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Team:Warsaw/EXTRAS
2014-10-18T01:47:11Z
<p>ASamsel: </p>
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<a href="https://2014.igem.org/Team:Warsaw">Project</a><br />
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<li><a href="https://2014.igem.org/Team:Warsaw/Project#proof_of_concept">Proof of concept</a></li><br />
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<br />
<h1>Technology</h1> </br><br />
<hr noshade="noshade" /><br />
<br />
<br />
<a name="bioprocess"><h2>Bioprocess</h2></a></br><br />
<p align="justify">Electronic equipment is the fastest growing waste category of waste in many developed countries. Amount of electronic waste grows rapidly because markets in which electronic is produced cross the other side of the ‘Digital Divide’. We stand in front of the following problem: what we are we going to do with all those computers, smartphones which we buy? All these products become obsolete or just unwanted within 1-3 years of purchase. Where will we find a new source of metals necessary to fabricate electronic equipment?</br><br />
Using old and broken WEEE in our project not only gives us a source of metals to produce new smartphones or computers, but also solves problem of storing WEEE.</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/6/6e/Recycling.jpg" alt="recyclin_itw" /></center><br />
<p align="justify">This figure shows a view on the context of re-use of EEE or its components. At the end of the use EEE returns to producers or to companies, which specialize in reuse of e-waste. <br />
The next step is preparation for re-use. On this step wasted electronic equipment is deconstructed. Not only computer memories or other parts which contains valuable metals are re-use. We also want to recover such materials as plastic or glass. That step requires labor work or special machines which will prepare WEE for another point of the plan. <br />
Some parts of the TVs or computers may not be useful in another manufactory. Sometimes we need to remarked parts which we need and work in place with special equipment. <br />
In the future we can use our bacteria with lanthanides binding sequences to re-use lanthanides.</br><br />
1) Removing from WEEE plastic and stirring parts which are abundant of rare earth metals such as lanthanides, especially computer memories. Stirring increase availability WEEE with acid which is produced by <i>Thiobacillus ferrooxidans</i>. It display terbium from alloy.</br><br />
2) Effluent from our bioreactor has low pH (about 2,5) what is a lethal for <i>E.coli</i>. This is the reason why we add Ca(OH)<sub>2</sub> to effluent. It increases our pH to a level which is optimal to <i>E.coli</i> (about 7). Ca(OH)<sub>2</sub> is also cheap and it is not increasing cost of the process very much.</br> <br />
3) Effluent goes to packed column bioreactor in which <i>E.coli</i> is immobilized. It gets across whole column. Ions of terbium binds and senses trough periplasmic domain.</br><br />
4) Column (with no initial effluent) is flushed by NaCl. PmrB can easily denaturate in NaCl solution losing its conformation. Ions of terbium do not precipitate with NaCl aq and do not make insoluble components with it.</br><br />
5) Ions of terbium are recovered from solution by electrolysis</br></br><br />
References:</br> <br />
Borrman Jeff et al., "One Global Understanding of Re-Use <br />
— Common Definitions", StEP, 2009 </br> <br />
Schluep Mathias et al., "Recycling from e-waste to resources" , StEP, 20 <br />
<a href="https://2014.igem.org/Team:Warsaw/EXTRAS"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<hr noshade="noshade" /><br />
<br />
<a name="discussion"><h2>Discussion</h2></a></br><br />
Our process finds not only a way to recovery the lanthanides, but also other metals such as copper ( it takes place at first point of it) and other rare metals.We not only gain rare metals, but also solve the problem of WEEE. There are alternative ways to recycle metals such as cooper or iron by using bacteria, but lanthanides are usually extraced from motherlode, which nowdays is really costly.</br><br />
Our way of recovery lanthanides does not produce pollution but it also is a safe way of managing with WEEE. Other methods of gaining lanthanides use mix of concentrated acids such as HCl or HNO<sub>3</sub>. The most common problems with these methods are difficult to neutralise byproducts. Byproducts, especially Ca(NO<sub>3</sub>)<sub>2</sub> are environmental-threatening. Their storing is very difficult and costly.Mining of lanthanides by old methods need to be placed on big areas, but our process takes less place. Area which will be taken to run it strictly depends on scale (in contrast to minors which always will take more space).</br><br />
Method which we presented is eco-friendly and solves the problem of full landfills with WEEE.</br> In our opinion using microorganisms to recycle e-waste will be a standard way of raising lanthanides.<br />
<a href="https://2014.igem.org/Team:Warsaw/EXTRAS"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<hr noshade="noshade" /><br />
<br />
<a name="alternative_methods"><h2>Alternative methods</h2></a></br><br />
<p align="justify">Our final system was of course not the only possibility. There were some points where we had to decide...<br />
<h4>Reporter protein</h4><br />
Finally we decided for GFP protein because of its prevalence, simplicity and fact that GFP does not require any additional reagents (eg. IPTG).</br><br />
We could have used other fluorescent proteins, for instance superfolder fluorescent proteins constructed by iGEM Warsaw 2013 Team, but regular GFP was the simpliest choice.</br><br />
<h4>Binding agent</h4><br />
Although we were unable to implement lanthanide binding system because of lack of time, we had several ideas how to accomplish this goal.</br><br />
<u>Poly-LBT peptide</u></br><br />
First was to construct a poly-LBT peptide which would be then anchored in the outer membrane of <i>E. coli</i> or transported to periplasm. We discarded this idea due to problems with modelling of behaviour of this polymer and problems with wet-lab design.</br><br />
<u>PmrB over-expression</u></br><br />
Another idea consisted of PmrB dependent over-expression of PmrB(LBT) protein. In such system we would have pmr<sup>C</sup> promoter - some logical device to boost the signal - PmrB(LBT), so in presence of lanthanides amount of PmrB(LBT) protein per cell would rise sharply, which should allow effective binding of lanthanides.</br><br />
<u>Small peptide fused with LBT</u></br><br />
Our final and probably best idea was to create a construct peptide of such composition:</br><br />
BBa_J32015 (<i>E. coli</i> periplasm signal peptide)-structure peptide (ubiquitin or 1L2Y [BBa_K1459015]) - lanthanide binding tag.</br><br />
The plan was to create a small, 'rubbish' protein which would only bind lanthanides without having any physiological function in cell (since we were afraid whether over-expression of PmrB would be cytotoxic).</br><br />
<a href="https://2014.igem.org/Team:Warsaw/EXTRAS"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<hr noshade="noshade" /></div>
ASamsel
http://2014.igem.org/Team:Warsaw
Team:Warsaw
2014-10-18T01:19:01Z
<p>ASamsel: </p>
<hr />
<div>{{:Team:Warsaw/Templates/2014Page|}}<br />
[[File:Members_itw.jpg|center|500px|link=https://static.igem.org/mediawiki/2014/5/53/Members_itw.jpg]]<br />
<div class="lang-pl"><br />
'''CeLuLaRE – System Recyklingu Lantanowców''' <br><br />
<br />
Ostatnio, wraz ze znacznym ubytkiem naturalnych zasobów, zaczęło wzrastać zainteresowanie odzyskiem metali ziem rzadkich, zwłaszcza lantanowców. Z tego powodu, naszym celem było zaprojektowanie systemu bioremediacji opartego na bakteriach, które wykrywałyby i odzyskiwały kationy lantanowców. W naszym wyobrażeniu, zaproponowany system mógłby umożliwić odzyskiwanie cennych metali (np. ze ścieków wodnych). Opiera się on na systemie wiążącym żelazo u Salmonella, który zmodyfikowaliśmy zastępując pętlę wiążącą żelazo różnymi tagami wiążącymi lantanowce (Lanthanide Binding Tags, LBTs).<br />
Wykorzystaliśmy również wcześniej zaprojektowany superfolder GFP by zmierzyć stężenie jonów lantanowców. W skrócie: kiedy jony zostają związane przez LBT, zostaje aktywowany krótki szlak sygnałowy, który indukuje ekspresję fluorescentnego białka. Nasz projekt przedstawia przyjazną środowisku technologię wydobywania, która potencjalnie może być wykorzystywana w przemyśle. Wydobyte metale są niezbędne w sprzęcie elektrycznym, w tym w sprzęcie medycznym. Co więcej, by zaproponować możliwe zastosowania projektu w medycynie, przygotowaliśmy raport na temat kilku fascynujących wyzwań w tej dziedzinie, przedstawiając model Szpitala Przyszłości (Future Lanthan Hospital).<br />
</div><br />
<br />
<div class="lang-en"><!--- class="wrapper"----><br />
'''CeLuLaRE – the Lanthanide Recycling System'''<br><br />
<br />
Recently, due to significant depletion of natural resources a growing interest was put in waste recovery of rare elements, especially lanthanides. Therefore, our goal was to design a bacteria-based bioremediating system capable of detecting and binding lanthanide cations. In our belief the proposed system could facilitate the recycling of these valuable metals (e.g. from wastewater). It is based on <i>Salmonella</i> iron-binding system, which we modified by replacing a iron-binding protein loop by various lanthanide binding tags (LBTs). <br><br />
<br />
We also used previously designed superfolder GFP to measure the concentration of lanthanide ions. Making a long story short: when an ion binds to LBT, a short signalling pathway is activated, inducing expression of the fluorescent protein. Our project introduces an eco-friendly retrieval technology, which potentially could be find industrial applications. Since extracted metals will are necessary in electronics, including medical devices. Furthermore, to propose possible applications in medicine we prepared a report about some fascinating challenges in that field proposing the model of Future Lantan Hospital.<br />
</div><br />
<br />
<br />
<div class="lang-ne"><!--- class="wrapper"----><br />
<br />
'''CeLuLaRE – Lanthanide Recycling Systeem''' <br><br />
<br />
Recentelijk groeit de interesse in het terughalen van chemische elementen uit afval, aangespoord door de depletie van sommige ertsen, lanthanide ertsen in het bijzonder. Ons doel omvat daarom het ontwerpen van een bacterieel systeem dat in staat is om lanthanides te binden. Vervolgens stellen we een methode voor om deze ionen te extraheren en dus effectief deze waardevolle metalen (bijv. uit afwalwater) te hergebruiken. We hebben lanthanide bindende tags, naar model van het Salmonella ijzer-bindende systeem, gemodificeerd om van toepassing te zijn op ionen van verschillende groottes. <br><br />
Ook gebruiken we een superfolder GFP om de concentratie van gewonnen ionen te meten. Als een ion aan LBT bind wordt er een signaaltransductieroute geactiveerd en wordt fluorescentie geïnduceerd.<br />
Ons project introduceert een ecologisch verantwoorde herwinnings technologie voor industrieel significante elementen. We hopen dat de geëxtraheerde metalen bruikbaar zullen zijn voor gebruik in electronica, waaronder medische apparatuur. Om mogelijke toepassingen in geneeskunde voor te stellen hebben wij een rapport voorbereid over enkele fascinerende uitdagingen in dat veld, en werken hierdoor aan de toekomst van het Lantan Ziekenhuis. <br><br />
</div><br />
<br />
<br />
<div class="lang-fr"><!--- class="wrapper"----><br />
<br />
''' CeLuLaRe - Système de Recyclage des Lanthanides ''' <br><br />
<br />
En raison de l’épuisement de certain minerais, comme les lanthanides, l’intérêt pour le recyclage de ces éléments chimiques à subitement augmenté. Notre but était de créer un système bactérien capable de reconnaître et de fixer les lanthanides. Nous avons proposé une méthode pour extraire ces ions(par exemple des eaux usées) qui permet un recyclage efficace de ces métaux . En s’appuyant sur le système de fixation du fer de Salmonella, nous avons modifié les sites de liaison des lanthanides pour qu'ils puissent s'adapter aux différentes tailles d'ions. <br><br />
Nous avons également utilisé une GFP superfolder pour mesurer la concentration d'ions collectés. Lorsqu'un ion se lie à son site de liaison, une voie de signalisation est activé, ce qui induit la fluorescence. Notre projet introduit une technologie de récupération écologique pour les éléments à fort potentiel industriel. Nous espérons que les métaux extraits seront viables pour des applications dans l'électronique comme des dispositifs médicaux.Pour proposer des applications possibles en médecine, nous avons préparé un rapport sur certains des défis passionnants dans ce domaine, créant ainsi le modèle d’un future hôpital Lantan. <br />
</div></div>
ASamsel
http://2014.igem.org/Team:Warsaw/Project
Team:Warsaw/Project
2014-10-18T01:02:36Z
<p>ASamsel: </p>
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<a href="https://2014.igem.org/Team:Warsaw">Project</a><br />
<ul><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#background">Background</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#proof_of_concept">Proof of concept</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#detailed_explanation">Detailed explanation</a></li> <br />
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<br />
<br />
<h1>The Project</h1></br><br />
<hr noshade="noshade" /><br />
<br />
<a name="parts"><h2>Background</h2></a></br><br />
<p align="justify"><br />
