Team:Heidelberg/Favorite Parts

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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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<h1><span style="font-size:150%; color:#666666">Favorite 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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<center><h1>Best BioBrick Part or Device, Natural.</h1></center>
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                   <div class="bronzebox" >
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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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                   <h2><span style="font-size:170%;">BBa_K1152013.</span style="font-size:170%;"> <span style="font-size":50%">IndC Blue Pigment Production Device.</h2>
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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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<li> The indigoidine synthetase IndC is a <b>single-module NRPS</b>
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<li> Catalyzes the formation of the <b>blue pigment indigoidine by circularizing glutamine</b>
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<li> Indigoidine is <b>visible to the naked eye</b>
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<li> Indigoidine production can <b>easily be quantified</b> photometrically and is thus an <b>ideal reporter</b>
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<li> In constrast to many other pigment production devices, <b>IndC does not need any external substrate supply</b>
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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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<center><h1>Best BioBrick Part or Device, Engineered.</h1></center>
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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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                   <h2><span style="font-size:170%;">BBa_K1152007.</span style="font-size:170%;"> <span style="font-size":50%">The GFP for Non-Ribosomal Peptides.</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 favorite part.
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<li> Enables labeling of <b>custom non-ribosomal peptides</b> with a blue pigment in vivo (see <b><a href="https://2013.igem.org/Team:Heidelberg/Project/Indigoidine-Tag">Indigoidine-Tag</a></b> page)
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<li> NRPS modules of choice are introduced in front of an <b>engineered IndC module</b> containing an additional C-domain for communication with the previous module
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<li> ccdB is replaced during cloning; close to <b>100 % cloning efficiency</b>
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<li> Peptide production <b>can be seen with the naked eye</b> or quantified photometrically
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<li> Labelled peptides can <b>easily be purified and characterized</b> by thin-layer chromatography
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<li> Helper-construct for high-throughput cloning of custom NRPSs for production of synthetic peptides (see <b><a href="https://2013.igem.org/Team:Heidelberg/RFCs">RFC 100</a></b> for details)
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<center><h1>Most improved Registry Part.</h1></center>
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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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                   <h2><span style="font-size:170%;">BBa_K1152009.</span style="font-size:170%;"> <span style="font-size":50%">Highly-Efficient NRPS-activating PPTase.</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 by domain exchange which lead to altered and in some cases enhanced efficiency of the NRP-production which could be measured by optical density. We submit the collection of alternate T-domains, of which we created several functional, synthetic ones as Best Part Range.
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<li> Helper plasmid for <b>efficient peptide production by engineered or natural NRPSs</b>
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<li> Transfers a 4'-PPT residue from CoA to a conserved serine residue in the T-Domain of NRPS modules, thereby <b>activating the NRPS module</b>
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<li> <b>Improves parts</b> BBa_K802006 and BBa_K302010 which are PPTases that were present in the registry, but neither annotated nor characterized as such
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<center><h1>Best Part Collection.</h1></center>
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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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                   <h2><span style="font-size:170%;">BBa_K1152015 - 2019.</span style="font-size:170%;"> <span style="font-size":50%">A Collection of Synthetic Indigoidine Synthetases Engineered by T-domain Exchange of IndC.</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 ...
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<li> T-domain is important for <b>NRPS efficiency</b>
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<li> IndC was used as scaffold and the native IndC T-domain was exchanged by <b>different natural and synthetic T-domains</b>
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<li> Differing in <b>indigoidine production efficiency</b>
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<li> <b>BBa_K1152015 outperforms the native IndC</b> (BBa_K1152013) when combined with the Sfp or EntD PPTase (red square)
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<li> Collection can now be applied for tagging synthetic peptides produced by engineered NRPSs (see <b><a href="https://2013.igem.org/Team:Heidelberg/Project/Indigoidine-Tag">Indigoidine-Tag</a></b> subproject)
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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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    <img style="width:200px; margin-bottom:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/2/27/Slider_kuvette.png" ></img>
 
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    <figcaption style="width:200px;"> Indigoidine synthetase activated by the different PPTases that we submitted</figcaption>
 
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</a>
 
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<p>
 
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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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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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<p>
 
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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 id="Engineered Parts">Engineered Parts</h2>
 
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<p>
 
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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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    </a>
 
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<p>
 
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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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    <img style="width:200px; margin-bottom:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/7/75/Slider_plattentabelle.png"></img>
 
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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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    </a>
 
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<p>
 
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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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</p>
 
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<p>
 
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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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    <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_ind_slider_6.png" ></img>
 
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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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</a>
 
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<p>
 
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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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<p>
 
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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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</p>
 
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<p>
 
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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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Revision as of 20:01, 28 October 2013

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


Best BioBrick Part or Device, Natural.

BBa_K1152013. IndC Blue Pigment Production Device.

  • The indigoidine synthetase IndC is a single-module NRPS
  • Catalyzes the formation of the blue pigment indigoidine by circularizing glutamine
  • Indigoidine is visible to the naked eye
  • Indigoidine production can easily be quantified photometrically and is thus an ideal reporter
  • In constrast to many other pigment production devices, IndC does not need any external substrate supply



    • Best BioBrick Part or Device, Engineered.

    BBa_K1152007. The GFP for Non-Ribosomal Peptides.

  • Enables labeling of custom non-ribosomal peptides with a blue pigment in vivo (see Indigoidine-Tag page)
  • NRPS modules of choice are introduced in front of an engineered IndC module containing an additional C-domain for communication with the previous module
  • ccdB is replaced during cloning; close to 100 % cloning efficiency
  • Peptide production can be seen with the naked eye or quantified photometrically
  • Labelled peptides can easily be purified and characterized by thin-layer chromatography
  • Helper-construct for high-throughput cloning of custom NRPSs for production of synthetic peptides (see RFC 100 for details)



    • Most improved Registry Part.

    BBa_K1152009. Highly-Efficient NRPS-activating PPTase.

  • Helper plasmid for efficient peptide production by engineered or natural NRPSs
  • Transfers a 4'-PPT residue from CoA to a conserved serine residue in the T-Domain of NRPS modules, thereby activating the NRPS module
  • Improves parts BBa_K802006 and BBa_K302010 which are PPTases that were present in the registry, but neither annotated nor characterized as such



    • Best Part Collection.

    BBa_K1152015 - 2019. A Collection of Synthetic Indigoidine Synthetases Engineered by T-domain Exchange of IndC.

  • T-domain is important for NRPS efficiency
  • IndC was used as scaffold and the native IndC T-domain was exchanged by different natural and synthetic T-domains
  • Differing in indigoidine production efficiency
  • BBa_K1152015 outperforms the native IndC (BBa_K1152013) when combined with the Sfp or EntD PPTase (red square)
  • Collection can now be applied for tagging synthetic peptides produced by engineered NRPSs (see Indigoidine-Tag subproject)


    • Thanks to

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