Team:Heidelberg/Parts

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<h1 style="margin-top:10%"><span style="font-size:150%">Our Parts.</span><span style="font-size:90%" class="text-muted"> Now it is Your turn. Start working with NRPS!</span></h1>
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<h1><span style="font-size:150%; color:#666666">Our Parts.</span><span style="font-size:90%" class="text-muted"> Now it is Your turn. Start working with NRPS!</span></h1>
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<h2 id="Natural Parts">Natural Parts</h2>
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<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/2/27/Slider_kuvette.png" style="float:left; margin-right: 10px;" title="Indigoidine synthetase activated by the different PPTases that we submitted.">
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                  <h2><span style="font-size:170%;">Synthetic Peptides</span style="font-size:170%;"> - <span style="font-size":50%">BBa_K1152000 - 2005</h2>
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    <figcaption style="width:200px;"> Indigoidine synthetase activated by the different PPTases that we submitted</figcaption>
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                            We provide the constructs for the novel Dipeptide- and Tripeptide-Synthetases. Furthermore, we submitted the single modules that they are comprised of. These parts should serve as the basis of a future library for standardized work with NRPS. The modules are specific for different amino acids: Phenylalanine (tycA), Proline (tycB1) and Leucine (tycC6).
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We offer several natural parts to the library. Firstly, three modules from the Tyrocidine cluster from <em>B. parabrevis</em> that are specific for three different amino acids: <a href="http://parts.igem.org/Part:BBa_K1152000">BBa_K1152000</a> (tycA) for Phenylalanine, <a href="http://parts.igem.org/Part:BBa_K1152001">BBa_K1152001</a> (tycB1) for Proline and <a href="http://parts.igem.org/Part:BBa_K1152003">BBa_K1152003</a> (tycC6) for Leucine. Secondly, we have submitted the Indigiodine synthetase indC from <em>P. luminescens</em> (cds: <a href="http://parts.igem.org/Part:BBa_K1152008">BBa_K1152008</a>, integratable device: <a href="http://parts.igem.org/Part:BBa_K1152013">BBa_K1152013</a>). And thirdly, we offer a collection of PPTases (see image) which are required enzymes in order to activate NRPSs and turn them from the apo- into the hoho-form: <a href="http://parts.igem.org/Part:BBa_K1152009">BBa_K1152009</a>, <a href="http://parts.igem.org/Part:BBa_K1152010">BBa_K1152010</a>, <a href="http://parts.igem.org/Part:BBa_K1152011">BBa_K1152011</a>, <a href="http://parts.igem.org/Part:BBa_K1152012">BBa_K1152012</a>.
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                                    <img src="https://static.igem.org/mediawiki/2013/2/26/Heidelberg_Parts_Gallery_1.png" style="width:70%; padding:1%" />
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We propose <a href="http://parts.igem.org/Part:BBa_K1152013">BBa_K1152013</a> as Best new BioBrick Part, Natural, because it encodes the original NRPS module from <i>Photorhabdus luminescens</i> capable of synthesizing Indigoidine, which offers the opportunity to combining it with modules from other NRPSs to yield fusion peptides with a blue pigment tag. Accompanying this BioBrick, we propose two standards (<a href="http://hdl.handle.net/1721.1/81332">RFC 99</a> & <a href="http://hdl.handle.net/1721.1/81333">RFC 100</a>) and established protocols which allow for BioBrick assembly or Gibson assembly and through this to a high-throughput creation of synthesized, tagged peptides. Besides this, Indigoidine is belived to have antimicrobial and anti-cancerous effects.
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We used Indigoidine in the subprojects that were dealing with the tagging of NPRs and, in the course of this, characterized its functionality thoroughly. Hence more information on Indigoidine is available on the <a href="https://2013.igem.org/Team:Heidelberg/Project/Indigoidine-Tag">Peptide-Tagging</a> page and the <a href="https://2013.igem.org/Team:Heidelberg/Project/Tag-Optimization">Tag-Optimization</a> page.
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                  <h2><span style="font-size:170%;">Indigoidine-Tag</span style="font-size:170%;"> - <span style="font-size":50%">BBa_K1152006 & 2007</h2>
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                            One of our major achievements is the establishment of an easily detectable, inert and universal tag - the <a href="https://2013.igem.org/Team:Heidelberg/Project/Indigoidine-Tag">Indigoidine-Tag</a>. With this tag, purification and validation of novel NRPs is significantly eased. The characteristics of the tag compare to those of the GFP-tag for proteins. We submit the helper-construct for tagging any desired Non-Ribosomal Peptide as our <a href="https://2013.igem.org/Team:Heidelberg/Favorite_Parts">favorite part</a>.
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                  <h2><span style="font-size:170%;">Tag-Optimization</span style="font-size:170%;"> - <span style="font-size":50%">BBa_K1152008 & 2013-2019</h2>
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                            Besides establishing the <a href="https://2013.igem.org/Team:Heidelberg/Project/Indigoidine-Tag">Indigoidine-Tag</a>, we focussed on <a href="https://2013.igem.org/Team:Heidelberg/Project/Tag-Optimization">optimizing</a> its functionality and efficiency. Therefore, we modified the natural sequence of the Indigoidine synthetase indC (which is our <a href="https://2013.igem.org/Team:Heidelberg/Favorite_Parts">Best Natural BioBrick</a> by domain exchange which lead to altered and in some cases enhanced efficiency of the NRP-production. We submit the collection of alternate T-domains, of which we created several functional, synthetic ones as <a href="https://2013.igem.org/Team:Heidelberg/Favorite_Parts">Best Part Range</a>.
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                  <h2><span style="font-size:170%;">Tag-Characterization</span style="font-size:170%;"> - <span style="font-size":50%">BBa_K1152009 - 2012</h2>
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                            During <a href="https://2013.igem.org/Team:Heidelberg/Project/Tag-Optimization">Tag-Optimization</a>, we also focussed on the enzymes that are required for the activation of NRPSs - the PPTases. We characterized several PPTases and assessed their compatibility and efficiency. We thereby improved parts <b><a href="http://parts.igem.org/Part:BBa_K802006">BBa_K802006</a></b> and <b><a href="http://parts.igem.org/Part:BBa_K302010">BBa_K302010</a></b> from the registry, which are PPTase-encoding parts not annotated or characterized.
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<h2 id="Engineered Parts">Engineered Parts</h2>
 