Lanthanides are a series of fifteen chemical elements with atomic numbers 57 through 71, from lanthanum to lutetium. All but one of the lanthanides are f-block elements, corresponding to the filling of the 4f electron shell (lutetium is a d-block element). The presence of f orbitals is responsible for their unique properties such as strong paramagnetism.</br></br><br />
<center><img src="https://static.igem.org/mediawiki/2014/8/8f/600px-Lanthanides1.jpg" alt="Something went straight to Hell" width="600" height="169"/></center></p></br></br><br />
<br />
They are required in a variety of modern technologies, such as electronics, aviation (eg. jet engines) and superconductors</br><br />
In spite of their name 'rare earths metals' their deposits on Earth are not scarce but very dispersed. Hence, there are only a few places (eg. China) where their concentration is high enough to be exploited on a commercial scale. Due to that situation the lanthanides market is not getting any better, especially taking into account that demand on these metals is still on increase.</br></br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/6/63/REE_world_deposits_map.jpeg" alt="Something went straight to Hell" width="751" height="307" /></center></br></br><br />
<br />
<br />
1. Worldwide deposits of rare earths elements - source: [1]</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/3/37/World_deposits_of_REE_graph.png" alt="Something went straight to Hell" width="601" height="401" /><center></br><br />
2. Total deposits of rare earth metals - source: [1]</br><br />
Other problems with lanthanides avaibility are their difficult extraction from the ore. Most of lanthanides appear together and very similar physical properties make extraction and purification difficult. Despite heavy costs and technical difficulties lanthanides are usually purified by ion-exchange chromatography.</br><br />
[1] Chapter 6, Kołodyńska D., Hubicki Z., <i>Investigation of Sorption and Separation of Lanthanides on the Ion Exchangers of Various Types</i>, <i>Ion exchange technologies</i>, edited by Ayben Kilislioğlu, Published: November 7, 2012 under CC BY 3.0 license</br><br />
</p><br />
<hr noshade="noshade" /><br />
<br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<br />
<a name="proof_of_concept"><h2>Proof of concept</h2></a></br><br />
<p align="justify"><br />
In 2013 group of prof. He from the University of Chicago published paper in <i>Journal of American Chemical Society</i> (<i>J. Am. Chem. Soc.</i> 2013 Feb 13;135(6):2037-9) in which they described thr devised lanthanide detecting system.<br />
</br><br />
To accomplish this, they engineered two-component system from <i>Salmonella enterica</i> creating the first bacteria capable of detecting lanthanides.<br />
These findings inspired us to create our bioremediating system.</br></br></br><br />
<center><img src="https://static.igem.org/mediawiki/2014/thumb/2/2e/ZDJ%C4%98CIE_WIKI_PMRA-PMRB.jpg/702px-ZDJ%C4%98CIE_WIKI_PMRA-PMRB.jpg" alt="Something went straight to Hell" width="350" height="300" /></center> <br></br><br />
A general scheme of PmrA-PmrB system.<br />
</p><br />
<hr noshade="noshade" /><br />
<br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<br />
<a name="detailed_explanation"><h2>Detailed explanation</h2></a></br><br />
<p align="justify"><br />
Initially, our project was intended to have two different parts. First being a lanthanide detecting system in BioBrick standard, much like the one constructed by group of prof. He<br />
and the second being lanthanide binding/recovery system, which would bind lanthanides much more effectively than the detecting system.</br><br />
Both of these systems were based on PmrA-PmrB two-component system, native to <i>Salmonella enterica</i>. This system consists of two proteins, PmrA and PmrB. PmrB is a transmembrane kinase with iron (III) binding motif on its extracellular loop.<br />
When iron (III) is bound to this tag, PmrB gains kinase activity and phosphorylates PmrA. PmrA is a transctriptoral factor and, upon activation, binds to pmr<sup>C</sup> promoter and induces expression of CheZ, a chemotaxis protein.</br><br />
So much for native systems.</br><br />
</p><br />
<br />
<h4>Design</h4><br />
<p align="justify"><br />
<b>Detecting system</b></br><br />
Our detecting system is planned as follows:</br><br />
Iron binding tag would be replaced with lanthanide binding tag (of which a various collection can be find in literature) and a reporter protein would be inserted downstream of pmr<sup>C</sup>. Thus, in the presence of lanthanides, fluorescence of GFP should be observed.</br><br />
<b>Binding system</b></br><br />
Binding system has more complicated design. PmrA-PmrB is not changed significantly, the only modification was introduction of LBT (lanthanide binding tag) instead of iron binding motif. The difference is downstream the pmrC promoter. First of all, we need to introduce some sort of binding agent, presumably a small protein.<br />
We decided to use ubiquitin or an artificial structurised peptide and combine it with a LBT to create synthetic protein capable of binding lanthanide ions. Since lanthanide cations are not transported to the bacterium cell the binding agent need to be secreted outside the cytoplasm. Hence, we planned to add a signal peptide to the N or C terminus of the protein. Such modification could allow the protein to be located in the bacterial periplasmic space.</br><br />
Another possible problem is connected with pmr<sup>C</sup>, which is a very weak promoter (even if induced by PmrA). So, even in the presence of lanthanides, expression of a binding agent could be inefficient. To overcome that, we planned to use some activating sequences to boost the expression from upon the pmr<sup>C</sup>. Our first idea was to put two subsequent inverters (based on different proteins, eg. tetR and lacI), which should alleviate the problem. Expression of binding agent is expected to be high in the presence of lanthanides and low in their absence.</br><br />
</p><br />
<table border="1"><br />
<tr><br />
<td></td><br />
<td colspan="3">Binding agent expression</td><br />
</tr><br />
<tr><br />
<td>Lanthanide presence</td><br />
<td>pmr<sup>C</sup></td><br />
<td>pmr<sup>C</sup>-inverter1</td><br />
<td>pmr<sup>C</sup>-inverter1-inverter2</td><br />
</tr><br />
<tr><br />
<td>none</td><br />
<td>zero (very low)</td><br />
<td>high</td><br />
<td>low</td><br />
</tr><br />
<tr><br />
<td>present</td><br />
<td>low</td><br />
<td>low</td><br />
<td>high</td><br />
</tr><br />
</table><br />
</br><br />
<p align="justify"><br />
This may seem like an excessive mean, but we could not have invented anything subtler.</br><br />
</p><br />
<h4>Project goals</h4><br />
<ol><br />
<li>Construction of a lanthanide sensor in the BioBrick standard</li><br />
<li>Cloning of PmrA/PmrB parts into pSB1C3 in the BioBrick standard</li><br />
<li>Construction of a lanthanide sensoring system with<br />
other LBT described in the literature </li><br />
<li>Construction of a lanthanide binding system</li><br />
</ol><br />
<hr noshade="noshade" /><br />
<br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<br />
<a name="modelling"><h2>Modelling</h2></a></br><br />
<br />
<p> Two-component systems (TCSs) are the most prevalent mechanism of transmembrane signal transduction. They control gene expression thus make bacteria respond to environmental changes and drive pathogen-host interactions. A typical TCS consists of a membrane-bound histidine kinase and<br />
a partner response regulator protein. The pmrA/pmrB system, which our team used in the project, also belongs to this class. pmrB is a histidine kinase and pmrA is a response regulator which strongly enhances expression upon binding to Pmr<sup>C</sup>. In order to understand better the mechanism of the system and to prevent any problems before starting the experiments in the wetlab we decided to create a simple model of this signaling pathway. Some other TCSs were successfully modeled before, but not the pmrA/pmrB.<br />
</p><br />
<br />
<b>The model</b><br />
<br />
<p> When designing our model we assumed the following pathway:<br />
<ol><br />
<li> lanthanide ion binds to the pmrB protein which leads to its autophosphorylation, </li><br />
<li> phosphorylated pmrB transfers the phosphate group onto pmrA </li><br />
<li> phosphorylated pmrA binds to pmrC and initiate expression of the reporter GFP protein </li><br />
<li> dephosphorylated pmrB induces pmrA dephoshporylation </li><br />
Additionally for model to work properly feedback loop in which phoshporylated pmrA induces pmrA expression is needed.<br />
</p><br />
<p><br />
The model diagram looks as follows:<br />
<br />
<img src="https://static.igem.org/mediawiki/2014/b/b4/Warsaw_pathway.png" width=780px alt="Signaling pathway" /><br />
<br />
</ol><br />
</p><br />
<br />
<p> We concluded that quantities of observed species change according to these equations:<br><br><br><br />
<img src="https://static.igem.org/mediawiki/2014/3/34/Warsaw_dpmrnaprmb.gif" alt="Equation 1" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/1/1f/Mrnapmra2.gif" alt="Equation 2" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/3/33/Warsaw_eq3.gif" alt="Equation 3" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/5/52/Warsaw_eq4.gif" alt="Equation 4" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/2/20/Warsaw_eq5.gif" alt="Equation 5" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/4/48/Warsaw_eq6.gif" alt="Equation 6" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/3/3b/Warsaw_eq7.gif" alt="Equation 7" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/c/c1/Mrnarp2.gif" alt="Equation 8" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/c/c3/Warsaw_eq9.gif" alt="Equation 9" /><br><br><br></br><br />
<br />
<br />
where:<br />
<ul><br />
<li> <i>mRNApmrB</i> is concentration of pmrB mRNA, the same goes for <i>mRNApmrA</i> and <i>mRNARP</i>, </li><br />
<li> <i>L</i> is lanthanide concentration, </li><br />
<li> <i>RP</i> is reporter protein concentration, </li><br />
<li> <i>pmrB.bound</i> is <i>pmrB</i> with lanthanide ion bound, </li><br />
<li> <i>prmB.bound.ph</i> is phosphorylated <i>pmrB</i> with lanthanide ion bound, </li><br />
<li> <i>pmrA.ph</i> is phosphorylated pmrA, </li><br />
<li> <i>ABComplex</i> is complex of <i>pmrA</i> and <i>pmrB.bound.ph</i> during <i>pmrA</i> phosphorylation, </li><br />
<li> <i>AComplex</i>, <i>RPComplex</i> are <i>pmrA.ph</i> inductors bound to respective promoters, </li><br />
<li> <i>ABRevComplex</i> is complex of <i>pmrA.ph</i> and <i>pmrB</i> during <i>pmrA</i> dephosphorylation </li><br />
</ul><br />
</p><br />
<br><br><br />
<br />
<br />
<b> The parameters </b><br />
<p><br />
Initial parameters were found in literature as we did not make independent component measures.<br />
</p><br />
<b> Simulation and results </b><br />
<p><br />
Deterministic simulations were performed using TinkerCell software. There are few bugs in it, but it allows for fast model building and makes changes to the model quite easy. <br />
Simulation showed that signal greatly enhances GFP expression, the its growth is exponential and correlate positively with increased concentration of lanthanide ions.<br />
</p><p><br />
GFP level when there is no lanthanide ions:<br />
<img src="https://static.igem.org/mediawiki/2014/f/f8/Warsaw_noL.png" width=780px alt="Chart" /><br />
</p><br />
<p><br />
GFP levels with 100 um of ions:<br />
<img src="https://static.igem.org/mediawiki/2014/c/c0/Warsaw_yesL.png" width=780px alt="Chart" /><br />
</p><br />
</br><br />
</br><br />
<b> References </b><br />
<p><br />
<i> Kierzek AM, Zhou L, Wanner BL. Stochastic kinetic model of two compo-<br />
nent system signalling reveals all-or-none, graded and mixed mode stochastic switching responses. Mol Biosyst. 2010;6(3):531-42<br />
</i><br />
</p><br />
<hr noshade="noshade" /><br />
<br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<br />
<a name="weeestudy"><h2>WEEE study</h2></a></br><br />
<p align="justify"><br />
WEEE stands for ‘Waste Electrical and Electronic Equipment’ such as computers, mobile phones, TV-sets and fridges.</br> <br />
Modern electronic products contain up to 60 elements, many of them are very valuable. The most complex of it is usually presented in printed wiring boards. Metals represent on average 23% of weight of the phone, in majority copper. Single mobile phone can contain up to 9 g Cu, 250 mg Ag, 24mg Au and 0,5 mg Tb. <br />
<center><img src="https://static.igem.org/mediawiki/2014/b/b9/Periodic_table.jpg" alt="periodic_table_itw"width="751"height="350" /></center></br></br><br />
<i>Material content mobile phone [Umicore 2008]</i></br> <br />
It seems to be not too much, but we have to remember how much WEEE average European citizen products.</br> <br />
<center><img src="https://static.igem.org/mediawiki/2014/7/71/Weee_collection-page-001.jpg" alt="weee_collection"width="550"height="500" /></center></br><br />
<p align="justify"><br />
By 2014 number of active cell phones will reach 7.3 billion. It gives 375 000 tons of Tb. All of it can be stored on landfill or recycled. Many of e-waste is transported to China, Ghana or Pakistan where fly dumpling is cheaper and the law is not as strict as European or American one.</br> <br />
The developing countries become toxic yard for e-waste. Heavy metals and toxins leak trough landfills into waterways, poisoing local people.<br />
<hr noshade="noshade" /><br />
<br />
<br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<br />
<a name="safety"><h2>Safety</h2></a></br><br />
<h3>1. Introduction</h3><br />
<p align="justify"><br />
We at Team Warsaw understand the need for good safety training and biosafe conduct in the lab.<br />
