flexible- and rigid-linked-DNMT1 could be circularized. <br />
<br />
To prove circularization by inteins, we cooperated with the core facility Protein Analysis of the German Cancer Research Center (Dkfz.). In the facility, circularized and linear DNMT1 were extracted from a provided gel, digested with trypsin and analyzed by ESI mass spectrometry. Unfortunately, we do not have the final results yet but are confident to be able to present them at the jamboree.<br />
<br />
As against that mDNMT1(731-1602) with sortase recognition sequences needs to be further processed before a circular protein is obtained. Unfortunately, TEV cleavage of the purified protein, which is necessary for subsequent circularization with sortase was not very efficient. Therefore sortase treatment did not yield in a detectable amount of processed mDNMT1(731-1602). Therefore, we fully concentrated on the promising intein approach for further mDNMT1(731-1602) circularization.<br />
<br />
<div style="clear:both;"></div><br />
<br />
==Thermal stability of circular mDNMT1(721-1602)==<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 8) Methylation after heat shock|<br />
descr=Circular DNMT1 with rigid linker (RC), circular DNMT1 with flexible linker (FC) and linear DNMT1 were heat shocked for 5 sec at 55°C and 60°C. |<br />
file=heatshock55-60.jpg}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 9) Persistance of methylation activity at high temperatures|<br />
descr=Circular DNMT1 with rigid linker (RC), circular DNMT1 with flexible linker (FC) and linear DNMT1 were heat shocked for 5 sec at 65°C and 72°C.Methylation after one hour was measured. A darker upper band for FC is even visible by eye, indication higher methylation activity.|<br />
file=heatshock65-72.jpg}}<br />
<br />
In our final assay we examined the effect of an heat shock on the remaining activity of the different Dnmt1 enzymes to proof the ability of circular enzyme to withstand higher temperatures. We wanted to produce evidence for the feasibility of a PCR 2.0 using circularisation.<br />
Behaviour after heat treatment of pre-used linear Dnmt1 (L) was compared to Dnmt1 with rigid linker (RC) and flexible linker (FC)<br />
The proteins were exposed to temperatures between 55°C and 72°C, temperatures that would also be reached in a PCR. The heat shock was conducted for 5 sec and DNA substrate was added afterwards at 37°C for methylation to occur.<br />
Looking at the gel picture (Figure?) a darker upper band indicating more methylated substrate for the circular Dnmt1 with flexible Linker at all temperatures is even visible by eye, suggesting more remained stability. To confirm this results, the intensity of the gel bands were analysed using Image J. Figure ? highlights the remaining activity over temperature after normalisation to the control at 37°C the ration of methylated DNA was normalised to activity at 37°C. Indeed activity of circular DNMT1 with the flexible linker (black) is exciding the linear and ciruclar with rigid linker at least double. Very surprisingly, the methylation activity of circular DNMT1 with a flexible linker seems to increase with higher temperatures. <br />
<br />
For significant evaluation, more data has to be collected. When applying Michael Menten kinetics , the methylation reaction for the controls already approaches saturation therefore the normalisation to the original activity might be defective. For further improvement of the assay, less volume of DNMT1 has to be taken for the enzyme reaction.<br />
<br />
=Discussion=<br />
<br />
== Achievements and impact==<br />
Taking everything together we showed that introduction of inteins represents a feasable approach for circularisation of mDNMT1(731-1602). The post translational modification is conceivably increasing heat stability of the protein without impeding its specific methylation activity. <br />
<br />
We proved that circularization with inteins is a rather easy method to increase heat stability of complex and sensitive proteins like mDNMT1(731-1602). In comparison, other methods for stabilization of proteins such as introduction of disulfide bonds require sophisticated structure analysis as well as non-physiological reaction conditions[[#References|[20]]] [[#References|[21]]]. <br />
<br />
The development and optimization of our CRAUT linker software has been an essential element in linking C- and N-terminus of mDNMT1(731-1602) while bypassing the active site of the protein and preserving its natural function. The two different linker compositions resulted in divergent heat stabilities of the protein. Therefore, software optimized linkers with designed properties have resulted in successful narrowing down of the infinite amount of possibilities to circularize mDNMT1(731-1602). Further experimental data of enzyme-specific inker variants will help to optimize the approach.<br />
<br />
Circularized mDNMT1(731-1602) represents a valuable tool for the realization of a methylation maintaining PCR2.0. This foundational advance aims to enable broad functional analysis of DNA methylation patterns despite limited amounts of primary material. Even though detection of methylation patterns has been established in a high-throughput manner by using the principle of bisulfite sequencing, handling of limited amounts of primary material still represents a major challenge. Approaches including single molecule sequencing have been reported, but have not been established in an affordable and efficient manner yet [[#References|[27]]]. Furthermore, these methods are limited to the detection of methylation patterns and do not allow functional analysis without complete analysis and cost-intensive synthesis. Therefore, our PCR2.0 aims to revolutionize the field of epigenetics by linking complex methylation patterns to their actual function. <i>In vitro</i> as well as <i>in vivo</i> experiments, requering large amounts of primary material will be feasible and more reproducible by using our PCR2.0. <br />
<br />
A very interesting application for the pharmaceutical use of our PCR2.0 could be the large scale production of methylated gene therapy vectors. Since, methylation of gene transfer vectors has been reported to extend transgene product expression by lowering cellular immunogenicity, the efficiency gene therapies could be easily improved with our method. [[#References|[22]]].<br />
<br />
==Further possible improvements==<br />
Additionally to the circularization of mDNMT1(731-1602), further approaches can be taken into account to realize a methylation maintaining PCR2.0. Those include further stabilization of the protein either by enhancing the hydrophobicity of the proteins core or direct links to limit possible conformations [[#References|[23]]].<br />
<br />
Another upcoming topic is the method of low temperature PCRs that exploit the function of helicases to segregate DNA strands. Even though these PCRs are not performed at high temperatures comparable to conventional PCRs, none of them has reached widespread establishment yet. Main reasons are that isothermal PCRs require either too long incubation times per cycle [[#References|[24]]] or still exceed physiological temperatures of most enzymes [[#References|[25]]].<br />
<br />
A still challenging factor for PCR2.0 is the DNMT1 cofactor S-adenosylmethionine (SAM) that exhibits only low stability at high temperatures Matos, J. & Wong, C. S-adenosylmethionine: Stability and stabilization. Bioorg. Chem. 80, 71–80 (1987). During our experiments, there has not been observed any limitation due to this instability. Nevertheless, addition of the quite cheap methyl-donor after each cycle of replication could circumvent this problem until a more stable variant of the cofactor is available. <br />
<br />
Moreover, the PCR2.0 reaction could be complemented with other enzymes such as UHRF1, that have been reported to increase the specificity of mDNMT1(731-1602) [[#References|[19]]] Bashtrykov, P., Jankevicius, G., Jurkowska, R. Z., Ragozin, S. & Jeltsch, A. The UHRF1 protein stimulates the activity and specificity of the maintenance DNA methyltransferase DNMT1 by an allosteric mechanism. J. Biol. Chem. 289, 4106–15 (2014). The wild type DNMT1 is known to be associated with the replication complex for direction of its activity. Therefore design of an appropriate Polymerase-DNMT-fusion protein could imitate this connection and therefore further reduce stochastic methylation events. [[#References|[26]]].<br />
<br />
Overall, the iGEM Team Heidelberg’s intein toolbox in combination with the CRAUT linker software have contributed strongly to the foundational advance of establishing a PCR2.0.<br />
<br />
=References=<br />
[1] Meissner, A. et al. Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature 454, 766–70 (2008).<br />
<br />
[2] Watanabe, M., Yuzawa, H., Handa, N., & Kobayashi, I.. Hyperthermophilic DNA methyltransferase M.PabI from the archaeon Pyrococcus abyssi. Applied and Environmental Microbiology, 72(8), 5367–75 (2006).<br />
<br />
[3] Vieille, C. & Zeikus, G. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65, 1–43 (2001).<br />
<br />
[4] Antos, J. M., Popp, M. W.-L., Ernst, R., Chew, G.-L., Spooner, E., & Ploegh, H. L.. A straight path to circular proteins. The Journal of Biological Chemistry, 284(23), 16028–36 (2009).<br />
<br />
[5] Tam, J. P. & Wong, C. T. T. Chemical synthesis of circular proteins. J. Biol. Chem. 287, 27020–5 (2012).<br />
<br />
[6] Robertson, K. D. DNA methylation and human disease. Nat. Rev. Genet. 6, 597–610 (2005).<br />
<br />
[7] Jaenisch, R. & Bird, A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat. Genet. 33 Suppl, 245–54 (2003).<br />
<br />
[8] Ehrlich, M.. Amount and distribution of 5-methycytosine in human DNA from different types of tissues or cells. Nucleic Acids Res. 10: 2709–2721 (1982).<br />
<br />
[9] Lister, R. & Ecker, J. Finding the fifth base: genome-wide sequencing of cytosine methylation. Genome Res. 959–966 (2009).<br />
<br />
[10] Bird, A.. DNA methylation patterns and epigenetic memory. Genes & Development 16, 6-21 (2002).<br />
<br />
[11] Jaenisch, R., and Bird, A.. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet 33, 245-254 (2003).<br />
<br />
[12] Weichenhan, D., and Plass, C.. The evolving epigenome. Human Molecular Genetics 22, R1 (2013).<br />
<br />
[13] Robertson, K. D. DNA methylation and human disease. Nat. Rev. Genet. 6, 597–610 (2005).<br />
<br />
[14] Jurkowska, R. Z., Jurkowski, T. P. & Jeltsch, A. Structure and function of mammalian DNA methyltransferases. Chembiochem 12, 206–22 (2011).<br />
<br />
[15] Song, J., Teplova, M., Ishibe-Murakami, S. & Patel, D. J. Structure-based mechanistic insights into DNMT1-mediated maintenance DNA methylation. Science 335, 709–12 (2012).<br />
<br />
[16] Bashtrykov, P. et al. Specificity of Dnmt1 for methylation of hemimethylated CpG sites resides in its catalytic domain. Chem. Biol. 19, 572–8 (2012).<br />
<br />
[17] Lee, C.-D., Sun, H.-C., Hu, S.-M., Chiu, C.-F., Homhuan, A., Liang, S.-M., Wang, T.-F. An improved SUMO fusion protein system for effective production of native proteins. Protein Science : A Publication of the Protein Society, 17(7), 1241–8 (2008).<br />
<br />
[18] Benov, L. & Al-ibraheem, J. Disrupting Escherichia coli : A Comparison of Methods. 35, 428–431 (2002).<br />
<br />
[19] Song, J., Rechkoblit, O., Bestor, T. H. & Patel, D. J. Structure of DNMT1-DNA complex reveals a role for autoinhibition in maintenance DNA methylation. Science 331, 1036–40 (2011).<br />
<br />
[20] Betz, S. F. Disulfide bonds and the stability of globular proteins. 1551–1558 (1993).<br />
<br />
[21] Bulaj, G. Formation of disulfide bonds in proteins and peptides. Biotechnol. Adv. 23, 87–92 (2005).<br />
<br />
[22] Reyes-Sandoval, a & Ertl, H. C. J. CpG methylation of a plasmid vector results in extended transgene product expression by circumventing induction of immune responses. Mol. Ther. 9, 249–61 (2004).<br />
<br />
[23] Vieille, C. & Zeikus, G. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65, 1–43 (2001).<br />
<br />
[24] Vincent, M., Xu, Y. & Kong, H. Helicase-dependent isothermal DNA amplification. EMBO Rep. 5, 795–800 (2004).<br />
<br />
[25] Xu, G. et al. Cross priming amplification: mechanism and optimization for isothermal DNA amplification. Sci. Rep. 2, 246 (2012).<br />
<br />
[26] Vilkaitis, G., Suetake, I., Klimasauskas, S. & Tajima, S. Processive methylation of hemimethylated CpG sites by mouse Dnmt1 DNA methyltransferase. J. Biol. Chem. 280, 64–72 (2005).<br />
<br />
[27] Krueger, F., Kreck, B., Franke, A. & Andrews, S. R. DNA methylome analysis using short bisulfite sequencing data. Nat. Methods 9, 145–51 (2012).<br />
<br />
[28] Smith, Z. D. & Meissner, A. DNA methylation: roles in mammalian development. Nat. Rev. Genet. 14, 204–20 (2013).</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/Project/BackgroundTeam:Heidelberg/Project/Background2014-10-17T19:46:41Z<p>Huhny: </p>
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<div>{{:Team:Heidelberg/templates/wikipage_new|<br />
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<hr />
<div><br/><br />
In the variety of proteins that exist, inteins are clearly among the most mysterious.<br />
They behave in such a peculiarly way that scientists are still puzzle about their initial role in the host organisms and are even more astonished when discovering their endless seeming abilities.<br />
<br />
Inteins are integrated as extraneous polypeptide sequences into ordinary proteins. They do not contribute to the original protein function but perform an autocatalytic splicing reaction after protein translation. Analog to intron splicing on RNA level, this posttranslational modification was named protein splicing. Consequentially the protein segments were called inteins, derived from “internal protein sequence”, and the flanking protein chains exteins, “external protein sequences”. Inteins excise themselves out of the host protein while reconnecting the remaining N and C exteins via a new peptide bond. Despite this immense invasion the original protein regains its normal structure and function after splicing [[#References|[1]]]. <br />
<br />
<div><br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 1) Trans-splicing mechanism reaction by split inteins. |<br />
descr=The N-Intein is fused to the C’ terminal end of the N-extein. Complementary, C-Intein is located at the N’ end of the C-extein. After assembly of the two intein fragments, a splicing reaction takes place, where the intein removes itself from the precursor protein and simultaneously ligates the exteins via a peptide bond.|<br />
file=InteinSplicingReaction.png}}<br />
</div><br />
<br />
==History==<br />
<br />
Inteins were discovered in 1990 when dissimilarities between the mature protein sequence of the yeast vacuolar ATPase (Vma1) and its corresponding mRNA in were investigated. Surprisingly the mature protein had a lower molecular weight than expected from the encoding sequence, indicating the loss of one part of the protein after translation. <br />
Indeed, a region of 454 amino acids was found to be translated and subsequently removed from he Vma1 protein [[#References|[2]]] [[#References|[3]]]. Since the discovery of the Vma1 intein, over 600 different inteins have been reported in all three domains of life and viruses [[#References|[4]]]. <br />
<br />
Dependent on the organism they belong to, the intein’s name consists out of the genus and species abbreviation followed by the host gene [[#References|[5]]].<br />
<br />
==Evolution: Inteins as parasitic genes==<br />
Even after many years of research the initial role of inteins in their host organism remains still unclear.<br />
Many natural inteins contain a homing endonuclease (HEN) domain, an enzyme that cleaves DNA and includes new sequences at homing sites via homologous recombination or reverse transcription. This characteristic led to the perception of the intein as selfish DNA sequence - a gene generating copies of itself without an advantage for the host organism [[#References|[6]]]. This allows horizontal gene transfer of inteins. [[#References|[7]]]. Supporting this hypothesis: 27% of the inteins host proteins are related to DNA metabolism and involved in DNA replication or repair. This ensures expression of the inteins and the HEN domain during DNA replication, where they can take advantage of the DNA replication and repair system for introducing changes in the DNA [[#References|[8]]].<br />
<br />
However recent work on conditional protein splicing has shown sensitivity of some inteins to redox state, temperature and small molecules (reviewed in [[#References|[9]]], [[#References|[10]]]). <br />
This might be a sign for inteins having a role in post-translational protein regulation upon environmental signals. Inserted close to the catalytic core of the protein the protein remains inactive until the splicing reaction has taken place. Evolutionary said, once selfish inteins might have adapted due to selection pressure to be a beneficial mechanism for the host cell [[#References|[8]]].<br />
<br />
==Classification and Structure==<br />
<br />
Inteins are divided into three groups: bifunctional (large) inteins, mini inteins and split inteins.<br />
Large inteins carry both a splicing domain and an endonuclease (HEN) domain whereas mini inteins lack the HEN domain [[#References|[11]]]. <br />
The most promising inteins for biotechnology are split inteins, which are basically mini-inteins, but divided in two fragments and expressed separately with different proteins. After translation, they assemble with high affinity to be catalytic active. Split enzymes can be found naturally [[#References|[12]]] but can also be artificially designed [[#References|[13]]]. <br />
<br />
In our project we concentrated on split inteins, as they present a powerful tool for inserting posttranslational modifications in biotechnology. We have characterized the most promising inteins on our Intein database.<br />
<br />