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We built different BioBricks in the course of investigating the modularity of NRPS. We offer two novel Non-Ribosomal Peptide Synthetases, one which is an assembly line for a Proline-Leucine Dipeptide (<a href="http://parts.igem.org/Part:BBa_K1152004">BBa_K1152004</a>), the other one is a synthetase for a Phenylalanine-Ornithine-Leucine Tripeptide (<a href="http://parts.igem.org/Part:BBa_K1152005">BBa_K1152005</a>). You can learn more about these parts on our <a href="https://2013.igem.org/Team:Heidelberg/Project/Tyrocidine">Synthetic Peptides</a> page.
 
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<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/e/ef/Heidelberg_Val-Ind_NRPS_Protein.png" style="float:right; margin-left: 10px;" title="Predicted tertiary structure of the Valine-Indigoidine-Synthetase.">
 
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    <img style="width:200px; margin-bottom:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/e/ef/Heidelberg_Val-Ind_NRPS_Protein.png"></img>
 
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    <figcaption style="width:200px">Predicted tertiary structure of the Valine-Indigoidine-Synthetase. </figcaption>
 
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Besides this, we created an inter-species fusion-peptide of the TycC4-module and the Indigoidine synthetase IndC, thereby giving a proof of concept for both, inter-species flexibility of modules and domains, and tagging of short peptides with the blue pigment Indigoidine. This is explained in detail on our <a href="https://2013.igem.org/Team:Heidelberg/Project/Indigoidine-Tag">Peptide-Tagging</a> page and in the parts' registry: <a href="http://parts.igem.org/Part:BBa_K1152006">BBa_K1152006</a>.
 
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<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/7/75/Slider_plattentabelle.png" style="float:left; margin-right: 10px;" title="Comparison of the activity of different T-domains activated by different PPTases.">
 
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    <figcaption style="width:200px">Comparison of the activity of different T-domains activated by different PPTases. </figcaption>
 
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Furthermore, we created several synthetic variants of the IndC-module by introducing a T-domain from another synthetase or by introducing entirely synthetic T-domains: <a href="http://parts.igem.org/Part:BBa_K1152015">BBa_K1152015</a>, <a href="http://parts.igem.org/Part:BBa_K1152016">BBa_K1152016</a>, <a href="http://parts.igem.org/Part:BBa_K1152017">BBa_K1152017</a>, <a href="http://parts.igem.org/Part:BBa_K1152018">BBa_K1152018</a>, <a href="http://parts.igem.org/Part:BBa_K1152019">BBa_K1152019</a>, <a href="http://parts.igem.org/Part:BBa_K1152020">BBa_K1152020</a>. We propose this set of parts as Best Parts Collection as they represent a valuable ensemble of various T-domains with different properties and besides this, proof the extent of flexibility of NRPS. Being designed semi-rationally based on multiple sequence alignments, we discovered that modified IndC-variants exhibit distinct synthesis rates for Indigoidine depending on the T-domains of this part collection. We therefore used them for <a href="https://2013.igem.org/Team:Heidelberg/Project/Tag-Optimization">Optimization of the Indigoidine-Tag</a>.
 