In the following sections, we will show you how we went about making sure we didn't put anyone at an unnecessary risk either at our faculty or in the outside world.<br />
</p><br />
<h3>2. (Bio)safe conduct</h3><br />
<p align="justify"><br />
Before summer, our work in the lab began with a safety training provided by our instructors. We were trained <br />
in accordance with the biosafety guidelines of our institution, focusing on lab-practical aspects of biosafety,<br />
i.e. where to work with bacteria, to always do it in the same place, to account for where the bacteria-containing material is being put, to always disinfect the immediate vicinity of your workbench once the work is finished, etc. We were also taught to properly store biological material, such as bacterial broths imbued with colonies, waste agar plates, or pipette tips and plastic tubes, and handle them in a manner suitable for preventing the spread of bacteria. Whenever some biological material-containing glass, as flasks or tubes, was broken into pieces, the adjacent area was mopped dry and disinfected with ethanol,<br />
and broken glass was stored in a separate container for glass, but only after possible remains of liquids have been removed by mopping and the pieces were disinfected by spraying with 70% ethanol.<br />
</p><br />
<p align="justify"><br />
Usually, we worked with DNA constructs so it was a matter of keeping everything else out the workplace (i.e. every type of contamination). Therefore, we had a set place for bacterial work on a bench, which was cleaned after each use, always worked with bacteria under conditions of closed windows and burner turned on, always worked in non-reusable gloves, which were disinfected with 70% ethanol at the start of work, proceeded to disinfect them regularly, stored contaminated plastic and liquids in separate containers suitable for autoclaving under standard conditions (which is taken care of by our Institute) and removed contaminated materials from our lab on a weekly basis. Whenever working with bacteria, we also refrained from touching objects outside the workbench (to prevent the possible spread of bacteria) and disinfected the working place using 70% ethanol.Of course, since our DNA constructs often carried antibiotic resistance gene, we took particular care to make sure all of our liquids remained in their respective tubes and cleaned the leakage places with 70% ethanol whenever these happened. In terms of work with hazardous substances, the only one we encountered was the ethidium bromide (used when working with agarose gels): to avoid any skin contact we always used gloves and we worked on a separate bench, devoted to ethidium bromide. Gels after visualization were stored in a separate container to be destroyed.<br />
</p><br />
<h3>3. Biosafety level</h3><br />
<p align="justify"><br />
In this year's project, we used the K-12 <i>E. coli</i> bacteria as chassis for our proteins: which are PmrA, PmrB and GFP. All the proteins we worked on, as well as with <i>Escherichia</i> chassis are harmless (non-pathogenic)<br />
to humans, which allowed for classification of our work at biosafety level 1 according to<br />
<a href="http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf?ua=1">WHO</a>.<br />
We therefore worked with our bacteria and constructs on ordinary open top benches, using Bunsen burners for sterility whenever aliquoting media, imbuing overnight cultures or sowing onto agar plates.<br />
We used <i>Salmonella enterica</i> subsp. <i>enterica</i> ser. Typhimurium, which is technically a biosafety level 2 organism, as source of parts isolated by PCR. However, we worked with a non-pathogenic, attenuated<br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/15063560">&#967;3987</a> <i>Salmonella enterica</i> subsp. <i>enterica</i> ser. Typhimurium carrying an <i>asd</i> (aspartate dehydrogenase) gene from <i>E.coli</i> on the p3342 plasmid, as derived from the <i>Salmonella Typhimurium</i> <a href="http://www.ncbi.nlm.nih.gov/pubmed/21622747">UK-1 strain</a>. This strain is non-virulent (&#916;crp, &#916;cya), hence provides no risk to personal or community health. Even though the genomic DNA isolation was a one-time operation, we still performed all manual activities under a suitable biological safety cabinet.</p><p align="justify"><br />
As regarding the parts isolated, i.e. proteins coded by the BasR (PmrA) and BasS (PmrB) genes, they are said to partake in the <i>Salmonella</i> virulence. Nonetheless, these two proteins merely regulate expression of the genes, whose products (usually LPS-modifyingenzymes) take part in virulence processes and so, "our" proteins are not involved in these processes themselves. We also used a PmrC/GFP construct-carrying plasmid (which we were glad to have received from<br />
<a href="http://he-group.uchicago.edu/">Prof. Chuan He's group</a> at the University of Chicago), whereby GFP was expressed from a PmrA-induced PmrC promoter. The PmrC gene codes for the phosphoethanolaminetransferase enzyme, which is required for Salmonella resistance to polymyxin. However, the plasmid we used was not carrying PmrC gene, but only the mentioned promoter which [the promoter] does not constitute a biosafety risk and neither does the GFP protein.<br />
</p><br />
<h3>4. Safety forms</h3><br />
<p align="justify"><br />
We submitted our <a href="https://igem.org/Safety/About_Our_Lab?team_id=1459">About Our Lab</a> form as well as the<br />
<a href="https://igem.org/Safety/Safety_Form?team_id=1459">Safety Form</a>, which can be found by clicking<br />
the hyperlinks on their respective names. We did not, however, need to fill out any Check-Ins, as neither of our parts nor the chassis fell under the required categories. The White List of parts<br />
and organisms and their Check-In necessity status can be found on the website of the<br />
<a href="https://2014.igem.org/Safety/White_List"> Safety Hub</a>.<br />
</p><br />
<h3>5. Environmental concerns</h3><br />
<p align="justify"><br />
The question about potential environmental concerns of our project was central to our attempts. However, due to the nature of the proteins expressed, our chassis bacteria have not acquired any characteristics that would enable them to compromise human immune system/other systems<br />
or evade detection and destruction by the former or facilitate spread between people/animals, which makes them harmless from both a personal and public health point of view.<br />
At the same time, neither the proteins encoded themselves, nor the functionality of the lanthanide detecting/binding system as a whole, imbue the bacteria with characteristics that would convey<br />
an evolutionary advantage against other organisms in the environment, both microorganisms and plants or animals, or act as toxins against the aforementioned, making the bacteria modified with the PmrA/PmrB system environmentally biosafe with no risk or them dominating any ecological niche. Our modified bacteria have, however, survival capabilities comparable to the wildtype ones.<br />
There must be the point stressed, however, that since the transformed bacteria carry<br />
a chloramphenicol resistance-encoding plasmid, the actual biosafety of the detection/binding system (i.e. prevention of HGT of the antibiotic resistance between the modified and wildtype bacteria) and so - the potential impact on the environment - depends greatly on the design of the bioreactor and the technological process, to minimize, or best prevent, the influx and efflux<br />
of non-transformed bacteria, and microorganisms in general, into the reactor.<br />
To sum up, our bacteria are not toxic towards either humans, plants, animals or other microorganisms, making them both biosafe both environmentally and health-wise. However, they can survive in the environment just as well as the wildtype bacteria, therefore the potential technological process of lanthanide detection and recycling must be optimized (esp. in the terms of preventing GMO bacteria efflux from the bioreactor) to prevent HGT and efflux of the acceptor bacteria back into the environment.<br />
</p><br />
<br />
<hr noshade="noshade" /><br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<br />
<a name="possibilities_of_development"><h2>Possibilities of development</h2></a></br><br />
<p align="justify"><br />
We envisage two opportunities allowing our project to be improved. First, to test more LBTs described in literature (or even design new ones) and second to create more effective binding systems.</br><br />
Furthermore, we considered utilising some kind of sulphur bacterias instead of <i>E. coli</i>. Their sulphur-based metabolism and ability to survive in low pH (in which metal leaching is more efficient) makes them excellent candidates for industrial application of our project.</br><br />
Another thing which is worth investigating: our system should not be present in bacterias as plasmids. It could be interesting to integrate it with bacteria genome, so it would be more stable within bacteria. We briefly investigated applying pMAT plasmid (known for it's remarkable stability in bacterial cells) in our project to fix the problem of 'deevolution' and eradication of our construct plasmids from bacterias. We also planned to construct a BioBrick compatible version of pMAT, but had to scratch the idea because of lack of time.</br><br />
</p><br />
<hr noshade="noshade" /><br />
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ASamsel
http://2014.igem.org/Team:Warsaw/HP
Team:Warsaw/HP
2014-10-18T00:55:00Z
<p>ASamsel: </p>
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<li><a href="https://2014.igem.org/Team:Warsaw/Project#proof_of_concept">Proof of concept</a></li><br />
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<li><a href="https://2014.igem.org/Team:Warsaw/Notebook">Notebook</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Protocoles">Protocoles</a></li><br />
<br />
<br />
</ul><br />
</li><br />
<li><br />
<a href="/Team:Warsaw/Team">Team</a><br />
<ul><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Team#members">Members</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Team#advisors">Advisors</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Team#sponsors">Sponsors</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Team#acknowledgements">Acknowledgements</a></li><br />
</ul><br />
</li><br />
<br />
<!-- iGEM link --><br />
<li class="igem-logo"><br />
<a href="https://2014.igem.org/"></a><br />
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</ul><br />
<div class="main-content"><br />
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<li class="igem-logo"><br />
<a href="https://2014.igem.org/"></a><br />
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</ul><br />
<div class="main-content"><br />
<br />
<h1>Human Practice</h1></br><br />
<hr noshade="noshade" /><br />
<br />
<a name="overview"><h2>Overview</h2></a></br><br />
<h2> Human Practice Philosophy: big dreams with applications </h2></br><br />
<br />
<p align="justify"> Behind our Human Practice we have one simple idea. We have a vision to transform our dreams in new applications to excel in achieving social goals. Our vision for Human Practice has been shown in report on Future Lanthan Hospital Model. It is a bottom-up strategy to transform healthcare by launching collaboration on the boundaries of several disciplines aimed at inducing openness and sharing, not only in business and academia but also in the society itself. In Human Practice we would like to show the perspective and context for Cellulare. To tell the truth we would be pleased show you big dreams with application and encourage you to collaborate with us! <br />
<br />
<br> </br><br />
<br />
<p align="justify"> We believe that the person with a great idea behind is more powerful than one who depend merely on facts. So, everything what we have done was based on that philosophy. We do not give you complete solutions, but a vision and a strategy to develop and apply. Our first goal is not to be the best iGEM project, our goal is to initiate the project which could be developed further, outside iGEM by people of different skills and different potential to solve complex problems on the larger scale. We are aimed at design both Cellulare itself and its Human Practice to serve social goals which could be developed far beyond synthetic biology.<br />
<br> </br><br />
<br />
<font size="4"><p align=" center"> <i>“Some look at things that are, and ask why. I dream of things that never were and ask why not?” </br> George Bernard Shaw </i> </p> </font><br />
<br> </br><br />
<img src="https://static.igem.org/mediawiki/2014/f/f6/Dream.JPG" alt="Smiley face" align="left" height="300" width="410" ><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/9/9d/Hand.JPG" alt="Smiley face" align="right" height="300" width="410" ><br />
<font size="2">Photos: Piotr Migdał, used with permission </font> <br />
<br></br><br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p align="right"><h6>Up↑</h6></p></a><br />
<hr noshade="noshade" /><br />
<br />
<br />
<br />
<h2> Recycling ideas: recovery - transformation – new application </h2></br><br />
<br />