The structure of inteins contains several conserved motifs. The splicing domains are located at the N and C terminal ends. Before splicing can take place the intein rearranges itself from the linear form it has been translated into to a horseshoe like structure where the termini are broad in close proximity to form the catalytic core [[#References|[14]]]. <br />
<br />
==A detailed description of trans splicing reaction==<br />
<br />
In contrast to the mini and large inteins that mediate cis-splicing reactions, split inteins are responsible for trans-splicing and fusion of protein parts. The trans-splicing reaction can be divided into the following steps:<br />
<br />
# N-Intein and C-Intein first assemble together to form a dimer like structure with a newly formed catalytic core next to the exteins.<br />
<br />
# The tertiary structure of the intein, once correctly formed, facilitates an ''N&rarr;O/S'' acyl rearrangement at its N-terminal serine or cysteine residue. The result is an ester or thioester bond, respectively, between the side-chain and the peptide backbone of the N-extein.<br />
<br />
# The two exteins are then linked by trans(thio)esterification involving the N-terminal serine or cysteine residue of the C-extein. The C-terminus of the N-extein is now covalently bound to the N-terminal side-chain of the C-extein, while its backbone still retains its normal peptide bond to the intein.<br />
<br />
# The C-terminal asparagine of the intein undergoes self-cyclisation to form a succinimidyl moiety. The peptide bond between the intein and the extein is thereby broken, resolving the branched intermediate.<br />
<br />
# In a final reaction, an ''O/S&rarr;N'' acyl shift results in the two exteins now being linked by an amide bond, indistinguishable from a ribosome-assembled fusion protein.<br />
(as reviewed in [[#References|[15]]]) <br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 2) Trans-splicing mechanism reaction by split inteins. |<br />
descr=Picture taken from [[#References|[16]]]|<br />
file=Trans_splicing_OCreaction}}<br />
<br />
For intein efficiency not only the structure of the intein domain itself is important but also the nature of the flanking extein residues, as they are heavily involved in the splicing reaction. It has been shown, that the first residue has to be a nucleophilic amino acid, preferable Cysteine, Serine or Threonine. Effectiveness of the intein can be mold by changing the following amino acids [[#References|[17]]].<br />
<br />
==Use of inteins in molecular biology and biotechnology==<br />
<br />
Due to their distinct characteristics intein are a powerful tool for molecular biology and biotechnology. Performing an autocatalytic reaction, the inteins are neither dependent on their host protein nor on any other additional substrate. This makes it them broadly applicable for in vivo and in vitro applications. [[#References|[16]]].<br />
<br />
Inteins have been used for protein purification, achieving a higher yield of protein due to more specific targeting. The production of semi-synthetic proteins and the attachment of synthetic groups is of huge interest for recombinant proteins. [[#References|[16]]]. Split Intein-mediated Circular Ligation Of Peptides a ProteinS (SICLOPPS) is a method to produce circular peptides of eight amino acids length, which could be potent therapeutic drugs [[#References|[18]]]. Inteins, fused to split fluorescent proteins as reporter and to the protein of investigation have been used to detect protein-protein interactions in vivo (reviewed in [[#References|[10]]]).<br />
Nevertheless most of this applications are performed in vitro and do not exploit the full potential of inteins as regulatory element for post-translational modification. The iGEM Team Heidelberg employs this excellent mechanism of protein splicing to specifically change whole amino-acid sequences and thereby regulate proteins via (dis-) assembly, protein cleavage or circularization of enzymes in vivo. We provide a new foundational advance by introducing the full potential of post-translational modification and therefore a new dimension of genetic engineering to Synthetic Biology.<br />
<br />
=References=<br />
<br />
[1] Perler FB, Davis EO, Dean GE, Gimble FS, Jack WE, et al. (1994) Protein splicing elements: inteins and exteins–a definition of terms and recommended nomenclature. Nucleic Acids Res 22: 1125–1127<br />
<br />
[2] Kane P.M., et al. Protein Splicing Converts the Yeast TEPI Gene Product to the 69-kD Subunit of the Vacuolar. 13253, (1990)<br />
<br />
[3] Hirata, R. et al. Molecular structure of a gene, VMA1, encoding the catalytic subunit of H(+)-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J. Biol. Chem. 265 , 6726–6733 (1990)<br />
<br />
[4] Perler, F. B. InBase : the Intein Database. 30, 383–384 (2002)<br />
<br />
[5] Perler, F. B. et al. Protein splicing elements : inteins and exteins — a definition of terms and recommended nomenclature. 22, 1125–1127 (1994)<br />
<br />
[6] Barzel, A., Naor, A., Privman, E., Kupiec, M. & Gophna, U. Homing endonucleases residing within inteins: evolutionary puzzles awaiting genetic solutions. Biochem. Soc. Trans. 39, 169–73 (2011)<br />
<br />
[7] Pietrokovski, S. Intein spread and extinction in evolution. Trends Genet. 17, 465-472 (2001)<br />
<br />
[8] Novikova, O., Topilina, N. & Belfort, M. Enigmatic distribution, evolution and function of inteins. J. Biol. Chem. (2014)<br />
<br />
[9] Shah, N. H., and Muir, T. W. Inteins: nature's gift to protein chemists. Chem. Sci. 5, 446- 461 (2014) <br />
<br />
[10] Topilina, N. I. & Mills, K. V. Recent advances in in vivo applications of intein-mediated protein splicing. Mob. DNA 5, 5 (2014)<br />
<br />
[11] Eryilmaz, E., Shah, N., Muir, T. & Cowburn, D. Structural and Dynamical Features of Inteins and Implications on Protein Splicing. J. Biol. Chem. (2014)<br />
<br />
[12] Carvajal-Vallejos, P., Pallissé, R., Mootz, H. D. & Schmidt, S. R. Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. J. Biol. Chem. 287, 28686–96 (2012) <br />
<br />
[13] Lin, Y. et al. Protein trans-splicing of multiple atypical split inteins engineered from natural inteins. PLoS One 8, e59516 (2013). <br />
<br />
[14] Eryilmaz, E., Shah, N., Muir, T. & Cowburn, D. Structural and Dynamical Features of Inteins and Implications on Protein Splicing. J. Biol. Chem. (2014)<br />
<br />
[15] Mills, K. V, Johnson, M. a & Perler, F. B. Protein Splicing: How Inteins Escape from Precursor Proteins. J. Biol. Chem. (2014)<br />
<br />
[16] Mootz, H. D. Split inteins as versatile tools for protein semisynthesis. Chembiochem 10, 2579–89 (2009).<br />
<br />
[17] Amitai, G., Callahan, B. P., Stanger, M. J., Belfort, G. & Belfort, M. Modulation of intein activity by its neighboring extein substrates. Proc. Natl. Acad. Sci. U. S. A. 106, 11005–10 (2009).<br />
<br />
[18] Tavassoli, A. & Benkovic, S. J. Split-intein mediated circular ligation used in the synthesis of cyclic peptide libraries in E. coli. Nat. Protoc. 2, 1126–1133 (2007).</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/backgroundTeam:Heidelberg/pages/background2014-10-17T19:33:27Z<p>Huhny: </p>
<hr />
<div><br/><br />
In the variety of proteins that exist, inteins are clearly among the most mysterious.<br />
They behave in such a peculiarly way that scientists are still puzzle about their initial role in the host organisms and are even more astonished when discovering their endless seeming abilities.<br />
<br />
Inteins are integrated as extraneous polypeptide sequences into ordinary proteins. They do not contribute to the original protein function but perform an autocatalytic splicing reaction after protein translation. Analog to intron splicing on RNA level, this posttranslational modification was named protein splicing. Consequentially the protein segments were called inteins, derived from “internal protein sequence”, and the flanking protein chains exteins, “external protein sequences”. Inteins excise themselves out of the host protein while reconnecting the remaining N and C exteins via a new peptide bond. Despite this immense invasion the original protein regains its normal structure and function after splicing [[#References|[1]]]. <br />
<br />
<div><br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 1) Trans-splicing mechanism reaction by split inteins. |<br />
descr=The N-Intein is fused to the C’ terminal end of the N-extein. Complementary, C-Intein is located at the N’ end of the C-extein. After assembly of the two intein fragments, a splicing reaction takes place, where the intein removes itself from the precursor protein and simultaneously ligates the exteins via a peptide bond.|<br />
file=InteinSplicingReaction.png}}<br />
</div><br />
<br />
==History==<br />
<br />
Inteins were discovered in 1990 when dissimilarities between the mature protein sequence of the yeast vacuolar ATPase (Vma1) and its corresponding mRNA in were investigated. Surprisingly the mature protein had a lower molecular weight than expected from the encoding sequence, indicating the loss of one part of the protein after translation. <br />
Indeed, a region of 454 amino acids was found to be translated and subsequently removed from he Vma1 protein [[#References|[2]]] [[#References|[3]]]. Since the discovery of the Vma1 intein, over 600 different inteins have been reported in all three domains of life and viruses [[#References|[4]]]. <br />
<br />
Dependent on the organism they belong to, the intein’s name consists out of the genus and species abbreviation followed by the host gene [[#References|[5]]].<br />
<br />
==Evolution: Inteins as parasitic genes==<br />
Even after many years of research the initial role of inteins in their host organism remains still unclear.<br />
Many natural inteins contain a homing endonuclease (HEN) domain, an enzyme that cleaves DNA and includes new sequences at homing sites via homologous recombination or reverse transcription. This characteristic led to the perception of the intein as selfish DNA sequence - a gene generating copies of itself without an advantage for the host organism [[#References|[6]]]. This allows horizontal gene transfer of inteins. [[#References|[7]]]. Supporting this hypothesis: 27% of the inteins host proteins are related to DNA metabolism and involved in DNA replication or repair. This ensures expression of the inteins and the HEN domain during DNA replication, where they can take advantage of the DNA replication and repair system for introducing changes in the DNA [#References|[8]]].<br />
<br />
However recent work on conditional protein splicing has shown sensitivity of some inteins to redox state, temperature and small molecules (reviewed in [#References|[9]]], [#References|[10]]]). <br />
This might be a sign for inteins having a role in post-translational protein regulation upon environmental signals. Inserted close to the catalytic core of the protein the protein remains inactive until the splicing reaction has taken place. Evolutionary said, once selfish inteins might have adapted due to selection pressure to be a beneficial mechanism for the host cell [#References|[8]]].<br />
<br />
==Classification and Structure==<br />
<br />
Inteins are divided into three groups: bifunctional (large) inteins, mini inteins and split inteins.<br />
Large inteins carry both a splicing domain and an endonuclease (HEN) domain whereas mini inteins lack the HEN domain [#References|[11]]]. <br />
The most promising inteins for biotechnology are split inteins, which are basically mini-inteins, but divided in two fragments and expressed separately with different proteins. After translation, they assemble with high affinity to be catalytic active. Split enzymes can be found naturally [#References|[12]]] but can also be artificially designed [#References|[13]]]. <br />
<br />
In our project we concentrated on split inteins, as they present a powerful tool for inserting posttranslational modifications in biotechnology. We have characterized the most promising inteins on our Intein database.<br />
<br />
The structure of inteins contains several conserved motifs. The splicing domains are located at the N and C terminal ends. Before splicing can take place the intein rearranges itself from the linear form it has been translated into to a horseshoe like structure where the termini are broad in close proximity to form the catalytic core [#References|[14]]]. <br />
<br />
==A detailed description of trans splicing reaction==<br />
<br />
In contrast to the mini and large inteins that mediate cis-splicing reactions, split inteins are responsible for trans-splicing and fusion of protein parts. The trans-splicing reaction can be divided into the following steps:<br />
<br />
# N-Intein and C-Intein first assemble together to form a dimer like structure with a newly formed catalytic core next to the exteins.<br />
<br />
# The tertiary structure of the intein, once correctly formed, facilitates an ''N&rarr;O/S'' acyl rearrangement at its N-terminal serine or cysteine residue. The result is an ester or thioester bond, respectively, between the side-chain and the peptide backbone of the N-extein.<br />
<br />
# The two exteins are then linked by trans(thio)esterification involving the N-terminal serine or cysteine residue of the C-extein. The C-terminus of the N-extein is now covalently bound to the N-terminal side-chain of the C-extein, while its backbone still retains its normal peptide bond to the intein.<br />
<br />
# The C-terminal asparagine of the intein undergoes self-cyclisation to form a succinimidyl moiety. The peptide bond between the intein and the extein is thereby broken, resolving the branched intermediate.<br />
<br />
# In a final reaction, an ''O/S&rarr;N'' acyl shift results in the two exteins now being linked by an amide bond, indistinguishable from a ribosome-assembled fusion protein.<br />
(as reviewed in [#References|[15]]]) <br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 2) Trans-splicing mechanism reaction by split inteins. |<br />
descr=Picture taken from [#References|[16]]]|<br />
file=Trans_splicing_OCreaction}}<br />
<br />
For intein efficiency not only the structure of the intein domain itself is important but also the nature of the flanking extein residues, as they are heavily involved in the splicing reaction. It has been shown, that the first residue has to be a nucleophilic amino acid, preferable Cysteine, Serine or Threonine. Effectiveness of the intein can be mold by changing the following amino acids [#References|[17]]].<br />
<br />
==Use of inteins in molecular biology and biotechnology==<br />
<br />
Due to their distinct characteristics intein are a powerful tool for molecular biology and biotechnology. Performing an autocatalytic reaction, the inteins are neither dependent on their host protein nor on any other additional substrate. This makes it them broadly applicable for in vivo and in vitro applications. [#References|[16]]].<br />
<br />
Inteins have been used for protein purification, achieving a higher yield of protein due to more specific targeting. The production of semi-synthetic proteins and the attachment of synthetic groups is of huge interest for recombinant proteins. [#References|[16]]]. Split Intein-mediated Circular Ligation Of Peptides a ProteinS (SICLOPPS) is a method to produce circular peptides of eight amino acids length, which could be potent therapeutic drugs [#References|[18]]]. Inteins, fused to split fluorescent proteins as reporter and to the protein of investigation have been used to detect protein-protein interactions in vivo (reviewed in [#References|[10]]]).<br />
Nevertheless most of this applications are performed in vitro and do not exploit the full potential of inteins as regulatory element for post-translational modification. The iGEM Team Heidelberg employs this excellent mechanism of protein splicing to specifically change whole amino-acid sequences and thereby regulate proteins via (dis-) assembly, protein cleavage or circularization of enzymes in vivo. We provide a new foundational advance by introducing the full potential of post-translational modification and therefore a new dimension of genetic engineering to Synthetic Biology.<br />
<br />
=References=<br />
<br />
[1] Perler FB, Davis EO, Dean GE, Gimble FS, Jack WE, et al. (1994) Protein splicing elements: inteins and exteins–a definition of terms and recommended nomenclature. Nucleic Acids Res 22: 1125–1127<br />
<br />
[2] Kane P.M., et al. Protein Splicing Converts the Yeast TEPI Gene Product to the 69-kD Subunit of the Vacuolar. 13253, (1990)<br />
<br />
[3] Hirata, R. et al. Molecular structure of a gene, VMA1, encoding the catalytic subunit of H(+)-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J. Biol. Chem. 265 , 6726–6733 (1990)<br />
<br />
[4] Perler, F. B. InBase : the Intein Database. 30, 383–384 (2002)<br />
<br />
[5] Perler, F. B. et al. Protein splicing elements : inteins and exteins — a definition of terms and recommended nomenclature. 22, 1125–1127 (1994)<br />
<br />
[6] Barzel, A., Naor, A., Privman, E., Kupiec, M. & Gophna, U. Homing endonucleases residing within inteins: evolutionary puzzles awaiting genetic solutions. Biochem. Soc. Trans. 39, 169–73 (2011)<br />
<br />
[7] Pietrokovski, S. Intein spread and extinction in evolution. Trends Genet. 17, 465-472 (2001)<br />
<br />
[8] Novikova, O., Topilina, N. & Belfort, M. Enigmatic distribution, evolution and function of inteins. J. Biol. Chem. (2014)<br />
<br />
[9] Shah, N. H., and Muir, T. W. Inteins: nature's gift to protein chemists. Chem. Sci. 5, 446- 461 (2014) <br />
<br />
[10] Topilina, N. I. & Mills, K. V. Recent advances in in vivo applications of intein-mediated protein splicing. Mob. DNA 5, 5 (2014)<br />
<br />
[11] Eryilmaz, E., Shah, N., Muir, T. & Cowburn, D. Structural and Dynamical Features of Inteins and Implications on Protein Splicing. J. Biol. Chem. (2014)<br />
<br />
[12] Carvajal-Vallejos, P., Pallissé, R., Mootz, H. D. & Schmidt, S. R. Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. J. Biol. Chem. 287, 28686–96 (2012) <br />
<br />
[13] Lin, Y. et al. Protein trans-splicing of multiple atypical split inteins engineered from natural inteins. PLoS One 8, e59516 (2013). <br />