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During our work with NRPS, we did not only create several synthetic parts in order to characterize and improve the efficiency of Indigoidine and synthesize new peptides, but also improved the parts <a href="http://parts.igem.org/Part:BBa_K802006">BBa_K802006</a> and <a href="http://parts.igem.org/Part:BBa_K302010">BBa_K302010</a>, which encode for sfp (a PPTase) from <i>Bacillus subtilis</i> but were deviating in three amino acids from the sequence published on National Center for Biotechnology Information (<a href="http://www.ncbi.nlm.nih.gov/">NCBI</a>). We therefore propose <a href="http://parts.igem.org/Part:BBa_K1152009">BBa_K1152009</a> as Most Improved Registry Part, because it encodes for sfp, a 4'-Phosphopanthetheinyl-transferase (PPTase) crucial for the activation of T-domains and contains the aforementionned three divergent amino acids. It is the first BioBrick in the registry that is explicitly encoding sfp, and it is annotated and characterized. Learn more about PPTases and their characterization on the <a href="https://2013.igem.org/Team:Heidelberg/Project/Tag-Optimization">Tag-Optimization</a> page!
 
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<h2 id="Engineered Devices">Engineered Devices</h2>
 
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<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/e/ef/Heidelberg_ind_slider_6.png" style="float:left; margin-right: 10px;" title="Now it's your turn! Peptide-Tagging allows easy detection and screening.">
 
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    <figcaption style="width:200px;"><b>Now it's your turn! Peptide-Tagging allows easy detection and screening.</b></figcaption>
 
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The work mentionned above was significantly eased by two helper constructs that we designed and we would hence like to promote them to the entire community. As it is the central idea of our project to make the very potent NRPS-system accessible to the iGEM community, we believe that those two parts are of a very high value.
 
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We are therefore proud to promote our <b>favorite part</b>, namely <a href="http://parts.igem.org/Part:BBa_K1152007">BBa_K1152007</a>, which is a helper construct for NRP-Indigoidine-tagging. In order to tag peptides with Indigoidine, a C-domain has to be inserted in front of the IndC-module, in order to standardize this, and in order to allow anyone to create tagged constructs easily and with low background, we developed this ccdB-driven (as negative selection), highly efficient (i.e. 0 % false-positive rate) cloning tool. The plasmid is compatible with custom non-ribosomal peptide (NRP) synthesis due to the standardized C-domain from <em>B. parabrevis</em>. So in summary <a href="http://parts.igem.org/Part:BBa_K1152007">BBa_K1152007</a> provides optimized fusion of the NRP to Indigoidine that serves as a tag that eases detection of positive clones that synthesize the desired construct. We therefore propose <a href="http://parts.igem.org/Part:BBa_K1152007">BBa_K1152007</a> as Best new BioBrick Part or Device, Engineered, as mentioned above. We found it very helpful to work with this part in the <a href="https://2013.igem.org/Team:Heidelberg/Project/Indigoidine-Tag">Peptide-Tagging</a> project.
 
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Additionally, we provide the community with the helper-construct that we used for the T-domain shuffling. It is also ccdB-based and therefore minimizes background during <a href="https://2013.igem.org/Team:Heidelberg/Project/Tag-Optimization">Tag-Optimization</a>: <a href="http://parts.igem.org/Part:BBa_K1152014">BBa_K1152014</a>
 
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<h2><span style="font-size:170%;">List of Parts</span style="font-size:170%;"> - <span style="font-size":50%">BBa_K1152000 - 2019</h2>
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<h2 id="PartList">You can find more detailed information on the respective project pages or in the Parts' Registry:</h2>
 
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Latest revision as of 21:57, 28 October 2013

Our Parts. Now it is Your turn. Start working with NRPS!


Synthetic Peptides - BBa_K1152000 - 2005

We provide the constructs for the novel Dipeptide- and Tripeptide-Synthetases. Furthermore, we submitted the single modules that they are comprised of. These parts should serve as the basis of a future library for standardized work with NRPS. The modules are specific for different amino acids: Phenylalanine (tycA), Proline (tycB1) and Leucine (tycC6).



Indigoidine-Tag - BBa_K1152006 & 2007

One of our major achievements is the establishment of an easily detectable, inert and universal tag - the Indigoidine-Tag. With this tag, purification and validation of novel NRPs is significantly eased. The characteristics of the tag compare to those of the GFP-tag for proteins. We submit the helper-construct for tagging any desired Non-Ribosomal Peptide as our favorite part.



Tag-Optimization - BBa_K1152008 & 2013-2019

Besides establishing the Indigoidine-Tag, we focussed on optimizing its functionality and efficiency. Therefore, we modified the natural sequence of the Indigoidine synthetase indC (which is our Best Natural BioBrick by domain exchange which lead to altered and in some cases enhanced efficiency of the NRP-production. We submit the collection of alternate T-domains, of which we created several functional, synthetic ones as Best Part Range.



Tag-Characterization - BBa_K1152009 - 2012

During Tag-Optimization, we also focussed on the enzymes that are required for the activation of NRPSs - the PPTases. We characterized several PPTases and assessed their compatibility and efficiency. We thereby improved parts BBa_K802006 and BBa_K302010 from the registry, which are PPTase-encoding parts not annotated or characterized.




List of Parts - BBa_K1152000 - 2019

Thanks to

Retrieved from "http://2013.igem.org/Team:Heidelberg/Parts"