<p align="justify"> We have started from listening what others want to say and, asking them questions to understand better the context of our project (Figure 1). This is how new application for Celulare has emerged. Our ultimate goal of the Human Practice is to add valuable contribution to healthcare transition by proposing a more synthetic biology-oriented approach for medicine. At the same time we want to encourage you to become more resource-efficient through optimal allocation of scarce material. We would like to show new application for mechanism design (engineering part of the economics) and the stable matching algorithm allowing to better resource-management addressing the needs of the patients. We also aim to provide a contribution to the processing of medical data and open source science in the era of big data in medicine. Here, we see new application for Celulare, (and in futher perspective iGEM itself) to finally bring idea of transform healthcare with synthetic biology.<br />
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<br> </br><br />
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<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/2/20/General_scheme.png" alt="Smiley face" height="400" width="600"></center><br />
<br> </br><br />
<br />
<p align="center"> <b> Figure 1. Cycles of ideas with new applications. </b> Context for Celulare itself and its Human Practice </p><br />
<br> </br><br />
<br />
<h2>Video game</h2></br><br />
<p align="justify"><br />
This year, as we continue our HP long term strategy, we<br />
developed an educational<br />
computer game. We are willing to use it to popularize synthetic biology term and our<br />
CeLuLa-Re project. The game was written in HTML5 using Construct2 software.</br><br />
<a href="https://f6052cc3a5c084cb37f0b0f1639e817316d820f5.googledrive.com/host/0ByxTljcJDMLoLVlGVzc5OGVURnM/">Here</a> you will find a link to an external server</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/54/Prt_scr_1_gra.png" alt="Smiley face" height="323" width="682"></center></br><br />
The game is actually a demo as we plan to develop it. We believe we can reach and<br />
attract many people as the game is funny and everybody learns best, while playing.</br><br />
<a href="https://www.youtube.com/watch?v=-H8yeYqBvqo">Here</a> is a short film showing gameplay</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/e/ec/Prt_scra_2_gra.png" alt="Smiley face" height="323" width="682"></centert></br><br />
<p align="justify">The game consists of 3 platform levels and 2 quizzes about our project. At the<br />
beginning the two levels are locked as the player proceeds after collecting particular<br />
amount of lanthanides ions. You can especially earn<br />
a lot of them by giving correct<br />
answers in quiz.</br><br />
Platform levels are full of arcade challenges such<br />
as deadly spikes or enemies<br />
making it hard to reach<br />
ions. Both quizzes consist of 10 questions about synthetic<br />
biology, rare earths elements, our wet lab and HP part. All the correct answers can<br />
be found<br />
on our<br />
Wiki<br />
and on the official<br />
iGEM webpage.</br><br />
The main hero is a genetically engineered bacteria<br />
which wants to return home. Its<br />
space ship was destroyed due to collision with space rubbish. Now the bacteria<br />
searches a dangerous<br />
and unknown land for lacking lanthanides ions to build a laser<br />
to clear the planet's atmosphere and repair its space ship so it can safely return<br />
home.</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/7/7e/Prt_scr_3_gra.png" alt="Smiley face" height="323" width="682"></center></br><br />
<p align="justify">Well, that is the demo. We have loads of other ideas to expand the game such as<br />
gaining super powers by hero when obtaining special<br />
BioBrick, different types of<br />
enemies and obstacles, new levels<br />
and of course we plan on adding<br />
more<br />
educational value. We are planning to create a book<br />
of knowledge and make a<br />
drag&drop task to teach cloning in BioBrick standard.</br><br />
We are inviting you to play and share<br />
this<br />
game with your friends!</br><br />
</p><br />
<br />
<p align="justify"> In our persuits we have been inspired by and support mutually with <a href="http://www.ci.edu.pl/en/collegium/idea "> Collegium Invisibile </a> and <a href="http://offtopicarium.wikidot.com/en:about"> Offtopicarium </a>who also realize some bottom-up initiatives and have excellent Human Practice. We understand, however, that our contribution is only a droplet in the sea of knowledge. Still, we envisage project to be a small thing with greater perspectives. That is why we are searching for new ways of communication via <a href="http://crastina.se/about/ "> Scientia Crastina </a>(latin for ‘The Science of Tomorrow’). Our aim is to encourage to seek new possibilities of collaboration of the interdisciplinary projects through open communication in science. <br />
<br> </br><br />
<br />
<br> </br><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/c/c2/Warsaw_logo_collegium.jpg" alt="Smiley face" height="80" width="250" align=left><br />
<br />
<img style="float: center; padding: 0px 0px 0px 70px;;" src="https://static.igem.org/mediawiki/2014/e/e8/Scientia.png" alt="Smiley face" height="80" width="280"><br />
<img src="https://static.igem.org/mediawiki/2014/d/df/Platformscientia.png " alt="Smiley face" height="80" width="250" align=right> <br />
<br> </br><br />
<br> </br><br />
<br> </br><br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p align="right"><h6>Up↑</h6></p></a><br />
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<br />
<a name="lanthan_hospital"><h2>Lanthan Hospital</h2></a></br><br />
<hr noshade="noshade" /><br />
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<br />
</br><br />
<br />
<p align="justify"> Moving to implement of our dreams in practice we comprised thoughts derived from our Human Practice in the raport “Celulare: transforming healthcare" in which we present Future Lanthan Hospital Model. The basic idea behind Lanthan Hospital Model is to integrate synthetic biology lab with hospital. This is sort of start-up strategy with systems impact, which we set in the context of a broader healthcare transformation. As Celulare is focused on lanthanides bioremediation, we want to propose strategy to optimal allocation of these elements and other deficit resources on healthcare market. That is why we have indicated several possible applications for novel economic concepts of Nobel Prize Winners in ‘Nobel Lantan Economy and Nobel Human Practice’ section. We believe that synthetic biology approach, especially iGEM itself may "influence the world from protein, to policy, to pixels.” To make it happen we develop Future Lantan Hospital Model. <br />
<br />
<br></br><br />
<br></br><br />
<br />
<center><br />
<a href="https://static.igem.org/mediawiki/2014/b/bc/Cellulare_-_report_Final.pdf"><br />
<img src="https://static.igem.org/mediawiki/2014/3/36/Cellulare_cover.png" alt="Smiley face" align="center" height="552" width="395" ><br />
<br><br><br />
Download the report (pdf file)<br />
</a><br />
</center><br />
<br />
<br />
<br> </br><br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p align="right"><h6>Up↑</h6></p></a><br />
</br></br><br />
<br />
<h2> Report - table of contests </h2></br><br />
<br />
<br />
Summary </br><br />
1. Background – from systems biology through synthetic biology to big data in medicine </br><br />
2. New application of synthetic biology: transforming healthcare </br><br />
3. New application of iGEM registry: challenging healthcare for data analysis </br><br />
4. Solving optimal resource allocation problem with support of synthetic biology, economy and medicine. Step forward to open health </br><br />
5. Celulare: transforming healthcare for optimal allocation of deficit resources </br><br />
6. Future Lanthan Hospital - implementation strategy through ‘Cellulare’ </br><br />
7. Celulare Recommendations </br><br />
8. Nobel Lanthan Economy with Nobel Human Practice </br><br />
9. Stable matching and mechanism design through Celulare to Polish healthcare market </br><br />
10. Closing remarks </br><br />
Annex 1. Anti selfie mechanism design for smartphone MDs through Celulare </br><br />
Annex 2. KPD - optimal allocation of deficit resources need access to databases </br><br />
Annex 3. Celulare – future leaders start here </br><br />
Annex 4. Creative education - workshops. </br><br />
<br />
</p> <br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p align="right"><h6>Up↑</h6></p></a><br />
<br />
<h2> Report - summary </h2> </br><br />
<br />
<p align="justify" style="text-indent: 0.5in;"> Nowadays, in the big data era, medicine will be inevitably transformed by systems biology. Rapid developments in deep sequencing, metabolomics and so called "personal medicine" have already changed medical sciences. Hence, we would like to add a valuable contribution to that healthcare transition by proposing a more synthetic biology-oriented approach for medicine. Our project ‘Cellulare’ touches both healthcare transformation for data analysis and optimal allocation of deficit resources. Our question is how to design a hospital only having access to limited resources, equip it in modern technologies (biomedical and IT), and finally how to combine modern technology and pro-ecological design in order to improve the work of healthcare. To address this difficult issue, we developed Future Lanthan Hospital Model, which is aimed to show that recovery of lanthanides (rare earth elements) from electronic waste (smartphones, tablets etc.) by synthetic biology methods, could be a beneficial supporting strategy for a modern hospital. We have proposed integration of a hospital with a supplementary synthetic biology lab, in which lanthanide remediation project could take place. First, such a solution would contribute to the public image of the hospital as a rationally functioning institution with eco-friendly profile. Secondly, it could allow to equip the hospital in modern technologies based on rare-earth metals. Simultaneously, we suggest mechanism design (engineering part of the economy) and the stable matching algorithm allowing better resource-management that would address the needs of the patients. By Future Lanthan Hospital Model we would like to show beneficial changes for society resulted from implementation of modern technologies in medicine and educate people about applications, safety and some security problems related to cutting-edge technologies. In the end we also aim to provide a contribution to the processing of medical data and open source science, since in the era of big data in medicine such measures seems to be necessary for the rapid transformation of the healthcare. </p> <br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p align="right"><h6>Up↑</h6></p></a><br />
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<br />
<h2>Collaboration and future perspectives </h2><br />
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<font size="4"><p align="left"> <i>We have a bright vision of the future so we start from collaboration and sharing. </i> </p> </font><br />
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<br />
<br />
<h3> Science Polish Perspectives </h3> </br><br />
<br />
<p align="justify">We are extremely proud that just before Giant Jamboree we have passed through competitive selection process for talks and we are to present Future Lanthan Hospital Model at the <a href="http://www.polishperspectives.org/en/home-en"> Science Polish Perspectives 2014 at Oxford</a>! One of main aim of conference is to popularise science, whilst also enhancing the cooperation between its British, Polish and European branches. Taking in mind SPP agenda we hope that presenting our project help us to build a network of contacts and discuss Poland’s participation in the development of science and technology. We agree with the agenda that drawing inspiration from each other is one of the best ways of developing the huge potential as individuals and as members of a fast-growing scientific community. <br />
<br />
<br> </br><br />
<a href="http://www.polishperspectives.org/speaker-pr"> Here</a> see announcment on talk 'solving limited resources problem with engineering part of biology, economy and medicine. Step forward to open health' to be presented by a member of our team. </p><br />
<br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p align="right"><h6>Up↑</h6></p></a><br />
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<br />
<h3> Leaders of healthcare market and Collegium Invisibile </h3> </br><br />
<br />
<p align="justify"> We aim to improve our Future Lanthan Hospital Model by means of cooperation with members and Alumni of the program ‘Leaders of healthcare market’ supported by Foundation 2065 im. Lesław A. Pagi and Collegium Invisible. Members of iGEM Warsaw Team, ‘Leaders of healthcare market’ program and Collegium Invisibile wish to contribute to educational initiatives, hence our long term goal is to build a group of passionate young people who will continue spreading our ideas in the future. In these particular cases we feel an obligation to help in STEM education in medicine and to find some future leaders to take care of Lanthan Hospital Model. We also hope to perform several analyses related to our project in cooperation between iGEM team and members mentioned organizations. See annex 4 in the report 'Celulare - future leaders start here'. <br />
<br />
<br><a href="https://2014.igem.org/Team:Warsaw/Project"><p align="right"><h6>Up↑</h6></p></a></br></p><br />
<br />
<br />
<h3> Kidney Paired Donation project</h3> </br><br />
<br />
<p align="justify"> We also support Future Lanthan Hospital Model by estimation of potential gains from KPD to facilitate implementation of pilot program in Poland (in the raport we feature also preliminary results of the project 'estimation of potential gains from Kidney Paired Donation and comparative analysis of national and worldwide transplant law to facilitate implementation of a pilot KPD program in Poland' - see annex 2).This part of the Human Practice is based on idea of openness and sharing and touches the problem of optimal allocation deficit resources so is very in line with Celulare philosophy. <br />