<br />
[14] Eryilmaz, E., Shah, N., Muir, T. & Cowburn, D. Structural and Dynamical Features of Inteins and Implications on Protein Splicing. J. Biol. Chem. (2014)<br />
<br />
[15] Mills, K. V, Johnson, M. a & Perler, F. B. Protein Splicing: How Inteins Escape from Precursor Proteins. J. Biol. Chem. (2014)<br />
<br />
[16] Mootz, H. D. Split inteins as versatile tools for protein semisynthesis. Chembiochem 10, 2579–89 (2009).<br />
<br />
[17] Amitai, G., Callahan, B. P., Stanger, M. J., Belfort, G. & Belfort, M. Modulation of intein activity by its neighboring extein substrates. Proc. Natl. Acad. Sci. U. S. A. 106, 11005–10 (2009).<br />
<br />
[18] Tavassoli, A. & Benkovic, S. J. Split-intein mediated circular ligation used in the synthesis of cyclic peptide libraries in E. coli. Nat. Protoc. 2, 1126–1133 (2007).</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/backgroundTeam:Heidelberg/pages/background2014-10-17T19:33:09Z<p>Huhny: </p>
<hr />
<div><br/><br />
In the variety of proteins that exist, inteins are clearly among the most mysterious.<br />
They behave in such a peculiarly way that scientists are still puzzle about their initial role in the host organisms and are even more astonished when discovering their endless seeming abilities.<br />
<br />
Inteins are integrated as extraneous polypeptide sequences into ordinary proteins. They do not contribute to the original protein function but perform an autocatalytic splicing reaction after protein translation. Analog to intron splicing on RNA level, this posttranslational modification was named protein splicing. Consequentially the protein segments were called inteins, derived from “internal protein sequence”, and the flanking protein chains exteins, “external protein sequences”. Inteins excise themselves out of the host protein while reconnecting the remaining N and C exteins via a new peptide bond. Despite this immense invasion the original protein regains its normal structure and function after splicing [[#References|[1]]]. <br />
<br />
<div><br />
{{:Team:Heidelberg/templates/image-quarter|<br />
align=right|<br />
caption=Figure 1) Trans-splicing mechanism reaction by split inteins. |<br />
descr=The N-Intein is fused to the C’ terminal end of the N-extein. Complementary, C-Intein is located at the N’ end of the C-extein. After assembly of the two intein fragments, a splicing reaction takes place, where the intein removes itself from the precursor protein and simultaneously ligates the exteins via a peptide bond.|<br />
file=InteinSplicingReaction.png}}<br />
</div><br />
<br />
==History==<br />
<br />
Inteins were discovered in 1990 when dissimilarities between the mature protein sequence of the yeast vacuolar ATPase (Vma1) and its corresponding mRNA in were investigated. Surprisingly the mature protein had a lower molecular weight than expected from the encoding sequence, indicating the loss of one part of the protein after translation. <br />
Indeed, a region of 454 amino acids was found to be translated and subsequently removed from he Vma1 protein [[#References|[2]]] [[#References|[3]]]. Since the discovery of the Vma1 intein, over 600 different inteins have been reported in all three domains of life and viruses [[#References|[4]]]. <br />
<br />
Dependent on the organism they belong to, the intein’s name consists out of the genus and species abbreviation followed by the host gene [[#References|[5]]].<br />
<br />
==Evolution: Inteins as parasitic genes==<br />
Even after many years of research the initial role of inteins in their host organism remains still unclear.<br />
Many natural inteins contain a homing endonuclease (HEN) domain, an enzyme that cleaves DNA and includes new sequences at homing sites via homologous recombination or reverse transcription. This characteristic led to the perception of the intein as selfish DNA sequence - a gene generating copies of itself without an advantage for the host organism [[#References|[6]]]. This allows horizontal gene transfer of inteins. [[#References|[7]]]. Supporting this hypothesis: 27% of the inteins host proteins are related to DNA metabolism and involved in DNA replication or repair. This ensures expression of the inteins and the HEN domain during DNA replication, where they can take advantage of the DNA replication and repair system for introducing changes in the DNA [#References|[8]]].<br />
<br />
However recent work on conditional protein splicing has shown sensitivity of some inteins to redox state, temperature and small molecules (reviewed in [#References|[9]]], [#References|[10]]]). <br />
This might be a sign for inteins having a role in post-translational protein regulation upon environmental signals. Inserted close to the catalytic core of the protein the protein remains inactive until the splicing reaction has taken place. Evolutionary said, once selfish inteins might have adapted due to selection pressure to be a beneficial mechanism for the host cell [#References|[8]]].<br />
<br />
==Classification and Structure==<br />
<br />
Inteins are divided into three groups: bifunctional (large) inteins, mini inteins and split inteins.<br />
Large inteins carry both a splicing domain and an endonuclease (HEN) domain whereas mini inteins lack the HEN domain [#References|[11]]]. <br />
The most promising inteins for biotechnology are split inteins, which are basically mini-inteins, but divided in two fragments and expressed separately with different proteins. After translation, they assemble with high affinity to be catalytic active. Split enzymes can be found naturally [#References|[12]]] but can also be artificially designed [#References|[13]]]. <br />
<br />
In our project we concentrated on split inteins, as they present a powerful tool for inserting posttranslational modifications in biotechnology. We have characterized the most promising inteins on our Intein database.<br />
<br />
The structure of inteins contains several conserved motifs. The splicing domains are located at the N and C terminal ends. Before splicing can take place the intein rearranges itself from the linear form it has been translated into to a horseshoe like structure where the termini are broad in close proximity to form the catalytic core [#References|[14]]]. <br />
<br />
==A detailed description of trans splicing reaction==<br />
<br />
In contrast to the mini and large inteins that mediate cis-splicing reactions, split inteins are responsible for trans-splicing and fusion of protein parts. The trans-splicing reaction can be divided into the following steps:<br />
<br />
# N-Intein and C-Intein first assemble together to form a dimer like structure with a newly formed catalytic core next to the exteins.<br />
<br />
# The tertiary structure of the intein, once correctly formed, facilitates an ''N&rarr;O/S'' acyl rearrangement at its N-terminal serine or cysteine residue. The result is an ester or thioester bond, respectively, between the side-chain and the peptide backbone of the N-extein.<br />
<br />
# The two exteins are then linked by trans(thio)esterification involving the N-terminal serine or cysteine residue of the C-extein. The C-terminus of the N-extein is now covalently bound to the N-terminal side-chain of the C-extein, while its backbone still retains its normal peptide bond to the intein.<br />
<br />
# The C-terminal asparagine of the intein undergoes self-cyclisation to form a succinimidyl moiety. The peptide bond between the intein and the extein is thereby broken, resolving the branched intermediate.<br />
<br />
# In a final reaction, an ''O/S&rarr;N'' acyl shift results in the two exteins now being linked by an amide bond, indistinguishable from a ribosome-assembled fusion protein.<br />
(as reviewed in [#References|[15]]]) <br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 2) Trans-splicing mechanism reaction by split inteins. |<br />
descr=Picture taken from [#References|[16]]]|<br />
file=Trans_splicing_OCreaction}}<br />
<br />
For intein efficiency not only the structure of the intein domain itself is important but also the nature of the flanking extein residues, as they are heavily involved in the splicing reaction. It has been shown, that the first residue has to be a nucleophilic amino acid, preferable Cysteine, Serine or Threonine. Effectiveness of the intein can be mold by changing the following amino acids [#References|[17]]].<br />
<br />
==Use of inteins in molecular biology and biotechnology==<br />
<br />
Due to their distinct characteristics intein are a powerful tool for molecular biology and biotechnology. Performing an autocatalytic reaction, the inteins are neither dependent on their host protein nor on any other additional substrate. This makes it them broadly applicable for in vivo and in vitro applications. [#References|[16]]].<br />
<br />
Inteins have been used for protein purification, achieving a higher yield of protein due to more specific targeting. The production of semi-synthetic proteins and the attachment of synthetic groups is of huge interest for recombinant proteins. [#References|[16]]]. Split Intein-mediated Circular Ligation Of Peptides a ProteinS (SICLOPPS) is a method to produce circular peptides of eight amino acids length, which could be potent therapeutic drugs [#References|[18]]]. Inteins, fused to split fluorescent proteins as reporter and to the protein of investigation have been used to detect protein-protein interactions in vivo (reviewed in [#References|[10]]]).<br />
Nevertheless most of this applications are performed in vitro and do not exploit the full potential of inteins as regulatory element for post-translational modification. The iGEM Team Heidelberg employs this excellent mechanism of protein splicing to specifically change whole amino-acid sequences and thereby regulate proteins via (dis-) assembly, protein cleavage or circularization of enzymes in vivo. We provide a new foundational advance by introducing the full potential of post-translational modification and therefore a new dimension of genetic engineering to Synthetic Biology.<br />
<br />
=References=<br />
<br />
[1] Perler FB, Davis EO, Dean GE, Gimble FS, Jack WE, et al. (1994) Protein splicing elements: inteins and exteins–a definition of terms and recommended nomenclature. Nucleic Acids Res 22: 1125–1127<br />
<br />
[2] Kane P.M., et al. Protein Splicing Converts the Yeast TEPI Gene Product to the 69-kD Subunit of the Vacuolar. 13253, (1990)<br />
<br />
[3] Hirata, R. et al. Molecular structure of a gene, VMA1, encoding the catalytic subunit of H(+)-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J. Biol. Chem. 265 , 6726–6733 (1990)<br />
<br />
[4] Perler, F. B. InBase : the Intein Database. 30, 383–384 (2002)<br />
<br />
[5] Perler, F. B. et al. Protein splicing elements : inteins and exteins — a definition of terms and recommended nomenclature. 22, 1125–1127 (1994)<br />
<br />
[6] Barzel, A., Naor, A., Privman, E., Kupiec, M. & Gophna, U. Homing endonucleases residing within inteins: evolutionary puzzles awaiting genetic solutions. Biochem. Soc. Trans. 39, 169–73 (2011)<br />
<br />
[7] Pietrokovski, S. Intein spread and extinction in evolution. Trends Genet. 17, 465-472 (2001)<br />
<br />
[8] Novikova, O., Topilina, N. & Belfort, M. Enigmatic distribution, evolution and function of inteins. J. Biol. Chem. (2014)<br />
<br />
[9] Shah, N. H., and Muir, T. W. Inteins: nature's gift to protein chemists. Chem. Sci. 5, 446- 461 (2014) <br />
<br />
[10] Topilina, N. I. & Mills, K. V. Recent advances in in vivo applications of intein-mediated protein splicing. Mob. DNA 5, 5 (2014)<br />
<br />
[11] Eryilmaz, E., Shah, N., Muir, T. & Cowburn, D. Structural and Dynamical Features of Inteins and Implications on Protein Splicing. J. Biol. Chem. (2014)<br />
<br />
[12] Carvajal-Vallejos, P., Pallissé, R., Mootz, H. D. & Schmidt, S. R. Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. J. Biol. Chem. 287, 28686–96 (2012) <br />
<br />
[13] Lin, Y. et al. Protein trans-splicing of multiple atypical split inteins engineered from natural inteins. PLoS One 8, e59516 (2013). <br />
<br />
[14] Eryilmaz, E., Shah, N., Muir, T. & Cowburn, D. Structural and Dynamical Features of Inteins and Implications on Protein Splicing. J. Biol. Chem. (2014)<br />
<br />
[15] Mills, K. V, Johnson, M. a & Perler, F. B. Protein Splicing: How Inteins Escape from Precursor Proteins. J. Biol. Chem. (2014)<br />
<br />
[16] Mootz, H. D. Split inteins as versatile tools for protein semisynthesis. Chembiochem 10, 2579–89 (2009).<br />
<br />
[17] Amitai, G., Callahan, B. P., Stanger, M. J., Belfort, G. & Belfort, M. Modulation of intein activity by its neighboring extein substrates. Proc. Natl. Acad. Sci. U. S. A. 106, 11005–10 (2009).<br />
<br />
[18] Tavassoli, A. & Benkovic, S. J. Split-intein mediated circular ligation used in the synthesis of cyclic peptide libraries in E. coli. Nat. Protoc. 2, 1126–1133 (2007).</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/backgroundTeam:Heidelberg/pages/background2014-10-17T19:32:09Z<p>Huhny: </p>
<hr />
<div><br/><br />
In the variety of proteins that exist, inteins are clearly among the most mysterious.<br />
They behave in such a peculiarly way that scientists are still puzzle about their initial role in the host organisms and are even more astonished when discovering their endless seeming abilities.<br />
<br />
Inteins are integrated as extraneous polypeptide sequences into ordinary proteins. They do not contribute to the original protein function but perform an autocatalytic splicing reaction after protein translation. Analog to intron splicing on RNA level, this posttranslational modification was named protein splicing. Consequentially the protein segments were called inteins, derived from “internal protein sequence”, and the flanking protein chains exteins, “external protein sequences”. Inteins excise themselves out of the host protein while reconnecting the remaining N and C exteins via a new peptide bond. Despite this immense invasion the original protein regains its normal structure and function after splicing [[#References|[1]]]. <br />
<br />
<div><br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 1) Trans-splicing mechanism reaction by split inteins. |<br />
descr=The N-Intein is fused to the C’ terminal end of the N-extein. Complementary, C-Intein is located at the N’ end of the C-extein. After assembly of the two intein fragments, a splicing reaction takes place, where the intein removes itself from the precursor protein and simultaneously ligates the exteins via a peptide bond.|<br />
file=InteinSplicingReaction.png}}<br />
</div><br />
<br />
==History==<br />
<br />
Inteins were discovered in 1990 when dissimilarities between the mature protein sequence of the yeast vacuolar ATPase (Vma1) and its corresponding mRNA in were investigated. Surprisingly the mature protein had a lower molecular weight than expected from the encoding sequence, indicating the loss of one part of the protein after translation. <br />
Indeed, a region of 454 amino acids was found to be translated and subsequently removed from he Vma1 protein [[#References|[2]]] [[#References|[3]]]. Since the discovery of the Vma1 intein, over 600 different inteins have been reported in all three domains of life and viruses [[#References|[4]]]. <br />
<br />
Dependent on the organism they belong to, the intein’s name consists out of the genus and species abbreviation followed by the host gene [[#References|[5]]].<br />
<br />
==Evolution: Inteins as parasitic genes==<br />
Even after many years of research the initial role of inteins in their host organism remains still unclear.<br />
Many natural inteins contain a homing endonuclease (HEN) domain, an enzyme that cleaves DNA and includes new sequences at homing sites via homologous recombination or reverse transcription. This characteristic led to the perception of the intein as selfish DNA sequence - a gene generating copies of itself without an advantage for the host organism [[#References|[6]]]. This allows horizontal gene transfer of inteins. [[#References|[7]]]. Supporting this hypothesis: 27% of the inteins host proteins are related to DNA metabolism and involved in DNA replication or repair. This ensures expression of the inteins and the HEN domain during DNA replication, where they can take advantage of the DNA replication and repair system for introducing changes in the DNA [#References|[8]]].<br />
<br />
However recent work on conditional protein splicing has shown sensitivity of some inteins to redox state, temperature and small molecules (reviewed in [#References|[9]]], [#References|[10]]]). <br />
This might be a sign for inteins having a role in post-translational protein regulation upon environmental signals. Inserted close to the catalytic core of the protein the protein remains inactive until the splicing reaction has taken place. Evolutionary said, once selfish inteins might have adapted due to selection pressure to be a beneficial mechanism for the host cell [#References|[8]]].<br />
<br />
==Classification and Structure==<br />
<br />
Inteins are divided into three groups: bifunctional (large) inteins, mini inteins and split inteins.<br />
Large inteins carry both a splicing domain and an endonuclease (HEN) domain whereas mini inteins lack the HEN domain [#References|[11]]]. <br />
The most promising inteins for biotechnology are split inteins, which are basically mini-inteins, but divided in two fragments and expressed separately with different proteins. After translation, they assemble with high affinity to be catalytic active. Split enzymes can be found naturally [#References|[12]]] but can also be artificially designed [#References|[13]]]. <br />
<br />
In our project we concentrated on split inteins, as they present a powerful tool for inserting posttranslational modifications in biotechnology. We have characterized the most promising inteins on our Intein database.<br />
<br />
The structure of inteins contains several conserved motifs. The splicing domains are located at the N and C terminal ends. Before splicing can take place the intein rearranges itself from the linear form it has been translated into to a horseshoe like structure where the termini are broad in close proximity to form the catalytic core [#References|[14]]]. <br />
<br />
==A detailed description of trans splicing reaction==<br />
<br />
In contrast to the mini and large inteins that mediate cis-splicing reactions, split inteins are responsible for trans-splicing and fusion of protein parts. The trans-splicing reaction can be divided into the following steps:<br />
<br />
# N-Intein and C-Intein first assemble together to form a dimer like structure with a newly formed catalytic core next to the exteins.<br />
<br />
# The tertiary structure of the intein, once correctly formed, facilitates an ''N&rarr;O/S'' acyl rearrangement at its N-terminal serine or cysteine residue. The result is an ester or thioester bond, respectively, between the side-chain and the peptide backbone of the N-extein.<br />
<br />
# The two exteins are then linked by trans(thio)esterification involving the N-terminal serine or cysteine residue of the C-extein. The C-terminus of the N-extein is now covalently bound to the N-terminal side-chain of the C-extein, while its backbone still retains its normal peptide bond to the intein.<br />
<br />
# The C-terminal asparagine of the intein undergoes self-cyclisation to form a succinimidyl moiety. The peptide bond between the intein and the extein is thereby broken, resolving the branched intermediate.<br />
<br />
# In a final reaction, an ''O/S&rarr;N'' acyl shift results in the two exteins now being linked by an amide bond, indistinguishable from a ribosome-assembled fusion protein.<br />
(as reviewed in [#References|[15]]]) <br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 2) Trans-splicing mechanism reaction by split inteins. |<br />
descr=Picture taken from [#References|[16]]]|<br />
file=Trans_splicing_OCreaction}}<br />
<br />
For intein efficiency not only the structure of the intein domain itself is important but also the nature of the flanking extein residues, as they are heavily involved in the splicing reaction. It has been shown, that the first residue has to be a nucleophilic amino acid, preferable Cysteine, Serine or Threonine. Effectiveness of the intein can be mold by changing the following amino acids [#References|[17]]].<br />
<br />
==Use of inteins in molecular biology and biotechnology==<br />
<br />
Due to their distinct characteristics intein are a powerful tool for molecular biology and biotechnology. Performing an autocatalytic reaction, the inteins are neither dependent on their host protein nor on any other additional substrate. This makes it them broadly applicable for in vivo and in vitro applications. [#References|[16]]].<br />
<br />
Inteins have been used for protein purification, achieving a higher yield of protein due to more specific targeting. The production of semi-synthetic proteins and the attachment of synthetic groups is of huge interest for recombinant proteins. [#References|[16]]]. Split Intein-mediated Circular Ligation Of Peptides a ProteinS (SICLOPPS) is a method to produce circular peptides of eight amino acids length, which could be potent therapeutic drugs [#References|[18]]]. Inteins, fused to split fluorescent proteins as reporter and to the protein of investigation have been used to detect protein-protein interactions in vivo (reviewed in [#References|[10]]]).<br />
Nevertheless most of this applications are performed in vitro and do not exploit the full potential of inteins as regulatory element for post-translational modification. The iGEM Team Heidelberg employs this excellent mechanism of protein splicing to specifically change whole amino-acid sequences and thereby regulate proteins via (dis-) assembly, protein cleavage or circularization of enzymes in vivo. We provide a new foundational advance by introducing the full potential of post-translational modification and therefore a new dimension of genetic engineering to Synthetic Biology.<br />
<br />
=References=<br />
<br />
[1] Perler FB, Davis EO, Dean GE, Gimble FS, Jack WE, et al. (1994) Protein splicing elements: inteins and exteins–a definition of terms and recommended nomenclature. Nucleic Acids Res 22: 1125–1127<br />
<br />
[2] Kane P.M., et al. Protein Splicing Converts the Yeast TEPI Gene Product to the 69-kD Subunit of the Vacuolar. 13253, (1990)<br />
<br />
[3] Hirata, R. et al. Molecular structure of a gene, VMA1, encoding the catalytic subunit of H(+)-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J. Biol. Chem. 265 , 6726–6733 (1990)<br />
<br />
[4] Perler, F. B. InBase : the Intein Database. 30, 383–384 (2002)<br />
<br />
[5] Perler, F. B. et al. Protein splicing elements : inteins and exteins — a definition of terms and recommended nomenclature. 22, 1125–1127 (1994)<br />
<br />
[6] Barzel, A., Naor, A., Privman, E., Kupiec, M. & Gophna, U. Homing endonucleases residing within inteins: evolutionary puzzles awaiting genetic solutions. Biochem. Soc. Trans. 39, 169–73 (2011)<br />
<br />
[7] Pietrokovski, S. Intein spread and extinction in evolution. Trends Genet. 17, 465-472 (2001)<br />
<br />
[8] Novikova, O., Topilina, N. & Belfort, M. Enigmatic distribution, evolution and function of inteins. J. Biol. Chem. (2014)<br />
<br />
[9] Shah, N. H., and Muir, T. W. Inteins: nature's gift to protein chemists. Chem. Sci. 5, 446- 461 (2014) <br />
<br />
[10] Topilina, N. I. & Mills, K. V. Recent advances in in vivo applications of intein-mediated protein splicing. Mob. DNA 5, 5 (2014)<br />
<br />
[11] Eryilmaz, E., Shah, N., Muir, T. & Cowburn, D. Structural and Dynamical Features of Inteins and Implications on Protein Splicing. J. Biol. Chem. (2014)<br />
<br />
[12] Carvajal-Vallejos, P., Pallissé, R., Mootz, H. D. & Schmidt, S. R. Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. J. Biol. Chem. 287, 28686–96 (2012) <br />
<br />
[13] Lin, Y. et al. Protein trans-splicing of multiple atypical split inteins engineered from natural inteins. PLoS One 8, e59516 (2013). <br />
<br />
[14] Eryilmaz, E., Shah, N., Muir, T. & Cowburn, D. Structural and Dynamical Features of Inteins and Implications on Protein Splicing. J. Biol. Chem. (2014)<br />
<br />
[15] Mills, K. V, Johnson, M. a & Perler, F. B. Protein Splicing: How Inteins Escape from Precursor Proteins. J. Biol. Chem. (2014)<br />
<br />
[16] Mootz, H. D. Split inteins as versatile tools for protein semisynthesis. Chembiochem 10, 2579–89 (2009).<br />
<br />
[17] Amitai, G., Callahan, B. P., Stanger, M. J., Belfort, G. & Belfort, M. Modulation of intein activity by its neighboring extein substrates. Proc. Natl. Acad. Sci. U. S. A. 106, 11005–10 (2009).<br />
<br />
[18] Tavassoli, A. & Benkovic, S. J. Split-intein mediated circular ligation used in the synthesis of cyclic peptide libraries in E. coli. Nat. Protoc. 2, 1126–1133 (2007).</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/backgroundTeam:Heidelberg/pages/background2014-10-17T19:25:14Z<p>Huhny: </p>
<hr />
<div>In the variety of proteins that exist, inteins are clearly among the most mysterious.<br />
They behave in such a peculiarly way that scientists are still puzzle about their initial role in the host organisms and are even more astonished when discovering their endless seeming abilities.<br />
<br />
Inteins are integrated as extraneous polypeptide sequences into ordinary proteins. They do not contribute to the original protein function but perform an autocatalytic splicing reaction after protein translation. Analog to intron splicing on RNA level, this posttranslational modification was named protein splicing. Consequentially the protein segments were called inteins, derived from “internal protein sequence”, and the flanking protein chains exteins, “external protein sequences”. Inteins excise themselves out of the host protein while reconnecting the remaining N and C exteins via a new peptide bond. Despite this immense invasion the original protein regains its normal structure and function after splicing [[#References|[1]]]. <br />
<br />
==History==<br />
<br />
Inteins were discovered in 1990 when dissimilarities between the mature protein sequence of the yeast vacuolar ATPase (Vma1) and its corresponding mRNA in were investigated. Surprisingly the mature protein had a lower molecular weight than expected from the encoding sequence, indicating the loss of one part of the protein after translation. <br />
Indeed, a region of 454 amino acids was found to be translated and subsequently removed from he Vma1 protein [[#References|[2]]] [[#References|[3]]]. Since the discovery of the Vma1 intein, over 600 different inteins have been reported in all three domains of life and viruses [[#References|[4]]]. <br />
<br />
Dependent on the organism they belong to, the intein’s name consists out of the genus and species abbreviation followed by the host gene [[#References|[5]]].<br />
<br />
==Evolution: Inteins as parasitic genes==<br />
Even after many years of research the initial role of inteins in their host organism remains still unclear.<br />
Many natural inteins contain a homing endonuclease (HEN) domain, an enzyme that cleaves DNA and includes new sequences at homing sites via homologous recombination or reverse transcription. This characteristic led to the perception of the intein as selfish DNA sequence - a gene generating copies of itself without an advantage for the host organism [[#References|[6]]]. This allows horizontal gene transfer of inteins. [[#References|[7]]]. Supporting this hypothesis: 27% of the inteins host proteins are related to DNA metabolism and involved in DNA replication or repair. This ensures expression of the inteins and the HEN domain during DNA replication, where they can take advantage of the DNA replication and repair system for introducing changes in the DNA [#References|[8]]].<br />
<br />
However recent work on conditional protein splicing has shown sensitivity of some inteins to redox state, temperature and small molecules (reviewed in [#References|[9]]], [#References|[10]]]). <br />
This might be a sign for inteins having a role in post-translational protein regulation upon environmental signals. Inserted close to the catalytic core of the protein the protein remains inactive until the splicing reaction has taken place. Evolutionary said, once selfish inteins might have adapted due to selection pressure to be a beneficial mechanism for the host cell [#References|[8]]].<br />
<br />
==Classification and Structure==<br />
<br />
Inteins are divided into three groups: bifunctional (large) inteins, mini inteins and split inteins.<br />
Large inteins carry both a splicing domain and an endonuclease (HEN) domain whereas mini inteins lack the HEN domain [#References|[11]]]. <br />
The most promising inteins for biotechnology are split inteins, which are basically mini-inteins, but divided in two fragments and expressed separately with different proteins. After translation, they assemble with high affinity to be catalytic active. Split enzymes can be found naturally [#References|[12]]] but can also be artificially designed [#References|[13]]]. <br />
<br />
In our project we concentrated on split inteins, as they present a powerful tool for inserting posttranslational modifications in biotechnology. We have characterized the most promising inteins on our Intein database.<br />
<br />
The structure of inteins contains several conserved motifs. The splicing domains are located at the N and C terminal ends. Before splicing can take place the intein rearranges itself from the linear form it has been translated into to a horseshoe like structure where the termini are broad in close proximity to form the catalytic core [#References|[14]]]. <br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 1) Trans-splicing mechanism reaction by split inteins. |<br />
descr=The N-Intein is fused to the C’ terminal end of the N-extein. Complementary, C-Intein is located at the N’ end of the C-extein. After assembly of the two intein fragments, a splicing reaction takes place, where the intein removes itself from the precursor protein and simultaneously ligates the exteins via a peptide bond.|<br />
file=InteinSplicingReaction.png}}<br />
<br />
==A detailed description of trans splicing reaction==<br />
<br />
In contrast to the mini and large inteins that mediate cis-splicing reactions, split inteins are responsible for trans-splicing and fusion of protein parts. The trans-splicing reaction can be divided into the following steps:<br />
<br />
# N-Intein and C-Intein first assemble together to form a dimer like structure with a newly formed catalytic core next to the exteins.<br />
<br />
# The tertiary structure of the intein, once correctly formed, facilitates an ''N&rarr;O/S'' acyl rearrangement at its N-terminal serine or cysteine residue. The result is an ester or thioester bond, respectively, between the side-chain and the peptide backbone of the N-extein.<br />
<br />
# The two exteins are then linked by trans(thio)esterification involving the N-terminal serine or cysteine residue of the C-extein. The C-terminus of the N-extein is now covalently bound to the N-terminal side-chain of the C-extein, while its backbone still retains its normal peptide bond to the intein.<br />
<br />
# The C-terminal asparagine of the intein undergoes self-cyclisation to form a succinimidyl moiety. The peptide bond between the intein and the extein is thereby broken, resolving the branched intermediate.<br />
<br />
# In a final reaction, an ''O/S&rarr;N'' acyl shift results in the two exteins now being linked by an amide bond, indistinguishable from a ribosome-assembled fusion protein.<br />
(as reviewed in [#References|[15]]]) <br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 2) Trans-splicing mechanism reaction by split inteins. |<br />
descr=Picture taken from [#References|[16]]]|<br />
file=Trans_splicing_OCreaction}}<br />
<br />
For intein efficiency not only the structure of the intein domain itself is important but also the nature of the flanking extein residues, as they are heavily involved in the splicing reaction. It has been shown, that the first residue has to be a nucleophilic amino acid, preferable Cysteine, Serine or Threonine. Effectiveness of the intein can be mold by changing the following amino acids [#References|[17]]].<br />
<br />
==Use of inteins in molecular biology and biotechnology==<br />
<br />
Due to their distinct characteristics intein are a powerful tool for molecular biology and biotechnology. Performing an autocatalytic reaction, the inteins are neither dependent on their host protein nor on any other additional substrate. This makes it them broadly applicable for in vivo and in vitro applications. [#References|[16]]].<br />
<br />
Inteins have been used for protein purification, achieving a higher yield of protein due to more specific targeting. The production of semi-synthetic proteins and the attachment of synthetic groups is of huge interest for recombinant proteins. [#References|[16]]]. Split Intein-mediated Circular Ligation Of Peptides a ProteinS (SICLOPPS) is a method to produce circular peptides of eight amino acids length, which could be potent therapeutic drugs [#References|[18]]]. Inteins, fused to split fluorescent proteins as reporter and to the protein of investigation have been used to detect protein-protein interactions in vivo (reviewed in [#References|[10]]]).<br />
Nevertheless most of this applications are performed in vitro and do not exploit the full potential of inteins as regulatory element for post-translational modification. The iGEM Team Heidelberg employs this excellent mechanism of protein splicing to specifically change whole amino-acid sequences and thereby regulate proteins via (dis-) assembly, protein cleavage or circularization of enzymes in vivo. We provide a new foundational advance by introducing the full potential of post-translational modification and therefore a new dimension of genetic engineering to Synthetic Biology.<br />
<br />
[1] Perler FB, Davis EO, Dean GE, Gimble FS, Jack WE, et al. (1994) Protein splicing elements: inteins and exteins–a definition of terms and recommended nomenclature. Nucleic Acids Res 22: 1125–1127<br />
<br />
[2] Kane P.M., et al. Protein Splicing Converts the Yeast TEPI Gene Product to the 69-kD Subunit of the Vacuolar. 13253, (1990)<br />
<br />
[3] Hirata, R. et al. Molecular structure of a gene, VMA1, encoding the catalytic subunit of H(+)-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J. Biol. Chem. 265 , 6726–6733 (1990)<br />
<br />
[4] Perler, F. B. InBase : the Intein Database. 30, 383–384 (2002)<br />
<br />
[5] Perler, F. B. et al. Protein splicing elements : inteins and exteins — a definition of terms and recommended nomenclature. 22, 1125–1127 (1994)<br />
<br />
[6] Barzel, A., Naor, A., Privman, E., Kupiec, M. & Gophna, U. Homing endonucleases residing within inteins: evolutionary puzzles awaiting genetic solutions. Biochem. Soc. Trans. 39, 169–73 (2011)<br />
<br />
[7] Pietrokovski, S. Intein spread and extinction in evolution. Trends Genet. 17, 465-472 (2001)<br />
<br />
[8] Novikova, O., Topilina, N. & Belfort, M. Enigmatic distribution, evolution and function of inteins. J. Biol. Chem. (2014)<br />
<br />
[9] Shah, N. H., and Muir, T. W. Inteins: nature's gift to protein chemists. Chem. Sci. 5, 446- 461 (2014) <br />
<br />
[10] Topilina, N. I. & Mills, K. V. Recent advances in in vivo applications of intein-mediated protein splicing. Mob. DNA 5, 5 (2014)<br />
<br />
[11] Eryilmaz, E., Shah, N., Muir, T. & Cowburn, D. Structural and Dynamical Features of Inteins and Implications on Protein Splicing. J. Biol. Chem. (2014)<br />
<br />
[12] Carvajal-Vallejos, P., Pallissé, R., Mootz, H. D. & Schmidt, S. R. Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. J. Biol. Chem. 287, 28686–96 (2012) <br />
<br />
[13] Lin, Y. et al. Protein trans-splicing of multiple atypical split inteins engineered from natural inteins. PLoS One 8, e59516 (2013). <br />
<br />