<br />
<br><a href="https://2014.igem.org/Team:Warsaw/Project"><p align="right"><h6>Up↑</h6></p></a> </br><br />
<br />
<br />
<h3> Workshops for gifted high school students </h3> </br><br />
<br />
<br />
<p align="justify"> <br />
<br />
We aimed at spread the idea of Future Lanthan Hospital and potential applications of lanthanides, as we thinking of our long term goals from the beginning - we want our project Celulare to have real application and meet people needs. Together with members of Collegium Invisibile we prepared workshops introducing idea of Future Lanthan Hospital and simultaneously fascinating young people with synthetic biology and showing idea of startups. We also prepared workshops on advanced visualization in oncology (molecular imaging, MRI) to show applications on lanthanides in medicine and collaboration on the boundaries of science. <br />
<br> <a href="https://2014.igem.org/Team:Warsaw/Project"><p align="right"><h6>Up↑</h6></p></a></br><br />
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<br />
<br />
<h3> Offtopicarium</h3> </br><br />
<br />
<br />
<p align="justify"> As we have discovered that Offtopicarium gives unique opportunity to present our project to people from different fields: scientists, entrepreneurs, people involved in startups, who like to discuss, create and solve problems and are not afraid of unconventional and "off-topic" ideas, we wanted to contribute to this event by being active participants and by preparing supporting materials(see Figure 2). Important to say is that one of our members has coorganized the event this year with a view to mutually spread ideas between iGEM Team and Offtopicarium community (for more information on Offtopicarium see <a href="http://crastina.se/5th-offtopicarium-in-poland-26-28-sept-2014-apply/ "> article </a> in Scientia Crastina).<br></br><br />
<br />
<br />
<br />
<center><img src="<br />
https://static.igem.org/mediawiki/2014/3/3f/Offtopicarium.jpg" alt="Smiley face" height="350" width="700"></center><br />
<br />
<p align="center"> <b> Figure 2. </b> Event cover - supporting materials prepared by the member of IGEM Warsaw Team 2014. </p><br />
<br> </br><br />
<br />
<p align="justify"> Offtopicarium has been launched to spread ideas among people with passion, so we prepared comprehensive presentation of ideas related to our project in three talks. First talk titled "Synthetic life - beyond biology and chemistry" briefly summarized the recent discoveries and development in biological chemistry which are connected to the idea of synthetic life such as new synthetic analogues of DNA, extended genetic code and so called bioorthogonal chemistry which allow us to introduce chemical modifications to proteins, nucleic acids and other biomolecules <i>in vivo</i>. Second talk “Serious games in Education – was related to computer game connected with Celulare - game that would teach synthetic biology. It was also a story on ventures with starting up a small game-design company. The last talk, titled “How to make healthcare more open? How to allocate resources in healthcare? - are those mirror questions?” presented Future Lanthan Hospital Model in the context of open health. This was a talk on the possibilities and problems encountered in spreading ideas in context of society awareness, medical environemnent and lack of economical and law analysis. </p><br />
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<br> <a href="https://2014.igem.org/Team:Warsaw/Project"><p align="right"><h6>Up↑</h6></p></a></br><br />
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<a name="science_festival"><h2>Science Festival</h2></a></br><br><br />
<hr noshade="noshade" /><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/8/8b/Logo_FN.png" alt="Smiley face" height="142" width="386" style="padding:10px;"><br><br><br />
<p align="justify">At the end of September our iGEM Team took part in Festiwal Nauki (Science Festival)– an annual event held all over Warsaw, organized since 1997. The aim of the Festival is to talk, show and share science with the entire society, to gain social endorsement for further progress of science in Poland. It is also an opportunity for anyone to do science on their own – by taking part in discussions, lectures, workshops, shows and many more events held every day for 10 days. <br><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/f/f6/IMG_3334.JPG" alt="Smiley face" height="225" width="300" align="right" style="padding:10px;"><br><br><br><br />
As a Team, we prepared two things for this occasion: workshop called “Design your own gene” and a stand devoted to synthetic biology and recycling. On our stand we were showing in simple ways what is synthetic biology and what we can do with it. We were also talking about our project, showing that not only bacteria can be used for recycling, but that it is also possible to use some basic trash to create something useful or pretty – like newspaper baskets or crayon boxes from milk containers. Most of our listeners there where young children. <br><br><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/c/cf/IMG_3328.JPG" alt="Smiley face" height="225" width="300" align="left" style="padding:10px;"><br><br><br />
The bioinformatics workshop was dedicated to older listeners, who already knew a little about biology. We were trying to show them what synthetic biology is and how the process of genetical engineering really looks like. Listeners where given a basic program that would show them if the sequence created by them will work as a gene. <br><br><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/4/4f/IMG_3322.JPG" alt="Smiley face" height="225" width="300" align="right" style="padding:10px;"><br><br><br />
This event was a very important part of our Human Practice, where we could meet our target people face-to-face. Overall, Science Festival was a great experience for us and an amazing way to meet and talk with people about synthetic biology and our project. Both children and adults seemed fascinated by the idea of bacteria recycling lanthanides and many people said they are looking forward to see our project become everyday reality.<br><br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p align="right"><h6>Up↑</h6></p></a><br><br />
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<a name="media"><h2>Media</h2></a></br><br />
<hr noshade="noshade" /><br />
<h3>Media mentions</h3><br />
<p align="leftt"><br />
<ol type="1"><br />
<li><a href="http://www.naukawpolsce.pap.pl/aktualnosci/news,401374,bezpieczne-bakterie-pomoga-w-odzyskiwaniu-rzadkich-metali.html">Polish Press Agency</a></li><br />
<li><a href="http://www.biol.uw.edu.pl/pl/aktualnosci/196-wiadomosci/1588-druzyna-igem-warsaw-team">Department of Biology</a></li><br />
<li><a href="http://wawalove.pl/Bakterie-z-Uniwersytetu-Warszawskiego-pomoga-w-recyklingu-smartfonow-a14442">"Wawa Love" web site</a></li><br />
<li><a href="http://laboratoria.net/pdf/?get=L2FrdHVhbG5vc2NpL19pdGVtLDIxOTczLHJpZCwscHJpbnQsMSxwZGYsMS5odG1s">"laboratoria.net" web site</a></li><br />
<li><a href="http://portaltechniczny.pl/aktualnosci/pt/projekt-polskich-bakterii-odzyskujacych-pierwiastki-ziem-rzadkich.html">"portaltechniczny.pl" web site</a></li><br />
<li><a href="http://wiadomosci.wp.pl/kat,1019393,title,Bakterie-z-Uniwersytetu-Warszawskiego-pomoga-w-recyklingu-smartfonow,wid,16645457,wiadomosc.html?ticaid=113a3f">"Wirtualna Polska" web site</a></li><br />
<li><a href="http://www.focus.pl/przyroda/bakterie-z-uw-pomoga-w-recyklingu-smartfonow-11317">"Focus" magazine</a></li><br />
<li><a href="http://www.chip.pl/news/wydarzenia/nauka-i-technika/2014/06/studenci-z-uniwersytetu-warszawskiego-pracuja-nad-nowa-metoda-recyklingu-smartfonow?fb_action_ids=814772768533394&fb_action_types=og.likes">"Chip" magazine</a></li><br />
</ol><br />
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<a href="https://2014.igem.org/Team:Warsaw/Project"><p align="right"><h6>Up↑</h6></p></a></div>
ASamsel
http://2014.igem.org/Team:Warsaw/HP
Team:Warsaw/HP
2014-10-18T00:38:42Z
<p>ASamsel: </p>
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<a href="https://2014.igem.org/Team:Warsaw">Project</a><br />
<ul><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#background">Background</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#proof_of_concept">Proof of concept</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#detailed_explanation">Detailed explanation</a></li> <br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#modelling">Modelling</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#weeestudy">WEEE study</a></li><br />
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<a href="/Team:Warsaw/Achievements">Achievements</a><br />
<ul><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Achievements#parts">Parts</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Achievements#results">Results</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Achievements#cooperation">Cooperation</a></li><br />
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<br />
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<li><a href="https://2014.igem.org/Team:Warsaw/EXTRAS#discussion">Discussion</a></li><br />
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<a href="/Team:Warsaw/Team">Team</a><br />
<ul><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Team#members">Members</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Team#advisors">Advisors</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Team#sponsors">Sponsors</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Team#acknowledgements">Acknowledgements</a></li><br />
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<div class="main-content"><br />
<br />
<h1>Human Practice</h1></br><br />
<hr noshade="noshade" /><br />
<br />
<a name="overview"><h2>Overview</h2></a></br><br />
<h2> Human Practice Philosophy: big dreams with applications </h2></br><br />
<br />
<p align="justify"> Behind our Human Practice we have one simple idea. We have a vision to transform our dreams in new applications to excel in achieving social goals. Our vision for Human Practice has been shown in report on Future Lanthan Hospital Model. It is a bottom-up strategy to transform healthcare by launching collaboration on the boundaries of several disciplines aimed at inducing openness and sharing, not only in business and academia but also in the society itself. In Human Practice we would like to show the perspective and context for Cellulare. To tell the truth we would be pleased show you big dreams with application and encourage you to collaborate with us! <br />
<br />
<br> </br><br />
<br />
<p align="justify"> We believe that the person with a great idea behind is more powerful than one who depend merely on facts. So, everything what we have done was based on that philosophy. We do not give you complete solutions, but a vision and a strategy to develop and apply. Our first goal is not to be the best iGEM project, our goal is to initiate the project which could be developed further, outside iGEM by people of different skills and different potential to solve complex problems on the larger scale. We are aimed at design both Cellulare itself and its Human Practice to serve social goals which could be developed far beyond synthetic biology.<br />
<br> </br><br />
<br />
<font size="4"><p align=" center"> <i>“Some look at things that are, and ask why. I dream of things that never were and ask why not?” </br> George Bernard Shaw </i> </p> </font><br />
<br> </br><br />
<img src="https://static.igem.org/mediawiki/2014/f/f6/Dream.JPG" alt="Smiley face" align="left" height="300" width="410" ><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/9/9d/Hand.JPG" alt="Smiley face" align="right" height="300" width="410" ><br />
<font size="2">Photos: Piotr Migdał, used with permission </font> <br />
<br></br><br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p align="right"><h6>Up↑</h6></p></a><br />
<hr noshade="noshade" /><br />
<br />
<br />
<br />
<h2> Recycling ideas: recovery - transformation – new application </h2></br><br />
<br />
<p align="justify"> We have started from listening what others want to say and, asking them questions to understand better the context of our project (Figure 1). This is how new application for Celulare has emerged. Our ultimate goal of the Human Practice is to add valuable contribution to healthcare transition by proposing a more synthetic biology-oriented approach for medicine. At the same time we want to encourage you to become more resource-efficient through optimal allocation of scarce material. We would like to show new application for mechanism design (engineering part of the economics) and the stable matching algorithm allowing to better resource-management addressing the needs of the patients. We also aim to provide a contribution to the processing of medical data and open source science in the era of big data in medicine. Here, we see new application for Celulare, (and in futher perspective iGEM itself) to finally bring idea of transform healthcare with synthetic biology.<br />
<br> </br><br />
<br> </br><br />
<br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/2/20/General_scheme.png" alt="Smiley face" height="400" width="600"></center><br />
<br> </br><br />
<br />
<p align="center"> <b> Figure 1. Cycles of ideas with new applications. </b> Context for Celulare itself and its Human Practice </p><br />