[14] Eryilmaz, E., Shah, N., Muir, T. & Cowburn, D. Structural and Dynamical Features of Inteins and Implications on Protein Splicing. J. Biol. Chem. (2014)<br />
<br />
[15] Mills, K. V, Johnson, M. a & Perler, F. B. Protein Splicing: How Inteins Escape from Precursor Proteins. J. Biol. Chem. (2014)<br />
<br />
[16] Mootz, H. D. Split inteins as versatile tools for protein semisynthesis. Chembiochem 10, 2579–89 (2009).<br />
<br />
[17] Amitai, G., Callahan, B. P., Stanger, M. J., Belfort, G. & Belfort, M. Modulation of intein activity by its neighboring extein substrates. Proc. Natl. Acad. Sci. U. S. A. 106, 11005–10 (2009).<br />
<br />
[18] Tavassoli, A. & Benkovic, S. J. Split-intein mediated circular ligation used in the synthesis of cyclic peptide libraries in E. coli. Nat. Protoc. 2, 1126–1133 (2007).</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/PCR_2.0Team:Heidelberg/pages/PCR 2.02014-10-17T14:22:23Z<p>Huhny: /* Thermal stability of circular mDNMT1(721-1602) */</p>
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<p style="font-size:25px; font-weight:bold">Abstract</p><br />
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<p>Circularized mDNMT1(731-1602) represents a valuable tool for the realization of a methylation maintaining PCR2.0. This foundational advance aims to enable broad functional analysis of DNA methylation patterns despite limited amounts of primary material.</p><br />
<p>We showed that the introduction of inteins represents a feasable approach for circularisation of mDNMT1(731-1602). The posttranslational modification is conceivably increasing heat stability of the protein without impeding its specific methylation activity.</p><br />
<p>Therefore, the iGEM Team Heidelberg’s intein toolbox in combination with the CRAUT linker software have contributed strongly to the establishment a PCR2.0.</p><br />
<p style="font-size:25px; font-weight:bold">Highlights</p><br />
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<li>Heat-stabilization of mDNMT1(731-1602) as pivotal element of a methylation maintaining PCR2.0.</li><br />
<li>Biggest circularized protein so far.</li><br />
<li>Successful application of rational linker design with CRAUT linker software.</li><br />
<li>PCR2.0 as valuable tool for amplifying limited amounts of methylated DNA from primary material.</li><br />
<li>Use of amplified samples in functional assays to link the existing knowledge of methylation patterns to their physiological importance.</li><br />
<li>Possible pharmaceutical use for large scale production of improved gene transfer vectors.</li><br />
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<br />
=Introduction=<br />
<br />
==Motivation==<br />
<br />
The invention of the polymerase chain reaction in 1983 by Kary Mulliy revolutionized the modern world by enabling amplification of DNA in an exponential manner. Further improvements including the use of thermo-stable DNA-polymerases made the method even more efficient, allowing its widespread use in nearly every field of modern diagnostics and research. However, a major part of information is lost when using conventional PCR due to missing transfer of DNA modifications. The most abundant modification is DNA methylation, which is present in all kingdoms of life where it acts as prominent regulator of gene expression. Methylation and other modifications that influence the DNA function without changing its actual sequence are studied in the fast-growing field of epigenetics (greek: epi/επί-over, above, around).<br />
<br />
To empower epigenetics, we propose the PCR 2.0 as an easy and efficient way to amplify DNA templates in an exponential manner while maintaining their specific methylation pattern. The pivotal element of this PCR reaction is the use of a heat-stable DNA methyltransferase (DNMT). Although comparable enzymes exist in thermophile organisms [[#References|[1]]] until now no suitable protein has been found or synthesized that withstands the harsh conditions of a PCR.<br />
<br />
Even though the detection and mapping of methylation patterns has become feasible in a high-throughput manner by using bisulfite sequencing and array techniques [[#References|[2]]], further functional analysis is still hindered by the small amount of primary material. Our approach therefore aims to provide unlimited amounts of any methylated DNA sample of interest, without knowledge of methylation patterns or the need of expensive methods such as de-novo synthesis – just by running a PCR. <br />
<br />
To achieve this goal of a PCR2.0 that can be used to amplify DNA including its intrinsic methylation pattern, our main aim was the generation of a heat-stable Dnmt1 that can be produced in a larger scale and shows efficient and specific methylation of DNA. Our approach applies protein circularization by using self-splicing protein sequences, socalled inteins from our toolbox as well as our [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software CRAUT Linker Software]. Since the restriction of conformational changes through intramolecular bonds has been known to increase the stability of proteins [[#References|[3]]] [[#References|[4]]]<br />
and joining of C- and N- terminus were reported to increases in thermostability of smaller peptides [[#References|[5]]] we started the circularization of the biggest protein so far…<br />
<br />
==Epigenetics - there is more than just A, T, C and G…==<br />
In mammals and other vertebrates methylation occurs at cytosine nucleotides that are followed by guanines (CpG) and is a prominent key regulator of transcription, embryonic development, X chromosome inactivation and many other cellular functions. [[#References|[6]]] [[#References|[7]]]. Approximately 1% of the genetic code in human somatic cells constists of methylated cytosines, amounting to 70-80% of all present CpG dinucleotides [[#References|[8]]]. Due to the great prevalence and the heredity of this modification, the 5-methylcytosine (5mC) is also known as the “fifth base” of eukaryotic genomes [[#References|[9]]]. DNA methylation is preferentially occurring at intergenic regions and repetitive sequences, where it is known to silence gene expression [[#References|[10]]] [[#References|[11]]]. In contrast, GC-rich promoter regions are usually characterized by a low methylation status, allowing transcription of the associated genes [[#References|[12]]]. Deregulation of cytosine methylation has been reported to play a crucial role in the development of numerous diseases, including cancer, imprinting diseases and repeat-instability based diseases such Huntington's disease [[#References|[13]]].<br />
<br />
==A closer look at DNA Methyltransferases==<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 1) Principle of mDNMT1-mediated maintenance methylation|<br />
descr=The enzyme DNMT1 is of particular interest for methylation maintenance PCR, as it allows exact duplication of the original methylation pattern to every new DNA copy.|<br />
file=methylation_principle.png}}<br />
<br />
The family of enzymes called DNA methyltransferases (DNMTs) is responsible for the establishment and maintenance of cell-type specific DNA methylation patterns. Whereas the two enzymes DNMT3a and DNMT3b contribute to de novo methylation of DNA during development, DNMT1 is preserving existing methylation patterns throughout cell divisions. To do so, DNMT1 exploits the principle of semiconservative replication, using the parental strand as a template to create an exact copy of methylation patterns on the daughter strand. Subsequent to the recognition of a so called hemi-methylated CpG site, the enzymatic machinery transfers a methyl group from the methyl donor S-Adenosyl-methionine (SAM) to the C5 position of the complementary cytosine residue [[#References|[15]]] [[#References|[19]]]. <br />
<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 2) Crystal structure of full-length and truncated (731-1602) mDNMT1|<br />
descr=Structural overview of full length mDNMT1 and truncated mDNMT1(731–1602) (A) Color-coded schematic overview of domain structure and numbering of mDNMT1 sequence. (B) Ribbon presentation of full-length DNMT1 with the CXXC,BAH1, BAH2,and methyltransferase domain colored in light blue, yellow, orange and red, respectively. (C) Crystal structure of truncated DNMT1, missing the CXXC domain and schowing more adjacent protein termini thatn the full-length version.|<br />
file=dnmt1_structure.png}}<br />
<br />
The complex structure of DNMT1 and several truncated versions have been recently solved by X-ray crystallography [[#References|[19]]]. For our experiments we used the smallest truncated version of DNMT1 that has been reported to efficiently mediate methylation maintenance. This truncated version derives from <i>Mus musculus </i> and comprises amino acids 731-1602 (mDNMT1(731-1602) [[#References|[15]]] [[#References|[19]]] kindly provided by Dr. Bashtrykov after approval from Prof. Patel). mDNMT1(731-1602) contains the C-terminal catalytic methyltransferasedomain as well as the aligning bromo-adjacent homology (BAH) domains, which prevent the binding of unmethylated DNA to the catalytic core. The truncated enzyme is missing the CXXC domain, which has a high affinity to CpG hemi-methylated dinucleotides, but was shown to be dispensable for protein function [[#References|[16]]]. In contrast to the full-length DNMT1 that can only be expressed in insect cells using a baculovirus based system, mDNMT1(731-1602) has the great advantage of being efficiently expressed in <i>E.coli</i>. Another advantage of the truncated DNMT1 is, that the N- and C- terminus are closer together than in the full-lentgh DNMT1. Thus cirularization might cause less deformation and show lower impact on the overall activity, which makes this truncated mDNMT1 an ideal target for our purposes.<br />
<br />
==Circularization - The missing link== <br />
<br />
Heat stabilization of mDNMT1 as a pivotal element of our methylation-maintaining PCR2.0 was approached by circularization of the protein. Until now, only smaller proteins have been stabilized using this method. Therefore, to our knowledge, mDNMT1 – even in its shortest truncated version of 871 amino acids and a molecular weight of approximately 100kDa – is the largest protein that has ever been tried to circularize. According to the crystal structure, the distance between the termini of the truncated DNMT1 is 48 Å. We suspected that circularization of the protein by direct fusion of both termini might cause deformation of the protein structure. Therefore we used our own [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software CRAUT Linker Software] to create a linking amino acid sequence, adapted to the structure of DNMT1. The software is able to design protein linkers with the required length to bridge the gap between the protein termini while bypassing the catalytic core of the enzyme. <br />
<br />
Since we could not estimate the impact of the linker introduction, we focused on two different kinds of linkers: a so called rigid linker that has been calculated and optimized during the establishment of our linker software and a flexible peptide connection consisting mostly of glycine and serine. <br />
To perform circularization we have used the split NpuDnaE Intein since it is used as the <i> golden standard </i> for protein splicing and has been efficiently tested by us through circularization of GFP, Lysosyme and Xylanase. Moreover, we tried to implement protein circularization by using sortase A, which is recognizing and cleaving a carboxyterminal sorting signal followed by a transpeptidation reaction that can be exploited for protein circularization. This method has been reported to be very efficient, especially when used <i> in vitro </i> [[#References|[4]]] and was therefore included in our study as possible candidate for large scale productions of circular proteins.<br />
<br />
=Materials and Methods=<br />
<br />
==Constructs==<br />
<br />
We designed constructs in order to characterize the efficiency of sortase A as well as intein mediated circularization of DNMT1. Both approaches are based on the comparison of the circularized protein with a corresponding linear counterpart at different temperatures and incubation times.<br />
<br />
For circularization of DNMT1 with inteins we fused the obtained truncated version of DNMT1 (731-1602) [[#References|[15]]] with an appropriate linker and split NpuDnaE domains at either site of the construct. For efficient purification of the protein, all constructs contained a hexa-histidine tag. Moreover, we have cloned the ubiquitin-like Smt3 protein from <i> Saccharomyces cerevisiae </i> in front of the DNMT1-intein complex. This attachment is used to increase the yield when expressing large mammalian proteins like DNMT1 in <i>E.coli</i> [[#References|[17]]]. The constructs have been designed in a way that Smt3 is no longer included in the final circular protein, and therefore cannot interfere with its function. <br />
All cloning steps were carried out using our new RFC[i] standard procedure. <br />
<br />
The mDNMT1 (731-1602) constructs that have been designed to allow sortase A mediated cirularization are flanked by the sortase A recognition sequence LPETGG on the C–terminus as well as N-terminal glycines that that can be exposed through previous TEV treatment. Since the transpeptidation via sortase requires an additional <i> in vitro </i> reaction, subsequent purification is necessary. To facilitate this step and to enrich the circular product in the sample, a hexa-histidine tag is present in the initial protein that is lost during circularization. Hence, an additional affinity chromatography via His trap can be used to separate the histidine tag containing sortase and not circularized educt from the circular flow-through. <br />
All cloning steps were carried out using our sortase standard. <br />
<br />
The following linkers that had been optimized along with our [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software CRAUT Linker Software]:<br />
<br />
{| class="table table-hover"<br />
|-<br />
! Definition!! Sequence <br />
|-<br />
|Rigid linker|| GGAEAAAKEAAAKVNLTAAEAAAKEAAAKEAAAKEAAAKEAAAKAVNLTAAEAAAKAHHHHHHSGRGT <br />
|-<br />
|Flexible linker|| CWEGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSHHHHHHSGRGT<br />
|}<br />
<br />
== Expression and purification==<br />
<br />
{{:Team:Heidelberg/templates/image-quarter|<br />
align=right|<br />
caption=Figure 3) Purification of recombinant mDNMT1|<br />
descr= The purification of recombinantly expressed mDNMT1 is a challenging task including large scale induction, efficient cell lysis, reduction of sample volume via ultracentrifugation, immobilized Ni-ion affinity chromatography, dialysis and concentration through centrifugation. |<br />
file=Purification_procedure_new.png}}<br />
<br />
In comparison to the full length murine DNMT1, our modified mDNMT1(731-1602) can be expressed in <i>E.coli (Rosetta DE3)</i> and does not need the establishment of Baculovirus based system and the use of insect cell cultures. First experiments were conducted to determine the optimal conditions for protein induction and expression. We tested several concentrations of IPTG as well as different temperatures to maximize the yield of mDNMT1. Still, even though expression of the constructs can be simplified by using <i>E.coli</i>, purification of the recombinant protein remains a challenging task requiring multiple different steps that had to be adapted and established from published protocols [[#References|[19]]]. <br />
<br />
In a first step, we optimized the process of cell lysis, to be able to extract a maximum amount of active protein from our cultures. Therefore we compared the two commonly used methods sonication and disruption via French press. Subsequently, the sample was cleared from cell debris by ultracentrifugation. The protein of interest containing a hexa-histidine tag was further enriched via immobilized metal ion affinity chromatography (IMAC) using a His trap and eluted with imidazol. In a next step, low molecular weight solutes such as salts and imidazol that would interfere with protein function were removed from the sample by dialysis. Finally, the purified protein was concentrated by size-exclusion centrifugation using filters with an appropriate mass cutoff.<br />
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<br />
==Methylation activity assay==<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 4) Sequence of 40-mer DNA template for methylation activity assay|<br />
descr=Sequence of the 40-mer DNA template. CpG dinucleotides are shaded in gray, Sau3AI and HpaII recognition sites are depicted in red. The first CpG dinucleotide is methylated on the reverse strand.|<br />
file=DNMT1_substrate.png}}<br />
<br />
To measure the efficiency of mDNMT1 maintenance methylation, we used an assay that is based on two methylation-sensitive restriction enzymes Sau3AI and HpaII. It relies on the inhibition of restriction enzymes when methyl groups are attached to the cleavage site. We used a 40-mer DNA template with one hemi-methylated and one unmethylated CpG site which are located within the Sau3AI and HpaII restriction site, respectively (adapted from Bashtrykov et colleagues [[#References|[16]]].<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=left|<br />
caption=Figure 4) Principle of the mDNMT1 methylation activity assay|<br />
descr=Principle of mDNMT1 methylation assay using a 40-mer substrate with Sau3AI and HpaII cleavage sites that are blocked upon further CpG methylation. The lack of methylation activity leads to fragmentation of the DNA template with either of the two restriction enzymes. Whereas specific methylation of hemimethylated CpGs hampers only cleavage by Sau3AI, <i>de novo</i> methylation activity of mDNMT1 can be detected when HpaII cleavage is blocked as well.|<br />
file=MethylationAssay_overview.png}}<br />
<br />
The restriction enzyme Sau3AI is capable of cleaving the template despite of the hemimethylated state of the first CpG nucleotide. Similarly, HpaII can attack its unmethylated native site, leading to fragmentation of the template as long as no further methylation occurs. Accordingly, the methylation activity of circular and linear mDNMT1 can be measured by quantifying the cleavage efficiency of the restriction enzymes after incubation with the protein. Impairment of Sau3AI cleavage indicates maintenance methylation activity, whereas a decrease in HpaII activity would result from <i>de novo </i> methylation. Therefore the assay is not only detecting activity of the enzyme but also includes its specificity for hemimethylated DNA over unmethylated DNA. To quantify maintenance and de novo methylation activity, the 40-mer fragments remaining after DNMT1 incubation and restriction digest were separated by gel electrophoresis. The intensities of the detected bands corresponding to different fragments were measured using ImageJ Gel Analyzer and quantified using gaussian fitting after background substraction. <br />
<br />
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<br />
=Results=<br />
<br />
==Optimizing expression and purification of linear mDNMT1(731-602)==<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 5) Optimisation of induction|<br />
descr=As soon as transformed Rosetta (DE3) cultures reached an OD600 of approximately 0.6, induction of linear mDNMT1(731-1602) was perfomed over night at either 15°C or 25°C, using different concentrations of IPTG. Higest expression of DNMT1 was observed at 15°C using 0.7mM IPTG.|<br />
file=DNMT1-Induction2.png}}<br />
<br />
Before starting circularization of DNMT1, we first tested if we can actually express and purify active linear DNMT1. Since DNMT1 expression in E.coli normally results in low yields (<1mg/liter of culture)[[#References|[15]]], we started with an optimization of the expression conditions. <br />
<br />
In general, low temperatures and low IPTG concentrations are known to prevent the occurrence of insoluble inclusion bodies under conditions of overexpression. Thus, we tested different IPTG concentrations and temperatures during induction in order to maximize yields of active mDNMT1(731-1602). Generally, solubility tags such as the fused Smt3 as well as lower temperatures are known to prevent the occurrence of insoluble inclusion bodies under conditions of overexpression. Accordingly, we observed an optimal induction of our mDNMT1(731-1602) construct after incubation at 15 °C using 0.7mM IPTG (Figure 5).<br />
<br />
Even though the overall yield of recombinant protein is quite low, the presence of mDNMT1(731-1602) after induction with IPTG was detectable through Coomassie staining and specifically verified by Western blot (Figure 6A and 6B). In the western blot, several other peptides with lower molecular weight were detected (Figure 6B). These peptides possibly result from incomplete translation or degradation processes, since the construct used for initial mDNMT1 expression and purification comprised an N-terminal hexa-histidine tag. To minimize the amount of incomplete protein products that would be enriched in the following purification, we optimized the mDNMT1(731-1602) constructs for circularization by shifting the His-tag to the C-terminus.<br />
<br />
<div style="clear:both;"></div><br />
<br />
After successful expression, the next critical step for high yields of recombinant protein after purification is protein extraction. The lysis method has to be efficient but still gentle enough to preserve the proteins function - especially when dealing with sensitive and low expressed proteins like DNMT1. In order to optimize protein extraction we therefore compared cell lysis by sonification with disruption via French press (Figure 6A and 6B). Due to the low overall yield, mDNMT1 does not appear as a prominent band on the Coomassie-stained gel as we expected for an overexpressed recombinant proteins. <br />
Assuming similar amounts of overall protein that should be present in the consecutively generated samples, sonification seems to be less efficient for extraction of recombinant protein compared to usage of the French press. Moreover, the disruption of <i>E.coli</i> using French press has been reported to preserve the biological activity of susceptible proteins more efficiently than sonication [18]. Therefore we used the French press for further extraction of linear as well as circular mDNMT1.<br />
<br />
Further purification of the protein using a His-trap resulted in successful enrichment of mDNMT1f(731-1602) (Figure 6C). Corresponding eluates were pooled and further concentrated for use in functional assays. <br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
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caption=Figure 6A) Comparison of DNMT1 extraction from bacteria by French press and sonification|<br />
descr=mDNMT1(731-1602) expression in Rosetta (DE3) was performed over night at 15°C. Subsequently, the bacteria were lysed by sonification or French press and ultracentrifuged. After each step, samples were collected for analysis by SDS-PAGE.<br />
(Untransformed negative control (UnTra), intervals of cell disruption (1, 2), supernatant (SN)).|<br />
file=DNMT1-Celllysis.jpg}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 6B)Lysis via French press efficiently etracts mDNMT1(731-1602)|<br />
descr=Western Blot of lysed samples was perfomed with an anti-hexa-histidine antibody for specific detection of mDNMT1. Full length mDNMT1(731-1602) is indicated with an arrow.|<br />
file=DNMT1-Celllysis-WB.jpg}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
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caption=Figure 6C) Purification of linear mDNMT1(731-1602)|<br />
descr=Coomassie stained SDS-PAGE showing efficient purification of linear mDNMT1(731-1602) via His-column. Arrow indicates peptides corresponding to the molecular weight of mDNMT1(731-1602) (133kDa).|<br />
file=DNMT1-Purification-linear.png}}<br />
<br />
<div style="clear:both;"></div><br />
<br />
==Activity and specificity of linear mDNMT1(731-1602)==<br />
<br />
After successful expression and purification of linear DNMT1, we characterized the proteins activity and specificity for hemi-methylated DNA by performing the methylation activity assay described above. To test the maintenance methylation activity of our linear DNMT1, we performed the methylation assay with different incubation times of DNMT1. We observe an increasing amount of completely methylated template over time (Figure 7), represented by increasing amounts of template that are unsusceptible towards SauAI cleavage. <br />
The truncated DNMT1 (731-1602) showed maximal methylation of the template after approximately 1/2 to 1hour. Therefore, our data agrees with efficiencies, that have been reported in literature for wild type and truncated versions of mDNMT1 [[#References|[15]]] [[#References|[16]]] .Overall, the reaction kinetics of the linear protein seem to be promising for later use in a PCR2.0, assuming that circularization of the protein will not ablate its function. <br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
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caption=Figure 7) Maintenance Methylation deposition of linear Dnmt1(731-1602) over time|<br />
descr= Purified linear mDNMT1(731-1602) was incubated with 40mer DNA template (see Figure4) for indicated times at 37°C. Subsequently the samples were digested with Sau3AI. Fragments were separated by PAGE and stained with ethidium bromide. (Homo me= homomethylated substrate, control for enzyme inhibition through methylation, Hemi me=hemimethylated substrate positive control for digest).|<br />
file=activityovertime.jpg}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 8) <i>De novo</i> methylation activity of mDNMT1|<br />
descr=Purified linear mDNMT1(731-1602) was incubated with 40mer DNA template (see Figure4) for indicated times at 37°C. Subsequently the samples were digested with HpaII. Fragments were separated using gel-electrophoresis and visualized with ethidiumbromid staining. (Homo me= homomethylated substrate,control for enzyme inhibition through methylation, Hemi me=hemimethylated substrate positive control for digest).|<br />
file=specificityovertime.jpg}}<br />
<br />
In oder to be used in the targeted PCR2.0 mDNMT1 needs to be highly specific for hemi-methylated sites and should not display <i>de novo</i> methylation activity. Therefore, we included HpaII digestion in our assay to determine <i>de novo</i> methylation of CpG sites. The first results indicate that the purified mDNMT1(731-1602) does not exhibit de novo methylation activity (Figure 8). Even with increasing DNMT1 incubation time, HpaII is not repressed which indicates absence of <i>de novo</i> methylation. <br />
However, the analysis of the gel pictures is difficult since restriction efficiency of HpaII was not 100% as it can be seen from the hemimethylated control. Therefore, uncut template which is not resulting from unspecific methylation activity of the enzyme complicates quantitative analysis. Further tests, especially with the circularized protein will have to be performed to confirm these initial data.<br />
<div style="clear:both;"></div><br />
<br />
=Circular mDNMT1(731-1602) - The pivotal element of PCR2.0=<br />
<br />
==Expression and purification of mDNMT1(731-1602) for circularization==<br />
<br />
After we succeeded in purifying active and specific linear mDNMT1, we went on with cloning, expression and purification of the circular DNMTs. <br />
<br />
Expression and purification of mDNMT1(731-1602) for the intein as well as the sortase approach were conducted as they had been established for the linear DNMT1. Elutions from the His-Trap showed an enrichment and concentration of peptides that correlate with the expected molecular weight of the truncated mDNMT1(731-1602) (Figure 9A and 9B). Nevertheless, purification with a His-Trap alone does not seem to exclude a variety of impurities that either result from degraded mDNMT1 that still contains a His-Tag or unspecific binding to the affinity column. Advanced protocols as they were used by Song and collegues [[#References|[19]]] for analysis of the proteins crystal structure are therefore including several more steps. Nevertheless, additional steps of purification increase the risk of reducing the overall protein yield and activity, necessitating greater amounts of starting material. Since the active protein should already be included and efficiently enriched in our sample despite of present impurities, we continued with the functional analysis of the sample.<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 9A) Efficiency of mDNMT1(731-1602) purification for the intein approach|<br />
descr=Induction of Rosetta (DE3) and purification were performed as described above. After each step, samples were collected for analysis by Coomassie stained SDS-PAGE. Samples are numbered according to their chronology of collection. Arrow indicates mDNMT1(731-1602).|<br />
file=DNMT1-intein-Purification3.png}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 9B) Sortase approach - Efficiency of Purification|<br />
descr=Induction of Rosetta (DE3) and purification were performed as described above. After each step, samples were collected for analysis by Coomassie stained SDS-PAGE. Samples are numbered according to their chronology of collection. Arrow indicates mDNMT1(731-1602).|<br />
file=DNMT1-sortase-Purification2.png}}<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 10) Expression and Purification of intein and sortase constructs with different linkers|<br />
descr=Expression and Purification of different constructs in Rosetta (DE3) was performed as described above. Unpurified as well as purified samples were collected for analysis by Coomassie stained SDS-PAGE. Ctrl indicates the untransformed control. D= no linker, R= Rigid linker, F=Flexible linker, L= linear. Arrow indicates peptides with molecular weight corresponding to mDNMT1(731-1602). Asterix indicates possibly spliced Intein constructs.|<br />
file=DNMT1-Purification1.png}}<br />
<br />
Our approach of heat-stabilization includes the testing of different linkers that had been calculated by our [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software CRAUT Linker Software]. We tested a flexible linker, consisting mostly of glycines and serines and a rigid linker, which was introduced to maximize stabilization of the protein through cicularization. Purification of the mDNMT1(731-1602) variants with different linkers for circularization was successful (Figure 10), but the yields were lower compared to different batches of linear mDNMT1(731-1602) that had been produced in a similar way. <br />
Interestingly, a shift between mDNMT1(731-1602) expressed from the intein construct compared to the sortase or linear versions was detected. Since protein circularization via inteins is an autocatalytic process that takes place inside the bacteria, this shift could possibly result from successful splicing activity of the inteins. The resulting product lacks Smt3 and is therefore approximately 30kDa smaller than its precursor (Figure 10, indicated by an asterix). Since intein splicing likely occurred, flexible- and rigid-linked-DNMT1 could be circularized. <br />
<br />
To prove circularization by inteins, we cooperated with the core facility Protein Analysis of the German Cancer Research Center (Dkfz.). In the facility, circularized and linear DNMT1 were extracted from a provided gel, digested with trypsin and analyzed by ESI mass spectrometry. Unfortunately, we do not have the final results yet but are confident to be able to present them at the jamboree.<br />
<br />
As against that mDNMT1(731-1602) with sortase recognition sequences needs to be further processed before a circular protein is obtained. Unfortunately, TEV cleavage of the purified protein, which is necessary for subsequent circularization with sortase was not very efficient. Therefore sortase treatment did not yield in a detectable amount of processed mDNMT1(731-1602). Therefore, we fully concentrated on the promising intein approach for further mDNMT1(731-1602) circularization.<br />
<br />
<div style="clear:both;"></div><br />
<br />
==Thermal stability of circular mDNMT1(721-1602)==<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 8) Methylation after heat shock|<br />
descr=Circular DNMT1 with rigid linker (RC), circular DNMT1 with flexible linker (FC) and linear DNMT1 were heat shocked for 5 sec at 55°C and 60°C. |<br />
file=heatshock55-60.jpg}}<br />
{{:Team:Heidelberg/templates/image-half|<br />
align=right|<br />
caption=Figure 9) Persistance of methylation activity at high temperatures|<br />
descr=Circular DNMT1 with rigid linker (RC), circular DNMT1 with flexible linker (FC) and linear DNMT1 were heat shocked for 5 sec at 65°C and 72°C.Methylation after one hour was measured. A darker upper band for FC is even visible by eye, indication higher methylation activity.|<br />
file=heatshock65-72.jpg}}<br />
<br />
In our final assay we examined the effect of an heat shock on the remaining activity of the different Dnmt1 enzymes to proof the ability of circular enzyme to withstand higher temperatures. We wanted to produce evidence for the feasibility of a PCR 2.0 using circularisation.<br />
Behaviour after heat treatment of pre-used linear Dnmt1 (L) was compared to Dnmt1 with rigid linker (RC) and flexible linker (FC)<br />
The proteins were exposed to temperatures between 55°C and 72°C, temperatures that would also be reached in a PCR. The heat shock was conducted for 5 sec and DNA substrate was added afterwards at 37°C for methylation to occur.<br />
Looking at the gel picture (Figure?) a darker upper band indicating more methylated substrate for the circular Dnmt1 with flexible Linker at all temperatures is even visible by eye, suggesting more remained stability. To confirm this results, the intensity of the gel bands were analysed using Image J. Figure ? highlights the remaining activity over temperature after normalisation to the control at 37°C the ration of methylated DNA was normalised to activity at 37°C. Indeed activity of circular DNMT1 with the flexible linker (black) is exciding the linear and ciruclar with rigid linker at least double. Very surprisingly, the methylation activity of circular DNMT1 with a flexible linker seems to increase with higher temperatures. <br />
<br />
For significant evaluation, more data has to be collected. When applying Michael Menten kinetics , the methylation reaction for the controls already approaches saturation therefore the normalisation to the original activity might be defective. For further improvement of the assay, less volume of DNMT1 has to be taken for the enzyme reaction.<br />
<br />
=Discussion=<br />
<br />
== Achievements and impact==<br />
Taking everything together we showed that introduction of inteins represents a feasable approach for circularisation of mDNMT1(731-1602). The post translational modification is conceivably increasing heat stability of the protein without impeding its specific methylation activity. <br />
<br />
We proved that circularization with inteins is a rather easy method to increase heat stability of complex and sensitive proteins like mDNMT1(731-1602). In comparison, other methods for stabilization of proteins such as introduction of disulfide bonds require sophisticated structure analysis as well as non-physiological reaction conditions[[#References|[20]]] [[#References|[21]]]. <br />
<br />
The development and optimization of our CRAUT linker software has been an essential element in linking C- and N-terminus of mDNMT1(731-1602) while bypassing the active site of the protein and preserving its natural function. The two different linker compositions resulted in divergent heat stabilities of the protein. Therefore, software optimized linkers with designed properties have resulted in successful narrowing down of the infinite amount of possibilities to circularize mDNMT1(731-1602). Further experimental data of enzyme-specific inker variants will help to optimize the approach.<br />
<br />