<br> </br><br />
<br />
<h2>Video game</h2></br><br />
<p align="justify"><br />
This year, as we continue our HP long term strategy, we<br />
developed an educational<br />
computer game. We are willing to use it to popularize synthetic biology term and our<br />
CeLuLa-Re project. The game was written in HTML5 using Construct2 software.</br><br />
<a href="https://f6052cc3a5c084cb37f0b0f1639e817316d820f5.googledrive.com/host/0ByxTljcJDMLoLVlGVzc5OGVURnM/">Here</a> you will find a link to an external server</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/54/Prt_scr_1_gra.png" alt="Smiley face" height="323" width="682"></center></br><br />
The game is actually a demo as we plan to develop it. We believe we can reach and<br />
attract many people as the game is funny and everybody learns best, while playing.</br><br />
<a href="https://www.youtube.com/watch?v=-H8yeYqBvqo">Here</a> is a short film showing gameplay</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/e/ec/Prt_scra_2_gra.png" alt="Smiley face" height="323" width="682"></centert></br><br />
The game consists of 3 platform levels and 2 quizzes about our project. At the<br />
beginning the two levels are locked as the player proceeds after collecting particular<br />
amount of lanthanides ions. You can especially earn<br />
a lot of them by giving correct<br />
answers in quiz.</br><br />
Platform levels are full of arcade challenges such<br />
as deadly spikes or enemies<br />
making it hard to reach<br />
ions. Both quizzes consist of 10 questions about synthetic<br />
biology, rare earths elements, our wet lab and HP part. All the correct answers can<br />
be found<br />
on our<br />
Wiki<br />
and on the official<br />
iGEM webpage.</br><br />
The main hero is a genetically engineered bacteria<br />
which wants to return home. Its<br />
space ship was destroyed due to collision with space rubbish. Now the bacteria<br />
searches a dangerous<br />
and unknown land for lacking lanthanides ions to build a laser<br />
to clear the planet's atmosphere and repair its space ship so it can safely return<br />
home.</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/7/7e/Prt_scr_3_gra.png" alt="Smiley face" height="323" width="682"></center></br><br />
Well, that is the demo. We have loads of other ideas to expand the game such as<br />
gaining super powers by hero when obtaining special<br />
BioBrick, different types of<br />
enemies and obstacles, new levels<br />
and of course we plan on adding<br />
more<br />
educational value. We are planning to create a book<br />
of knowledge and make a<br />
drag&drop task to teach cloning in BioBrick standard.</br><br />
We are inviting you to play and share<br />
this<br />
game with your friends!</br><br />
</p><br />
<br />
<p align="justify"> In our persuits we have been inspired by and support mutually with <a href="http://www.ci.edu.pl/en/collegium/idea "> Collegium Invisibile </a> and <a href="http://offtopicarium.wikidot.com/en:about"> Offtopicarium </a>who also realize some bottom-up initiatives and have excellent Human Practice. We understand, however, that our contribution is only a droplet in the sea of knowledge. Still, we envisage project to be a small thing with greater perspectives. That is why we are searching for new ways of communication via <a href="http://crastina.se/about/ "> Scientia Crastina </a>(latin for ‘The Science of Tomorrow’). Our aim is to encourage to seek new possibilities of collaboration of the interdisciplinary projects through open communication in science. <br />
<br> </br><br />
<br />
<br> </br><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/c/c2/Warsaw_logo_collegium.jpg" alt="Smiley face" height="80" width="250" align=left><br />
<br />
<img style="float: center; padding: 0px 0px 0px 70px;;" src="https://static.igem.org/mediawiki/2014/e/e8/Scientia.png" alt="Smiley face" height="80" width="280"><br />
<img src="https://static.igem.org/mediawiki/2014/d/df/Platformscientia.png " alt="Smiley face" height="80" width="250" align=right> <br />
<br> </br><br />
<br> </br><br />
<br> </br><br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p align="right"><h6>Up↑</h6></p></a><br />
<br />
<br />
<a name="lanthan_hospital"><h2>Lanthan Hospital</h2></a></br><br />
<hr noshade="noshade" /><br />
<br />
<br />
</br><br />
<br />
<p align="justify"> Moving to implement of our dreams in practice we comprised thoughts derived from our Human Practice in the raport “Celulare: transforming healthcare" in which we present Future Lanthan Hospital Model. The basic idea behind Lanthan Hospital Model is to integrate synthetic biology lab with hospital. This is sort of start-up strategy with systems impact, which we set in the context of a broader healthcare transformation. As Celulare is focused on lanthanides bioremediation, we want to propose strategy to optimal allocation of these elements and other deficit resources on healthcare market. That is why we have indicated several possible applications for novel economic concepts of Nobel Prize Winners in ‘Nobel Lantan Economy and Nobel Human Practice’ section. We believe that synthetic biology approach, especially iGEM itself may "influence the world from protein, to policy, to pixels.” To make it happen we develop Future Lantan Hospital Model. <br />
<br />
<br></br><br />
<br></br><br />
<br />
<center><br />
<a href="https://static.igem.org/mediawiki/2014/b/bc/Cellulare_-_report_Final.pdf"><br />
<img src="https://static.igem.org/mediawiki/2014/3/36/Cellulare_cover.png" alt="Smiley face" align="center" height="552" width="395" ><br />
<br><br><br />
Download the report (pdf file)<br />
</a><br />
</center><br />
<br />
<br />
<br> </br><br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p align="right"><h6>Up↑</h6></p></a><br />
</br></br><br />
<br />
<h2> Report - table of contests </h2></br><br />
<br />
<br />
Summary </br><br />
1. Background – from systems biology through synthetic biology to big data in medicine </br><br />
2. New application of synthetic biology: transforming healthcare </br><br />
3. New application of iGEM registry: challenging healthcare for data analysis </br><br />
4. Solving optimal resource allocation problem with support of synthetic biology, economy and medicine. Step forward to open health </br><br />
5. Celulare: transforming healthcare for optimal allocation of deficit resources </br><br />
6. Future Lanthan Hospital - implementation strategy through ‘Cellulare’ </br><br />
7. Celulare Recommendations </br><br />
8. Nobel Lanthan Economy with Nobel Human Practice </br><br />
9. Stable matching and mechanism design through Celulare to Polish healthcare market </br><br />
10. Closing remarks </br><br />
Annex 1. Anti selfie mechanism design for smartphone MDs through Celulare </br><br />
Annex 2. KPD - optimal allocation of deficit resources need access to databases </br><br />
Annex 3. Celulare – future leaders start here </br><br />
Annex 4. Creative education - workshops. </br><br />
<br />
</p> <br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p align="right"><h6>Up↑</h6></p></a><br />
<br />
<h2> Report - summary </h2> </br><br />
<br />
<p align="justify" style="text-indent: 0.5in;"> Nowadays, in the big data era, medicine will be inevitably transformed by systems biology. Rapid developments in deep sequencing, metabolomics and so called "personal medicine" have already changed medical sciences. Hence, we would like to add a valuable contribution to that healthcare transition by proposing a more synthetic biology-oriented approach for medicine. Our project ‘Cellulare’ touches both healthcare transformation for data analysis and optimal allocation of deficit resources. Our question is how to design a hospital only having access to limited resources, equip it in modern technologies (biomedical and IT), and finally how to combine modern technology and pro-ecological design in order to improve the work of healthcare. To address this difficult issue, we developed Future Lanthan Hospital Model, which is aimed to show that recovery of lanthanides (rare earth elements) from electronic waste (smartphones, tablets etc.) by synthetic biology methods, could be a beneficial supporting strategy for a modern hospital. We have proposed integration of a hospital with a supplementary synthetic biology lab, in which lanthanide remediation project could take place. First, such a solution would contribute to the public image of the hospital as a rationally functioning institution with eco-friendly profile. Secondly, it could allow to equip the hospital in modern technologies based on rare-earth metals. Simultaneously, we suggest mechanism design (engineering part of the economy) and the stable matching algorithm allowing better resource-management that would address the needs of the patients. By Future Lanthan Hospital Model we would like to show beneficial changes for society resulted from implementation of modern technologies in medicine and educate people about applications, safety and some security problems related to cutting-edge technologies. In the end we also aim to provide a contribution to the processing of medical data and open source science, since in the era of big data in medicine such measures seems to be necessary for the rapid transformation of the healthcare. </p> <br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p align="right"><h6>Up↑</h6></p></a><br />
<br></br><br />
<br />
<br />
<h2>Collaboration and future perspectives </h2><br />
<br> </br><br />
<br />
<font size="4"><p align="left"> <i>We have a bright vision of the future so we start from collaboration and sharing. </i> </p> </font><br />
<br> </br><br />
<br />
<br />
<h3> Science Polish Perspectives </h3> </br><br />
<br />
<p align="justify">We are extremely proud that just before Giant Jamboree we have passed through competitive selection process for talks and we are to present Future Lanthan Hospital Model at the <a href="http://www.polishperspectives.org/en/home-en"> Science Polish Perspectives 2014 at Oxford</a>! One of main aim of conference is to popularise science, whilst also enhancing the cooperation between its British, Polish and European branches. Taking in mind SPP agenda we hope that presenting our project help us to build a network of contacts and discuss Poland’s participation in the development of science and technology. We agree with the agenda that drawing inspiration from each other is one of the best ways of developing the huge potential as individuals and as members of a fast-growing scientific community. <br />
<br />
<br> </br><br />
<a href="http://www.polishperspectives.org/speaker-pr"> Here</a> see announcment on talk 'solving limited resources problem with engineering part of biology, economy and medicine. Step forward to open health' to be presented by a member of our team. </p><br />
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<a href="https://2014.igem.org/Team:Warsaw/Project"><p align="right"><h6>Up↑</h6></p></a><br />
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<h3> Leaders of healthcare market and Collegium Invisibile </h3> </br><br />
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<p align="justify"> We aim to improve our Future Lanthan Hospital Model by means of cooperation with members and Alumni of the program ‘Leaders of healthcare market’ supported by Foundation 2065 im. Lesław A. Pagi and Collegium Invisible. Members of iGEM Warsaw Team, ‘Leaders of healthcare market’ program and Collegium Invisibile wish to contribute to educational initiatives, hence our long term goal is to build a group of passionate young people who will continue spreading our ideas in the future. In these particular cases we feel an obligation to help in STEM education in medicine and to find some future leaders to take care of Lanthan Hospital Model. We also hope to perform several analyses related to our project in cooperation between iGEM team and members mentioned organizations. See annex 4 in the report 'Celulare - future leaders start here'. <br />
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<br><a href="https://2014.igem.org/Team:Warsaw/Project"><p align="right"><h6>Up↑</h6></p></a></br></p><br />
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<h3> Kidney Paired Donation project</h3> </br><br />
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<p align="justify"> We also support Future Lanthan Hospital Model by estimation of potential gains from KPD to facilitate implementation of pilot program in Poland (in the raport we feature also preliminary results of the project 'estimation of potential gains from Kidney Paired Donation and comparative analysis of national and worldwide transplant law to facilitate implementation of a pilot KPD program in Poland' - see annex 2).This part of the Human Practice is based on idea of openness and sharing and touches the problem of optimal allocation deficit resources so is very in line with Celulare philosophy. <br />
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<br><a href="https://2014.igem.org/Team:Warsaw/Project"><p align="right"><h6>Up↑</h6></p></a> </br><br />
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<h3> Workshops for gifted high school students </h3> </br><br />
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<p align="justify"> <br />