Circularized mDNMT1(731-1602) represents a valuable tool for the realization of a methylation maintaining PCR2.0. This foundational advance aims to enable broad functional analysis of DNA methylation patterns despite limited amounts of primary material. Even though detection of methylation patterns has been established in a high-throughput manner by using the principle of bisulfite sequencing, handling of limited amounts of primary material still represents a major challenge. Approaches including single molecule sequencing have been reported, but have not been established in an affordable and efficient manner yet [[#References|[27]]]. Furthermore, these methods are limited to the detection of methylation patterns and do not allow functional analysis without complete analysis and cost-intensive synthesis. Therefore, our PCR2.0 aims to revolutionize the field of epigenetics by linking complex methylation patterns to their actual function. <i>In vitro</i> as well as <i>in vivo</i> experiments, requering large amounts of primary material will be feasible and more reproducible by using our PCR2.0. <br />
<br />
A very interesting application for the pharmaceutical use of our PCR2.0 could be the large scale production of methylated gene therapy vectors. Since, methylation of gene transfer vectors has been reported to extend transgene product expression by lowering cellular immunogenicity, the efficiency gene therapies could be easily improved with our method. [[#References|[22]]].<br />
<br />
==Further possible improvements==<br />
Additionally to the circularization of mDNMT1(731-1602), further approaches can be taken into account to realize a methylation maintaining PCR2.0. Those include further stabilization of the protein either by enhancing the hydrophobicity of the proteins core or direct links to limit possible conformations [[#References|[23]]].<br />
<br />
Another upcoming topic is the method of low temperature PCRs that exploit the function of helicases to segregate DNA strands. Even though these PCRs are not performed at high temperatures comparable to conventional PCRs, none of them has reached widespread establishment yet. Main reasons are that isothermal PCRs require either too long incubation times per cycle [[#References|[24]]] or still exceed physiological temperatures of most enzymes [[#References|[25]]].<br />
<br />
A still challenging factor for PCR2.0 is the DNMT1 cofactor S-adenosylmethionine (SAM) that exhibits only low stability at high temperatures Matos, J. & Wong, C. S-adenosylmethionine: Stability and stabilization. Bioorg. Chem. 80, 71–80 (1987). During our experiments, there has not been observed any limitation due to this instability. Nevertheless, addition of the quite cheap methyl-donor after each cycle of replication could circumvent this problem until a more stable variant of the cofactor is available. <br />
<br />
Moreover, the PCR2.0 reaction could be complemented with other enzymes such as UHRF1, that have been reported to increase the specificity of mDNMT1(731-1602) [[#References|[19]]] Bashtrykov, P., Jankevicius, G., Jurkowska, R. Z., Ragozin, S. & Jeltsch, A. The UHRF1 protein stimulates the activity and specificity of the maintenance DNA methyltransferase DNMT1 by an allosteric mechanism. J. Biol. Chem. 289, 4106–15 (2014). The wild type DNMT1 is known to be associated with the replication complex for direction of its activity. Therefore design of an appropriate Polymerase-DNMT-fusion protein could imitate this connection and therefore further reduce stochastic methylation events. [[#References|[26]]].<br />
<br />
Overall, the iGEM Team Heidelberg’s intein toolbox in combination with the CRAUT linker software have contributed strongly to the foundational advance of establishing a PCR2.0.<br />
<br />
=References=<br />
[1] Watanabe, M., Yuzawa, H., Handa, N., & Kobayashi, I.. Hyperthermophilic DNA methyltransferase M.PabI from the archaeon Pyrococcus abyssi. Applied and Environmental Microbiology, 72(8), 5367–75 (2006).<br />
<br />
[2] Meissner, A. et al. Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature 454, 766–70 (2008)<br />
<br />
[3] Vieille, C. & Zeikus, G. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65, 1–43 (2001).<br />
<br />
[4] Antos, J. M., Popp, M. W.-L., Ernst, R., Chew, G.-L., Spooner, E., & Ploegh, H. L.. A straight path to circular proteins. The Journal of Biological Chemistry, 284(23), 16028–36 (2009).<br />
<br />
[5] Tam, J. P. & Wong, C. T. T. Chemical synthesis of circular proteins. J. Biol. Chem. 287, 27020–5 (2012).<br />
<br />
[6] Robertson, K. D. DNA methylation and human disease. Nat. Rev. Genet. 6, 597–610 (2005).<br />
<br />
[7] Jaenisch, R. & Bird, A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat. Genet. 33 Suppl, 245–54 (2003).<br />
<br />
[8] Ehrlich, M.. Amount and distribution of 5-methycytosine in human DNA from different types of tissues or cells. Nucleic Acids Res. 10: 2709–2721 (1982).<br />
<br />
[9] Lister, R. & Ecker, J. Finding the fifth base: genome-wide sequencing of cytosine methylation. Genome Res. 959–966 (2009).<br />
<br />
[10] Bird, A.. DNA methylation patterns and epigenetic memory. Genes & Development 16, 6-21 (2002).<br />
<br />
[11] Jaenisch, R., and Bird, A.. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet 33, 245-254 (2003).<br />
<br />
[12] Weichenhan, D., and Plass, C.. The evolving epigenome. Human Molecular Genetics 22, R1 (2013).<br />
<br />
[13] Robertson, K. D. DNA methylation and human disease. Nat. Rev. Genet. 6, 597–610 (2005).<br />
<br />
[14] Jurkowska, R. Z., Jurkowski, T. P. & Jeltsch, A. Structure and function of mammalian DNA methyltransferases. Chembiochem 12, 206–22 (2011).<br />
<br />
[15] Song, J., Teplova, M., Ishibe-Murakami, S. & Patel, D. J. Structure-based mechanistic insights into DNMT1-mediated maintenance DNA methylation. Science 335, 709–12 (2012).<br />
<br />
[16] Bashtrykov, P. et al. Specificity of Dnmt1 for methylation of hemimethylated CpG sites resides in its catalytic domain. Chem. Biol. 19, 572–8 (2012).<br />
<br />
[17] Lee, C.-D., Sun, H.-C., Hu, S.-M., Chiu, C.-F., Homhuan, A., Liang, S.-M., Wang, T.-F. An improved SUMO fusion protein system for effective production of native proteins. Protein Science : A Publication of the Protein Society, 17(7), 1241–8 (2008).<br />
<br />
[18] Benov, L. & Al-ibraheem, J. Disrupting Escherichia coli : A Comparison of Methods. 35, 428–431 (2002).<br />
<br />
[19] Song, J., Rechkoblit, O., Bestor, T. H. & Patel, D. J. Structure of DNMT1-DNA complex reveals a role for autoinhibition in maintenance DNA methylation. Science 331, 1036–40 (2011).<br />
<br />
[20] Betz, S. F. Disulfide bonds and the stability of globular proteins. 1551–1558 (1993).<br />
<br />
[21] Bulaj, G. Formation of disulfide bonds in proteins and peptides. Biotechnol. Adv. 23, 87–92 (2005).<br />
<br />
[22] Reyes-Sandoval, a & Ertl, H. C. J. CpG methylation of a plasmid vector results in extended transgene product expression by circumventing induction of immune responses. Mol. Ther. 9, 249–61 (2004).<br />
<br />
[23] Vieille, C. & Zeikus, G. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65, 1–43 (2001).<br />
<br />
[24] Vincent, M., Xu, Y. & Kong, H. Helicase-dependent isothermal DNA amplification. EMBO Rep. 5, 795–800 (2004).<br />
<br />
[25] Xu, G. et al. Cross priming amplification: mechanism and optimization for isothermal DNA amplification. Sci. Rep. 2, 246 (2012).<br />
<br />
[26] Vilkaitis, G., Suetake, I., Klimasauskas, S. & Tajima, S. Processive methylation of hemimethylated CpG sites by mouse Dnmt1 DNA methyltransferase. J. Biol. Chem. 280, 64–72 (2005).<br />
<br />
[27] Krueger, F., Kreck, B., Franke, A. & Andrews, S. R. DNA methylome analysis using short bisulfite sequencing data. Nat. Methods 9, 145–51 (2012).</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/backgroundTeam:Heidelberg/pages/background2014-10-16T12:51:19Z<p>Huhny: </p>
<hr />
<div>=Introduction=<br />
<br />
=Scientific Background of Inteins=<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
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caption=Figure 1) Split-Intein splicing reaction|<br />
descr=Curly fries inteins -> Request for comments|<br />
file=InteinSplicingReaction.png}}<br />
<br />
==Splicing Reaction==<br />
<br />
<br />
=Outlook=</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/backgroundTeam:Heidelberg/pages/background2014-10-16T12:50:35Z<p>Huhny: /* Scientific Background of Inteins */</p>
<hr />
<div>=Introduction=<br />
<br />
=Scientific Background of Inteins=<br />
<br />
{{:Team:Heidelberg/templates/image-half|<br />
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caption=Figure 1) Split-Intein splicing reaction|<br />
descr=Curly fries inteins -> Request for comments|<br />
file=InteinSplicingReaction.png}}<br />
<br />
==Splicing Reaction==<br />
<br />
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=Outlook=</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/CollaborationsTeam:Heidelberg/pages/Collaborations2014-10-16T08:48:22Z<p>Huhny: </p>
<hr />
<div>Kurze Einleitung<br />
<br />
=iGEM Team Aachen=<br />
<br />
Michael from the iGEM team Aachen visited us in Heidelberg. Their team worked on the development of a real-time pathogen detection technique for what they have build their own fluorescence measurement camera. We provided our expression vectors to the iGEM team Aachen, in exchange they offered to test these expression vectors with their own designed analyser by measuring the amount of produced fluorescent proteins.<br />
<br />
=iGEM Team Tuebingen=<br />
<br />
<br />
=iGEM Team Freiburg=<br />
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}}<br />
<br />
Three of our team members, Anna, Caro, and Charlotte visited the iGEM team Freiburg. We met the team right after a big team meeting, therefore we were able to hear their latest project developments. We spend the evening talking about experiences in the lab and about iGEM. It was relieving to hear that they faced similar difficulties in the lab. <br />
The morning after we presented our project to the iGEM team Freiburg. Freiburg was very interested in our program iGEM@home as we offered them to promote their own human practise projects.<br />
Both our teams are working with light inducible systems, manly the LOV domain. Although working in different organisms, the constructs were compatible, therefore we could interchange protocols for induction and preliminary results. Furthermore we gazed at their professional light induction box - we can definitely improved ours. <br />
<br />
Gibson vs. Cpec?<br />
<br />
After cooperation with the iGEM Team Freiburg for two years in a row we are hoping to keep Freiburg as a reliable partner in the next years as well.<br />
<br />
<br />
=iGEM Team Imperial Collage London=</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/CollaborationsTeam:Heidelberg/pages/Collaborations2014-10-16T08:47:26Z<p>Huhny: </p>
<hr />
<div>Kurze Einleitung<br />
<br />
=iGEM Team Aachen=<br />
<br />
Michael from the iGEM team Aachen visited us in Heidelberg. Their team worked on the development of a real-time pathogen detection technique for what they have build their own fluorescence measurement camera. We provided our expression vectors to the iGEM team Aachen, in exchange they offered to test these expression vectors with their own designed analyser by measuring the amount of produced fluorescent proteins.<br />
<br />
=iGEM Team Tuebingen=<br />
<br />
<br />
=iGEM Team Freiburg=<br />
<br />
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}}<br />
<br />
Three of our team members, Anna, Caro, and Charlotte visited the iGEM team Freiburg. We met the team right after a big team meeting, therefore we were able to hear their latest project developments. We spend the evening talking about experiences in the lab and about iGEM. It was relieving to hear that they faced similar difficulties in the lab. <br />
The morning after we presented our project to the iGEM team Freiburg. Freiburg was very interested in our program iGEM@home as we offered them to promote their own human practise projects.<br />
Both our teams are working with light inducible systems, manly the LOV domain. Although working in different organisms, the constructs were compatible, therefore we could interchange protocols for induction and preliminary results. Furthermore we gazed at their professional light induction box - we can definitely improved ours. <br />
<br />
Gibson vs. Cpec?<br />
<br />
After cooperation with the iGEM Team Freiburg for two years in a row we are hoping to keep Freiburg as a reliable partner in the next years as well.<br />
<br />
<br />
=iGEM Team Imperial Collage London=</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/CollaborationsTeam:Heidelberg/pages/Collaborations2014-10-16T08:44:32Z<p>Huhny: </p>
<hr />
<div>Kurze Einleitung<br />
<br />
=iGEM Team Aachen=<br />
<br />
Michael from the iGEM team Aachen visited us in Heidelberg. Their team worked on the development of a real-time pathogen detection technique for what they have build their own fluorescence measurement camera. We provided our expression vectors to the iGEM team Aachen, in exchange they offered to test these expression vectors with their own designed analyser by measuring the amount of produced fluorescent proteins.<br />
<br />
=iGEM Team Tuebingen=<br />
<br />
<br />
=iGEM Team Freiburg=<br />
<br />
<br />
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}}<br />
<br />
Three of our team members, Anna, Caro, and Charlotte visited the iGEM team Freiburg. We met the team right after a big team meeting, therefore we were able to hear their latest project developments. We spend the evening talking about experiences in the lab and about iGEM. It was relieving to hear that they faced similar difficulties in the lab. <br />
The morning after we presented our project to the iGEM team Freiburg. Freiburg was very interested in our program iGEM@home as we offered them to promote their own human practise projects.<br />
Both our teams are working with light inducible systems, manly the LOV domain. Although working in different organisms, the constructs were compatible, therefore we could interchange protocols for induction and preliminary results. Furthermore we gazed at their professional light induction box - we can definitely improved ours. <br />
<br />
Gibson vs. Cpec?<br />
<br />
After cooperation with the iGEM Team Freiburg for two years in a row we are hoping to keep Freiburg as a reliable partner in the next years as well.<br />
<br />
<br />
=iGEM Team Imperial Collage London=</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/CollaborationsTeam:Heidelberg/pages/Collaborations2014-10-15T12:00:49Z<p>Huhny: </p>
<hr />
<div><!-- Collaborations --><br />
<br />
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<h1>iGEM Team Aachen</h1><br />
<br />
Michael from the iGEM team Aachen visited us in Heidelberg. Their team worked on the development of a real-time pathogen detection technique for what they have build their own fluorescence measurement camera. We provided our expression vectors to the iGEM team Aachen, in exchange they offered to test these expression vectors with their own designed analyser by measuring the amount of produced fluorescent proteins.<br />
<br />
<h1>iGEM Team Tuebingen</h1><br />
<br />
<h1>iGEM Team Freiburg</h1><br />
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</div><br />
Three of our team members, Anna, Caro, and Charlotte visited the iGEM team Freiburg. We met the team right after a big team meeting, therefore we were able to hear their latest project developments. We spend the evening talking about experiences in the lab and about iGEM. It was relieving to hear that they faced similar difficulties in the lab. <br />
The morning after we presented our project to the iGEM team Freiburg. Freiburg was very interested in our program iGEM@home as we offered them to promote their own human practise projects.<br />
Both our teams are working with light inducible systems, manly the LOV domain. Although working in different organisms, the constructs were compatible, therefore we could interchange protocols for induction and preliminary results. Furthermore we gazed at their professional light induction box - we can definitely improved ours. <br />
<br />
Gibson vs. Cpec?<br />
<br />
After cooperation with the iGEM Team Freiburg for two years in a row we are hoping to keep Freiburg as a reliable partner in the next years as well.<br />
<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|<br />
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<br />
<br />
<h1>iGEM Team Imperial Collage London</h1><br />
<br />
<br />
}}</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/pages/Public_RelationsTeam:Heidelberg/pages/Public Relations2014-10-14T22:11:59Z<p>Huhny: /* Tumblr Blog */</p>
<hr />
<div>=Tumblr Blog=<br />
Discussions on Biotechnology are often false biased because of a lack of detailed knowledge in this topic. Besides organising lectures and raising attention with facebook and twitter, we also established a [http://igem14-heidelberg.tumblr.com/ Tumblr blog], where we continuously upload articles about Synthetic Biology and Biotechnology to make science accessible for broad public we are write in understandable language. <br />
<br />
Articles are about the basic principles of biology, methods of gene technology like the PCR reaction or modelling of biologic processes, we give an overview of the huge topic genetically cell structure, processes, methods of gene technologie, pros and cons biotechnology, synthetic biology peppered with personal stories from our lab - fun and easy way to get information primary goal: no false or subjective articles, unbiased - information for broad public to increase discussion on a level of better knowledge.</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/Human_PracticeTeam:Heidelberg/Human Practice2014-10-14T22:07:58Z<p>Huhny: </p>
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<p><br />
Research and knowledge lead to responsibility for everyone of us. In Friedrich Dürrenmatt’s satiric drama “The Physicists” the main character Möbius has solved the world formula, which includes an explanation for gravity. This discovery turns out to be both blessing and curse and could decide the fate of humanity. Being aware of the impact his findings might have he chooses to spend the rest of his life in a sanatorium instead of having to bear the responsibility that comes with publication. </p><p><br />
Although written in 1962 the drama is still up to date as it reflects the questions of scientific ethics and humanity's ability to handle its intellectual responsibilities.</p><p><br />
With ongoing advances, the introduction to consequences of scientific research to the public is becoming an increasingly important task for an iGEM team, as it should be in every scientific community. In our Human Practice work we address this problem in a comprehensive program and discuss benefits as well as dangers of Synthetic Biology.<br />
The potential discoveries and applications in Synthetic Biology will have significant influences on everyday life, therefore a continuous and public dialogue on limitations and allowances is necessary.</p><p><br />
A key aspect of the work of the iGEM Team Heidelberg is therefore to increase the dialogue between science, society and industry. </p><p>Read about the different efforts we made in the last few months and dig deeper into our Human Practice concept, which includes also a new open source tool for the whole iGEM community.</p><br />
</div><br />
</div><br />
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<a href="/Team:Heidelberg/Human_Practice/igemathome" class="box cell" style="display:block;"><br />
<h2>iGEM@Home</h2><img src="/wiki/images/9/9f/Heidelberg_Igemathome_bg.png" alt="iGemathome Image" style="width:40%;float:right;" /><br />
Hier findet der Leser eine Erklärung. Hier findet der Leser eine kurze Erklärung. <br />
Hier findet der Leser eine Erklärung. Hier findet der Leser eine Erklärung. Hier findet der Leser eine kurze Erklärung. <br />
Hier findet der Leser eine Erklärung.<br />
<div class="clearfix"></div> <br />
</a><br />
<a href="/Team:Heidelberg/Human_Practice/Education" class="box cell"><br />
<h3><span>Education</span></h3><br />
Hier findet der Leser eine Erklärung. Hier findet der Leser eine Erklärung. Hier findet der Leser eine Erklärung.<br />
</a><br />
</div><br />
<div class="boxes-table"><br />
<a href="#" class="box cell" style="width:30%;"><br />
<h3>Religion, Ethics and Synthetic Biology</h3> <br />
Hier findet der Leser eine Erklärung. Hier findet der Leser eine Erklärung. Hier findet der Leser eine Erklärung.<br />
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<h3><span>Experts</span></h3><br />
To make sure our project will have an impact for both basic research and industry, we presented our project to different experts even in the developing phase. In the following short articles we talk about our experience and the feedback we got. <br />
</a><br />
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<h3><span>Public Relations</span></h3><br />
We – being „generation Y“ – know how much attention you can raise using the effect of social media. For content and idea sharing we used platforms like twitter, facebook and tumblr. Read more about our Public Relations and our weekly Tumblr blog articles to inform people about basics of Synthetic Biology. <br />
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<div>=Tumblr Blog=<br />
Discussions on Biotechnology are often false biased because of a lack of detailed knowledge in this topic. Besides organising lectures and raising attention with facebook and twitter, we also established a tumblr blog, where we continuously upload articles about Synthetic Biology and Biotechnology to make science accessible for broad public we are write in understandable language. <br />
<br />
Articles are about the basic principles of biology, methods of gene technology like the PCR reaction or modelling of biologic processes, we give an overview of the huge topic genetically cell structure, processes, methods of gene technologie, pros and cons biotechnology, synthetic biology peppered with personal stories from our lab - fun and easy way to get information primary goal: no false or subjective articles, unbiased - information for broad public to increase discussion on a level of better knowledge.</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/Human_Practice/Public_RelationsTeam:Heidelberg/Human Practice/Public Relations2014-10-14T22:03:24Z<p>Huhny: Created page with "{{:Team:Heidelberg/templates/wikipage_new| title=PUBLIC RELATIONS | white=true | red-logo= | header-img= | body-style=background-color:white; | header-bg=#DE4230 | subtitle= This..."</p>
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<p><br />
Research and knowledge lead to responsibility for everyone of us. In Friedrich Dürrenmatt’s satiric drama “The Physicists” the main character Möbius has solved the world formula, which includes an explanation for gravity. This discovery turns out to be both blessing and curse and could decide the fate of humanity. Being aware of the impact his findings might have he chooses to spend the rest of his life in a sanatorium instead of having to bear the responsibility that comes with publication. </p><p><br />
Although written in 1962 the drama is still up to date as it reflects the questions of scientific ethics and humanity's ability to handle its intellectual responsibilities.</p><p><br />
With ongoing advances, the introduction to consequences of scientific research to the public is becoming an increasingly important task for an iGEM team, as it should be in every scientific community. In our Human Practice work we address this problem in a comprehensive program and discuss benefits as well as dangers of Synthetic Biology.<br />
The potential discoveries and applications in Synthetic Biology will have significant influences on everyday life, therefore a continuous and public dialogue on limitations and allowances is necessary.</p><p><br />
A key aspect of the work of the iGEM Team Heidelberg is therefore to increase the dialogue between science, society and industry. </p><p>Read about the different efforts we made in the last few months and dig deeper into our Human Practice concept, which includes also a new open source tool for the whole iGEM community.</p><br />
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<h2>iGEM@Home</h2><img src="/wiki/images/9/9f/Heidelberg_Igemathome_bg.png" alt="iGemathome Image" style="width:40%;float:right;" /><br />
Hier findet der Leser eine Erklärung. Hier findet der Leser eine kurze Erklärung. <br />
Hier findet der Leser eine Erklärung. Hier findet der Leser eine Erklärung. Hier findet der Leser eine kurze Erklärung. <br />
Hier findet der Leser eine Erklärung.<br />
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<h3><span>Education</span></h3><br />
Hier findet der Leser eine Erklärung. Hier findet der Leser eine Erklärung. Hier findet der Leser eine Erklärung.<br />
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<h3>Religion, Ethics and Synthetic Biology</h3> <br />
Hier findet der Leser eine Erklärung. Hier findet der Leser eine Erklärung. Hier findet der Leser eine Erklärung.<br />
<img src="/wiki/images/1/10/Heidelberg_Religion.png"><br />
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<h3><span>Experts</span></h3><br />
To make sure our project will have an impact for both basic research and industry, we presented our project to different experts even in the developing phase. In the following short articles we talk about our experience and the feedback we got. <br />
</a><br />
<a href="#" class="cell box" ><br />
<h3><span>Public Relations</span></h3><br />
We – being „generation Y“ – know how much attention you can raise using the effect of social media. For content and idea sharing we used platforms like twitter, facebook and tumblr. Read more about our Public Relations and our weekly Tumblr blog articles to inform people about basics of Synthetic Biology. <br />
</a><br />
</div><br />
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Synthetic Biology is a very dynamic and still evolving field in science. It has been broad forward by young and creative scientist - iGEM is the best example of it. Therefore it is even more important to introduce young and motivated people to Synthetic Biology. We cooperated with the Heidelberg Life Science Lab of the German Cancer Research Center (DKFZ), a project dedicated to support extraordinarily gifted and talented high-school students in developing their scientific as well as personal skills and in gaining practical research experience. Parallel to our own iGEM Project we supervised a group of 15 pupils in their own project, with which they plan to participate at iGEM High school competition 2015. They have developed a biologic circle, team name: BioLogIC, using Quorum Sensing of bacteria. This project combines the decidedness of digital systems with the multiplex possibilities of biology. Before starting an own project the group was taught in the basic principle of Synthetic Biology. Flowing we had a long planning time, where we met the group once a month. In February 2014 they were able to do the first cloning experiments in the lab. Furthermore they have presented their work on the Schülersymposium and were invited to the Symposium on Synthetic Biology in Heidelberg.<br />
<br />
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The progress in natural sciences and technology has an impact on the life of each and every one within a society. Therefore it is enormously important to raise discussions in close dialogue with the general public about ethical issues, risks and benefits in Synthetic Biology. <br />
<br />
To participate in considerations and evaluations about Synthetic Biology, a basic comprehension of biological processes is essential. To spread knowledge about the practical handling of Synthetic Biology within an actual lab, we offered a three days practical course to pupils at the age of 15 to 18 within the Liege Science Lab in Heidelberg. The Heidelberg Life-Science Lab is part of the German Cancer Research Center (DKFZ). It is dedicated to support extraordinarily gifted and talented high-school students in developing their scientific as well as personal skills and in gaining practical research experience. The school students actually took over the experiments we were performing in the lab and tried to circularize GFP. The fact that our young scientist produced data, which were actually very crucial to us, motivated them to try very hard for the best results. Alternating in theoretical lectures and practical courses, the pupils learned the background of our project and the actual experiments first-hand:<br />
<br />
¶protocol<br />
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==Practical lab work==<br />
On the first day, the interns conducted a PCR from a plasmid containing GFP with primers containing BsaI overhangs. The PCR product was visualised on an agarose gel, purified and its concentration determined using a spectroscopical measurement technique. The purified construct was used for golden gate cloning using our toolbox construct for circularization.<br />
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The final construct, a positive control containing normal GFP on an expression vector and a negative control, were transformed in BL21 (DE3) and plated on agar containing ampicillin. Additionally liquid cultures were inoculated.<br />
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On the second day the liquid cultures were used to inoculate the main expression culture. Moreover a sample was taken for a colony PCR to verify the presence of our circularization construct and the linear GFP construct.<br />
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Upon the ID of 0.8 the school interns induced the cultures with 1 mM IPTG for one hour. To visualise the proteins- linear and circular GFP version- an SDS- PAGE and Western Blot was conducted.<br />
<br />
If our construct leads to the circularization of GFP a shift should be visible on the Western Blot between linear and circular protein. The circular construct runs faster on a gel, since its coiled nature experiences less resistance from the gel matrix.<br />
<br />
Unfortunately, the pupils experienced some difficulties in the experiments. To conduct an experiment properly it needs a lot of attention and at least some experience. Even though the results did not look like expected or desired, the pupils learned many new methods and quite some theoretical background. Altogether not only the school kids had much fun with their first-hand experience in the lab, but also we as supervisors and tutors.</div>Huhnyhttp://2014.igem.org/Team:Heidelberg/SoftwareTeam:Heidelberg/Software2014-10-14T21:15:55Z<p>Huhny: </p>
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<h3>iGEM@home</h3><br />
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<img src="/wiki/images/9/9f/Heidelberg_Igemathome_bg.png" class="img-responsive"/><br />
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<h3>Linker Software</h3><br />
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<div class="cell box nohover"><br />
<div style="display:block; padding:0; position:relative;"><br />
<img src="/wiki/images/9/9f/Heidelberg_Igemathome_bg.png" class="img-responsive"/><br />
<span style="position:absolute; padding:5px; bottom:0; left:0;"><br />
<h3>MidnightDoc</h3><br />
</span> <br />
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<a href="https://2014.igem.org/Team:Heidelberg/Software/igemathome" class="cell box"><br />
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<h3>iGEM@home</h3><br />
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<span style="position:absolute; padding:5px; bottom:0; left:0;"><br />
<h3>MidnightDoc</h3><br />
</span> <br />
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<a href="https://2014.igem.org/Team:Heidelberg/Software/igemathome" class="cell box"><br />
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<h3>The Software</h3><br />
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<span style="position:absolute; padding:5px; bottom:0; left:0;"><br />
<h3>iGEM@home</h3><br />
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<div style="display:block; padding:0; position:relative;"><br />
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<span style="position:absolute; padding:5px; bottom:0; left:0;"><br />
<h3>Linker-Software</h3><br />
</span> <br />
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</div><br />
<div class="cell box nohover"><br />
<div style="display:block; padding:0; position:relative;"><br />
<img src="/wiki/images/9/9f/Heidelberg_Igemathome_bg.png" class="img-responsive"/><br />
<span style="position:absolute; padding:5px; bottom:0; left:0;"><br />
<h3>MidnightDoc</h3><br />
</span> <br />
</div><br />
</div><br />
</div><br />
<div class="boxes-row"><br />
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<h3>The Software</h3><br />
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<span style="position:absolute; padding:5px; bottom:0; left:0;"><br />
<h3>iGEM@home</h3><br />
</span> <br />
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</div><br />
<div class="cell box nohover"><br />
<div style="display:block; padding:0; position:relative;"><br />
<img src="/wiki/images/9/9f/Heidelberg_Igemathome_bg.png" class="img-responsive"/><br />
<span style="position:absolute; padding:5px; bottom:0; left:0;"><br />
<h3>Linker-Software</h3><br />
</span> <br />
</div><br />
</div><br />
<div class="cell box nohover"><br />
<div style="display:block; padding:0; position:relative;"><br />
<img src="/wiki/images/9/9f/Heidelberg_Igemathome_bg.png" class="img-responsive"/><br />
<span style="position:absolute; padding:5px; bottom:0; left:0;"><br />
<h3>MidnightDoc</h3><br />
</span> <br />
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</div><br />
</div><br />
<div class="boxes-row"><br />
<a href="#" class="cell box"><br />
<h3>The Software</h3><br />
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<img src="/wiki/images/9/9f/Heidelberg_Igemathome_bg.png" class="img-responsive"/><br />
<span style="position:absolute; padding:5px; bottom:0; left:0;"><br />
<h3>iGEM@home</h3><br />
</span> <br />
</div><br />
</div><br />
<div class="cell box nohover"><br />
<div style="display:block; padding:0; position:relative;"><br />
<img src="/wiki/images/9/9f/Heidelberg_Igemathome_bg.png" class="img-responsive"/><br />
<span style="position:absolute; padding:5px; bottom:0; left:0;"><br />
<h3>Linker-Software</h3><br />
</span> <br />
</div><br />
</div><br />
<div class="cell box nohover"><br />
<div style="display:block; padding:0; position:relative;"><br />
<img src="/wiki/images/9/9f/Heidelberg_Igemathome_bg.png" class="img-responsive"/><br />
<span style="position:absolute; padding:5px; bottom:0; left:0;"><br />
<h3>MidnightDoc</h3><br />
</span> <br />
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<div class="boxes-row"><br />
<a href="#" class="cell box"><br />
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<div class="cell box nohover"><br />
<div style="display:block; padding:0; position:right;"><br />
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<h3>Linker-Software</h3><br />
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<div class="cell box nohover"><br />
<div style="display:block; padding:0; position:right;"><br />
<img src="/wiki/images/9/9f/Heidelberg_Igemathome_bg.png" class="img-responsive"/><br />
<span style="position:absolute; padding:5px; bottom:0; left:0;"><br />
<h3>MidnightDoc</h3><br />
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<div class="boxes-row"><br />
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<span style="position:absolute; padding:15px; bottom:0; left:0; color:white;"><br />
<h3>iGEM@home</h3><br />
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<div class="cell box nohover"><br />
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