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We aimed at spread the idea of Future Lanthan Hospital and potential applications of lanthanides, as we thinking of our long term goals from the beginning - we want our project Celulare to have real application and meet people needs. Together with members of Collegium Invisibile we prepared workshops introducing idea of Future Lanthan Hospital and simultaneously fascinating young people with synthetic biology and showing idea of startups. We also prepared workshops on advanced visualization in oncology (molecular imaging, MRI) to show applications on lanthanides in medicine and collaboration on the boundaries of science. <br />
<br> <a href="https://2014.igem.org/Team:Warsaw/Project"><p align="right"><h6>Up↑</h6></p></a></br><br />
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<h3> Offtopicarium</h3> </br><br />
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<p align="justify"> As we have discovered that Offtopicarium gives unique opportunity to present our project to people from different fields: scientists, entrepreneurs, people involved in startups, who like to discuss, create and solve problems and are not afraid of unconventional and "off-topic" ideas, we wanted to contribute to this event by being active participants and by preparing supporting materials(see Figure 2). Important to say is that one of our members has coorganized the event this year with a view to mutually spread ideas between iGEM Team and Offtopicarium community (for more information on Offtopicarium see <a href="http://crastina.se/5th-offtopicarium-in-poland-26-28-sept-2014-apply/ "> article </a> in Scientia Crastina).<br></br><br />
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<center><img src="<br />
https://static.igem.org/mediawiki/2014/3/3f/Offtopicarium.jpg" alt="Smiley face" height="350" width="700"></center><br />
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<p align="center"> <b> Figure 2. </b> Event cover - supporting materials prepared by the member of IGEM Warsaw Team 2014. </p><br />
<br> </br><br />
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<p align="justify"> Offtopicarium has been launched to spread ideas among people with passion, so we prepared comprehensive presentation of ideas related to our project in three talks. First talk titled "Synthetic life - beyond biology and chemistry" briefly summarized the recent discoveries and development in biological chemistry which are connected to the idea of synthetic life such as new synthetic analogues of DNA, extended genetic code and so called bioorthogonal chemistry which allow us to introduce chemical modifications to proteins, nucleic acids and other biomolecules <i>in vivo</i>. Second talk “Serious games in Education – was related to computer game connected with Celulare - game that would teach synthetic biology. It was also a story on ventures with starting up a small game-design company. The last talk, titled “How to make healthcare more open? How to allocate resources in healthcare? - are those mirror questions?” presented Future Lanthan Hospital Model in the context of open health. This was a talk on the possibilities and problems encountered in spreading ideas in context of society awareness, medical environemnent and lack of economical and law analysis. </p><br />
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<br> <a href="https://2014.igem.org/Team:Warsaw/Project"><p align="right"><h6>Up↑</h6></p></a></br><br />
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<a name="science_festival"><h2>Science Festival</h2></a></br><br><br />
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<img src="https://static.igem.org/mediawiki/2014/8/8b/Logo_FN.png" alt="Smiley face" height="142" width="386" style="padding:10px;"><br><br><br />
<p align="justify">At the end of September our iGEM Team took part in Festiwal Nauki (Science Festival)– an annual event held all over Warsaw, organized since 1997. The aim of the Festival is to talk, show and share science with the entire society, to gain social endorsement for further progress of science in Poland. It is also an opportunity for anyone to do science on their own – by taking part in discussions, lectures, workshops, shows and many more events held every day for 10 days. <br><br />
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<img src="https://static.igem.org/mediawiki/2014/f/f6/IMG_3334.JPG" alt="Smiley face" height="225" width="300" align="right" style="padding:10px;"><br><br><br><br />
As a Team, we prepared two things for this occasion: workshop called “Design your own gene” and a stand devoted to synthetic biology and recycling. On our stand we were showing in simple ways what is synthetic biology and what we can do with it. We were also talking about our project, showing that not only bacteria can be used for recycling, but that it is also possible to use some basic trash to create something useful or pretty – like newspaper baskets or crayon boxes from milk containers. Most of our listeners there where young children. <br><br><br />
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<img src="https://static.igem.org/mediawiki/2014/c/cf/IMG_3328.JPG" alt="Smiley face" height="225" width="300" align="left" style="padding:10px;"><br><br><br />
The bioinformatics workshop was dedicated to older listeners, who already knew a little about biology. We were trying to show them what synthetic biology is and how the process of genetical engineering really looks like. Listeners where given a basic program that would show them if the sequence created by them will work as a gene. <br><br><br />
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<img src="https://static.igem.org/mediawiki/2014/4/4f/IMG_3322.JPG" alt="Smiley face" height="225" width="300" align="right" style="padding:10px;"><br><br><br />
This event was a very important part of our Human Practice, where we could meet our target people face-to-face. Overall, Science Festival was a great experience for us and an amazing way to meet and talk with people about synthetic biology and our project. Both children and adults seemed fascinated by the idea of bacteria recycling lanthanides and many people said they are looking forward to see our project become everyday reality.<br><br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p align="right"><h6>Up↑</h6></p></a><br><br />
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<a name="media"><h2>Media</h2></a></br><br />
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<h3>Media mentions</h3><br />
<p align="justify"><br />
<ol type="1"><br />
<li><a href="http://www.naukawpolsce.pap.pl/aktualnosci/news,401374,bezpieczne-bakterie-pomoga-w-odzyskiwaniu-rzadkich-metali.html">Polish Press Agency</a></li><br />
<li><a href="http://www.biol.uw.edu.pl/pl/aktualnosci/196-wiadomosci/1588-druzyna-igem-warsaw-team">Department of Biology</a></li><br />
<li><a href="http://wawalove.pl/Bakterie-z-Uniwersytetu-Warszawskiego-pomoga-w-recyklingu-smartfonow-a14442">"Wawa Love" web site</a></li><br />
<li><a href="http://laboratoria.net/pdf/?get=L2FrdHVhbG5vc2NpL19pdGVtLDIxOTczLHJpZCwscHJpbnQsMSxwZGYsMS5odG1s">"laboratoria.net" web site</a></li><br />
<li><a href="http://portaltechniczny.pl/aktualnosci/pt/projekt-polskich-bakterii-odzyskujacych-pierwiastki-ziem-rzadkich.html">"portaltechniczny.pl" web site</a></li><br />
<li><a href="http://wiadomosci.wp.pl/kat,1019393,title,Bakterie-z-Uniwersytetu-Warszawskiego-pomoga-w-recyklingu-smartfonow,wid,16645457,wiadomosc.html?ticaid=113a3f">"Wirtualna Polska" web site</a></li><br />
<li><a href="http://www.focus.pl/przyroda/bakterie-z-uw-pomoga-w-recyklingu-smartfonow-11317">"Focus" magazine</a></li><br />
<li><a href="http://www.chip.pl/news/wydarzenia/nauka-i-technika/2014/06/studenci-z-uniwersytetu-warszawskiego-pracuja-nad-nowa-metoda-recyklingu-smartfonow?fb_action_ids=814772768533394&fb_action_types=og.likes">"Chip" magazine</a></li><br />
</ol><br />
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<a href="https://2014.igem.org/Team:Warsaw/Project"><p align="right"><h6>Up↑</h6></p></a></div>
ASamsel
http://2014.igem.org/File:Members_itw.jpg
File:Members itw.jpg
2014-10-18T00:11:23Z
<p>ASamsel: members_itw</p>
<hr />
<div>members_itw</div>
ASamsel
http://2014.igem.org/Team:Warsaw/Protocoles
Team:Warsaw/Protocoles
2014-10-18T00:03:16Z
<p>ASamsel: </p>
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<a href="https://2014.igem.org/Team:Warsaw">Project</a><br />
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<li><a href="https://2014.igem.org/Team:Warsaw/Project#background">Background</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#proof_of_concept">Proof of concept</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#detailed_explanation">Detailed explanation</a></li> <br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#modelling">Modelling</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#weeestudy">WEEE study</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#safety">Safety</a></li> <br />
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<li><a href="https://2014.igem.org/Team:Warsaw/EXTRAS#discussion">Discussion</a></li><br />
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<li><a href="https://2014.igem.org/Team:Warsaw/Team#members">Members</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Team#advisors">Advisors</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Team#sponsors">Sponsors</a></li><br />
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<div class="main-content"><br />
<br />
<h1>Protocoles</h1></br><br />
<hr noshade="noshade" /><br />
<h2>Synthetic biology protocols</h2><br><br><br />
<h3>Chemocompetent bacteria</h3><br><br />
<ol type="1"><br />
<li>Inoculate 500 ml LB with 5 ml of the overnight culture and incubate it shaking at 37 °C till the OD = 0.4.</li><br />
<li>Cool the culture for 10 min. in some ice.</li><br />
<li>Centifruge the sample at 6000 rpm at 4 °C for 3 minutes.</li><br />
<li>Suspend the precipitate gently in ~ 20 ml of cold solution of 0.1 M CaCl2, then add 0.1 M CaCl2 to a volume of 300 ml.</li><br />
<li>Centrifuge again as above.</li><br />
<li>Suspend the precipitate in 10 ml of cold 0.1 M CaCl2 and incubate it for 30 min.</li><br />
<li>Centrifuge again.</li><br />
<li>Suspend the sample in 6 ml of 0.1 M CaCl2 and 15% glycerol and Pipette 50 μl to an eppendorf (put them immediately in liquid nitrogen) and store at -80 °C.<br />
<a href="https://2014.igem.org/Team:Warsaw/Protocoles"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
</li></ol><br><br><br />
<h3>Transformation of chemocompetent bacteria</h3><br><br><br />
<ol type="1"><br />
<li>Put the bacteria into ice for 2-3 minutes (or until it melts)</li><br />
<li>Add a cooled plasmid or ligation in a volume not bigger than 20 μl and stir with a tip</li><br />
<li>Keep it in the ice for 20 to 30 minutes</li><br />
<li>Put it to a heating block set for 42°C for 1.5 min</li><br />
<li>Put it back to ice for 2 min</li><br />
<li>Add 900 μl of SOB and incubate at 37°C for 1 h</li><br />
<li>Sow on the appropriate selective deposit.</li><br />
<a href="https://2014.igem.org/Team:Warsaw/Protocoles"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
</ol><br><br><br />
<h3>Transformation of electrocompetent bacteria</h3><br><br><br />
<ol type="1"><br />
<li>Put the bacteria into some ice for 2-3 minutes (or until it melts).</li><br />
<li>50 μl of bacteria pipete to a dialised and cooled plasmid DNA or ligation</li><br />
<li>Put the transformation mixture to a cuvette (also previously cooled on ice) - it is important that the sample must be on the bottom of the cuvette and free of air bubbles. You can tap the cuvette several times on the table.</li><br />
<li>Transform the bacteria in an electroporator set on 2500 V (time constant should be around 5)</li><br />
<li>Immediately add 900 μl of SOB and incubate at 37°C for 1 h</li><br />
<li>Sow the appropriate selective deposit</li><br />
<a href="https://2014.igem.org/Team:Warsaw/Protocoles"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
</ol><br><br><br />
<br />
<h3>Alkaline Lysis</h3><br><br><br />
<ol type="1"><br />
<li>Pick single colony and inoculate 3 ml of LB broth. Shake at 37°C overnight.</li><br />
<li>Centrifuge two times 1.5 ml cells in the same 1.5 ml Eppendorf tube at 6000 rpm for 1 minute. Aspirate supernatant.</li><br />
<li>Resuspend cell pellet in 100 μl of GTE buffer (50 mM Glucose, 25 mM Tris-HCl, 10 mM EDTA, pH 8).</li><br />
<li>Add 200 μl of buffer II (0.2 M NaOH, 1% SDS). Invert gently tube 6-8 times.</li><br />
<li>Add 150 μl of buffer III (3M potassium acetate, 2M acetic acid, pH 5.5). This solution neutralizes NaOH in the previous lysis step while precipitating the genomic DNA and SDS in an insoluble, white, amorphous precipitate. </li><li>Incubate in ice for 5 min. Spin at top speed 10 min.</li><br />
<li>Transfer supernatant to a new tube, being careful not to pick up any white flakes. Precipitate the nucleic acids with 1ml of 96% ethanol for 5 minutes at room temperature and centrifuge at top speed for 10 minutes. Aspirate supernatant, add 200 &mul 70% ethanol and centrifuge at top speed for 1 minute.</li><br />
<li>Aspirate off all the ethanol supernatant. Dissolve the pellet in 40 μl of water. Store at -20°C.</li><br />
<a href="https://2014.igem.org/Team:Warsaw/Protocoles"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
</ol><br><br><br />
<br />
<h3>DNA Digestion</h3><br><br><br />
<ol type="1"><li>Add to Eppendorf tube (for 20 μl):</li><br />
<ul><br />
<li>15 μl water</li><br />
<li>2 μl buffer</li><br />
<li>2 μl DNA that you want digest (ex. plasmid)</li><br />
<li>0,5 μl enzyme X and 0,5 μl enzyme Y (or 1 μl enzyme Z)</li></ul><br><br />
(for 10 &mul [verification cloning]):<br />
<ul><br />
<li>7 μl plasmid</li><br />
<li>1 μl buffer</li><br />
<li>1 μl water</li><br />
<li>0,5 μl enzyme X and 0,5 μl enzyme Y (or 1 μl enzyme Z)</li></ul><br />
<li>Tubes incubate at 37°C for 1 h.</li><br />
<a href="https://2014.igem.org/Team:Warsaw/Protocoles"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
</ol><br><br><br />
<br />
<h3>DNA Ligation</h3><br />
<ol type="1"><br />
<li>Add to Eppendorf tube</li><br />
<ul><br />
<li>5 μl water</li><br />
<li>2 μl buffer</li><br />
<li>10 μl digest plasmids mix</li><br />
<li>1 μl T4 ligase</li></ul><br />
<li>Incubate 2-3 h at room temperature or O/N at 16 degrees Celsius</li><br />
<a href="https://2014.igem.org/Team:Warsaw/Protocoles"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
</ol><br><br><br />
<br />
<h3>PCR</h3><br><br><br />
<br />
Add to PCR tube (0,2 ml), do it on ice:<br />
<ul><br />
<li>34 μl water</li><br />
<li>10 μl buffer with MgCl2</li><br />
<li>1 μl 10mM dNTPs</li><br />
<li>2 μl primers</li><br />
<li>2 μl template DNA (200x attenuate plasmid)</li><br />
<li>1 μl Phusion polymerase</li></ul><br><br><br />
<li>PCR programme:</li><br />
<ul><br />
<li>Initialization step – 98°C – 3 min</li><br />
<li>Denaturation step – 98°C – 30 sec</li><br />
<li>Annealing step – 55°C – 30 sec</li><br />
<li>Elongation step – 72°C – 40 sec</li><br />
<li>Final elongation – 72°C – 5 min</li><br />
<li>Final hold – 4°C – to end of time</li><br />
<li>30 cycles</li></ul><br><br><br />
<br />
Total time: about 1 h 40 minutes. <br />
<a href="https://2014.igem.org/Team:Warsaw/Protocoles"><p style="text-align:right;"><h6>Up↑</h6></p></a></div>
ASamsel
http://2014.igem.org/Team:Warsaw/Achievements
Team:Warsaw/Achievements
2014-10-17T23:47:49Z
<p>ASamsel: </p>
<hr />
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<ul class="main-menu"><br />
<br />
<!-- Menu items --><br />
<li><br />
<a href="https://2014.igem.org/Team:Warsaw">Project</a><br />
<ul><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#background">Background</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#proof_of_concept">Proof of concept</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#detailed_explanation">Detailed explanation</a></li> <br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#modelling">Modelling</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#weeestudy">WEEE study</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#safety">Safety</a></li> <br />
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<a href="/Team:Warsaw/Achievements">Achievements</a><br />
<ul><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Achievements#parts">Parts</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Achievements#results">Results</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Achievements#cooperation">Cooperation</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Achievements#medal_criteria">Medal Criteria</a></li><br />
<br />
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<ul><br />
<li><a href="https://2014.igem.org/Team:Warsaw/HP#overview">Overview</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/HP#lanthan_hospital">Lanthan Hospital</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/HP#science_festival">Science Festival</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/HP#media">Media</a></li><br />
</ul><br />
</li><br />
<li><br />
<a href="/Team:Warsaw/EXTRAS">EXTRAS</a><br />
<ul><br />
<li><a href="https://2014.igem.org/Team:Warsaw/EXTRAS#bioprocess">Bioprocess</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/EXTRAS#discussion">Discussion</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/EXTRAS#alternative_methods">Alternative methods</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Notebook">Notebook</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Protocoles">Protocoles</a></li><br />
<br />
<br />
</ul><br />
</li><br />
<li><br />
<a href="/Team:Warsaw/Team">Team</a><br />
<ul><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Team#members">Members</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Team#advisors">Advisors</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Team#sponsors">Sponsors</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Team#acknowledgements">Acknowledgements</a></li><br />
</ul><br />
</li><br />
<!-- iGEM link --><br />
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<a href="https://2014.igem.org/"></a><br />
</li><br />
</ul><br />
<div class="main-content"><br />
<br />
<br />
<h1>Achievements</h1></br><br />
<hr noshade="noshade" /><br />
<br />
<a name="parts"><h2>Parts</h2></a></br><br />
<br />
<table border="1"><br />
<br />
<tr><br />
<td><b>Registry number</b></td><br />
<td><b>Construct name</b></td><br />
<td><b>Gene lenght [nts]</b></td><br />
<td><b>Protein lenght [aa]</b></td><br />
<td><b>Physical DNA sent</b></td><br />
<td><b>Construct type product</b></td><br />
<td><b>Native host</b></td><br />
<td><b>Plasmid</b></td><br />
<td><b>Standard</b></td><br />
</tr><br />
<br />
<tr><br />
<td>BBa_K1459001</td><br />
<td>PmrA</td><br />
<td>669</td><br />
<td>222</td><br />
<td><b>yes</b></td><br />
<td>protein</td><br />
<td><i>Salmonella spp.</i></td><br />
<td>pSB1C3</td><br />
<td>RFC 10</td><br />
</tr><br />
<br />
<tr><br />
<td>BBa_K1459016</td><br />
<td>PmrB WT (Fe<sup>3+</sup>)</td><br />
<td>1071</td><br />
<td>356</td><br />
<td>no</td><br />
<td>protein</td><br />
<td><i>Salmonella spp.</i></td><br />
<td>pSB1C3</td><br />
<td>RFC 10</td><br />
</tr><br />
<br />
<tr><br />
<td>BBa_K1459010</td><br />
<td>PmrB (MUT)</td><br />
<td>1029</td><br />
<td>343</td><br />
<td><b>yes</b></td><br />
<td>protein</td><br />
<td><i>Salmonella spp.</i></td><br />
<td>pSB1C3</td><br />
<td>RFC 10</td><br />
</tr><br />
<br />
<tr><br />
<td>BBa_K1459003</td><br />
<td>PmrA-PmrB</td><br />
<td>1749</td><br />
<td>222 + 356</td><br />
<td>no</td><br />
<td>2 proteins</td><br />
<td><i>Salmonella spp.</i></td><br />
<td>pSB1C3</td><br />
<td>RFC 10</td><br />
</tr><br />
<br />
<tr><br />
<td>BBa_K1459004</td><br />
<td>PmrA-PmrB(MUT)-terminator</td><br />
<td>1791</td><br />
<td>222+343(two proteins)</td><br />
<td><b>yes</b></td><br />
<td>proteins + transcription terminator </td><br />
<td><i>Salmonella spp.</i></td><br />
<td>pSB1C3</td><br />
<td>RFC 10</td><br />
</tr><br />
<br />
<tr><br />
<td>BBa_K1459011</td><br />
<td>PmrB N-terminus </td><br />
<td>102</td><br />
<td>34</td><br />
<td><b>yes</b></td><br />
<td>protein domain</td><br />
<td><i>Salmonella spp.</i></td><br />
<td>pSB1C3</td><br />
<td>RFC 25</td><br />
</tr><br />
<br />
<tr><br />
<td>BBa_K1459009</td><br />
<td>PmrB C-terminus </td><br />
<td>882</td><br />
<td>294</td><br />
<td><b>yes</b></td><br />
<td>protein domain</td><br />
<td><i>Salmonella spp.</i></td><br />
<td>pSB1C3</td><br />
<td>RFC 25</td><br />
</tr><br />
<br />
<tr><br />
<td>BBa_K1459005</td><br />
<td>PmrA-PmrB N-terminus </td><br />
<td>782 </td><br />
<td>220 + 34</td><br />
<td><b>yes</b></td><br />
<td>protein and protein domain</td><br />
<td><i>Salmonella spp.</i></td><br />
<td>pSB1C3</td><br />
<td>RFC 25</td><br />
</tr><br />
<br />
<tr><br />
<td>BBa_K1459006</td><br />
<td>pmr<sup>C</sup> promoter </td><br />
<td>404</td><br />
<td>-</td><br />
<td>no</td><br />
<td>promoter</td><br />
<td><i>Salmonella spp.</i></td><br />
<td>pSB1C3</td><br />
<td>RFC 10</td><br />
</tr><br />
<br />
<tr><br />
<td>BBa_K1459017</td><br />
<td>pmr<sup>C</sup>-GFP </td><br />
<td>1119</td><br />
<td>238</td><br />
<td>no</td><br />
<td>promoter and protein</td><br />
<td><i>Salmonella spp.</i></td><br />
<td>pSB1C3</td><br />
<td>RFC 10</td><br />
</tr><br />
<br />
<tr><br />
<td>BBa_K1459008</td><br />
<td>pmr<sup>C</sup>-GFP-terminator</td><br />
<td>1177</td><br />
<td>238</td><br />
<td><b>yes</b></td><br />
<td>promoter and protein and terminator</td><br />
<td><i>Salmonella spp.</i></td><br />
<td>pSB1C3</td><br />
<td>RFC 10</td><br />
</tr><br />
<br />
<tr><br />
<td>BBa_K1459012</td><br />
<td>SENG lanthanide binding tag</td><br />
<td>60</td><br />
<td>20</td><br />
<td>no</td><br />
<td>peptide</td><br />
<td>synthetic</td><br />
<td>pSB1C3</td><br />
<td>RFC 25</td><br />
</tr><br />
<br />
<tr><br />
<td>BBa_K1459013</td><br />
<td>wSE3 lanthanide binding tag</td><br />
<td>51</td><br />
<td>17</td><br />
<td>no</td><br />
<td>peptide</td><br />
<td>synthetic</td><br />
<td>pSB1C3</td><br />
<td>RFC 25</td><br />
</tr><br />
<br />
<tr><br />
<td>BBa_K1459014</td><br />
<td>Lanthanide Binding Tag</td><br />
<td>51</td><br />
<td>17</td><br />
<td>no</td><br />
<td>peptide</td><br />
<td>synthetic</td><br />
<td>pSB1C3</td><br />
<td>RFC 25</td><br />
</tr><br />
<br />
<tr><br />
<td>BBa_K1459015</td><br />
<td>1L2Y short peptide</td><br />
<td>66</td><br />
<td>22</td><br />
<td>no</td><br />
<td>peptide</td><br />
<td>synthetic</td><br />
<td>pSB1C3</td><br />
<td>RFC 25</td><br />
</tr><br />
<br />
</table><br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<br />
<br />
<br><br><br><br />
<h3>BBa_K1459001 - PmrA</h3></br><br />
<b>Protein name: </b>PmrA</br><br />
<b>Other names: </b>basR, parA</br><br />
<b>Gene name: </b>basR</br><br />
<b>Source organism for the data: </b><i>Salmonella enterica</i> subsp. enterica serovar Typhimurium str. <i>strain LT2 / SGSC1412 / ATCC 700720</i></br><br />
<b>UniProtKB signature: </b>P36556</br><br />
<b>Gene sequence RefSeq accession number: </b>NC_003197.1</br><br />
<b>Protein sequence RefSeq accession number: </b>NP_463157.1</br><br />
<b>Length: </b>222 aa</br><br />
<b>Molecular mass: </b>25,035 Da</br><br />
<b>Cellular localization: </b>cytoplasmic</br><br />
<b>Biological function: </b>transcription regulator</br><br />
<p align="justify"><br />
The PmrA protein is a cognate response regulator of<br />
the histidine kinase PmrB. Upon<br />
phosphorylation by PmrB, PmrA undergoes dimerization which dramatically increases its affinity for<br />
promoter DNA. This allows it to regulate expression<br />
of a number of genes, usually coding for LPS-<br />
modifying enzymes.</br><br />
In our project, we used the PmrA unchanged, for it<br />
to serve as an transcription inductor for our<br />
reporter - the GFP, expressed under the control of the<br />
PmrC promoter, i.e. the promoter of one of the<br />
genes involved in LPS modification. Notably, however, there is no PmrC gene under its promoter<br />
in our constructs, so that neither this LPS-modifyi<br />
ng enzyme, nor any other enzymes of this kind, are<br />
expressed. </br><br />
If you wish to study PmrA-PmrB system more closely, we suggest reading following papers:</br><br />
[1] H. Liang, X. Deng, M. Bosscher, Q. Ji, M. P. Jensen, C. He, <i>Engineering Bacterial Two-Component System PmrA/PmrB to Sense<br />
Lanthanide Ions</i>, <i>J.Am.Chem.Soc.</i> 2013, 135, 2037−2039</br><br />
[2] M. Wonsten, L. Kox, S. Chamnogpol, F. Soncini, E. Groisman,<i>A Signal Transduction System that Responds to Extracellular Iron</i>,<i>Cell</i>, Vol. 103, 113–125, September 29, 2000</br><br />
SENT TO REGISTRY</br><br />
</p><br />
<br />
<br><br><br><br />
<h3>BBa_K1459002 - C-term of PmrB from <i>Salmonella enterica</i></h3></br><br />
<p align="justify"><br />
PmrB is a transmembrane kinase. After binding iron (III) ion by binding peptide on extracellular loop, it's intracellular domain gains kinase activity and phosphorylates PmrA (BBa_K1459000).</br><br />
PmrB C-term is a part of two-component system. When fused with some binding tag, PmrB(N-term), PmrA and pmrC-reporter, it is a viable detecting system.</br><br />
If you wish to study PmrA-PmrB system more closely, we suggest reading following papers:</br><br />
[1] H. Liang, X. Deng, M. Bosscher, Q. Ji, M. P. Jensen, C. He, <i>Engineering Bacterial Two-Component System PmrA/PmrB to Sense<br />
Lanthanide Ions</i>, <i>J.Am.Chem.Soc.</i> 2013, 135, 2037−2039</br><br />
[2] M. Wonsten, L. Kox, S. Chamnogpol, F. Soncini, E. Groisman,<i>A Signal Transduction System that Responds to Extracellular Iron</i>,<i>Cell</i>, Vol. 103, 113–125, September 29, 2000</br><br />
SENT TO REGISTRY</br><br />
</p><br />
<br />
<br><br><br><br />
<h3>BBa_K1459003 - PmrA-PmrB(LBT) two-component system</h3></br><br />
<p align="justify"><br />
PmrA-PmrB two-component system is native to <i>Salmonella enterica</i> and in its native state the system is responsible for chemotaxis. PmrB is a transmembrane protein with iron binding peptide on its extracellular loop. When PmrB binds iron (III) iron, the intracellular domain gains kinase activity and phosphorylates PmrA, which subsequently binds to pmrC promoter and induces expression of chemotaxis CheZ protein. In this part iron binding tag on the extracellular loop was exchanged with a lanthanide binding tag (LBT), to allow PmrA-PmrB two-component system to respond to lanthanide ions.</br><br />
If you wish to study PmrA-PmrB system more closely, we suggest reading following papers:</br><br />
[1] H. Liang, X. Deng, M. Bosscher, Q. Ji, M. P. Jensen, C. He, <i>Engineering Bacterial Two-Component System PmrA/PmrB to Sense<br />
Lanthanide Ions</i>, <i>J.Am.Chem.Soc.</i> 2013, 135, 2037−2039</br><br />
[2] M. Wonsten, L. Kox, S. Chamnogpol, F. Soncini, E. Groisman,<i>A Signal Transduction System that Responds to Extracellular Iron</i>,<i>Cell</i>, Vol. 103, 113–125, September 29, 2000</br><br />
</p><br />
<br />
<br><br><br><br />
<h3>BBa_K1459004 - PmrA-PmrB(LBT) with terminator (BBa_B1006)</h3></br><br />
<p align="justify"><br />
PmrA-PmrB two-component system is native to <i>Salmonella enterica</i> and in its native state it is responsible for chemotaxis. PmrB is a transmembrane protein with iron binding peptide on its extracellular loop. When PmrB binds iron (III) iron, its intracellular domain gains kinase activity and phosphorylates PmrA, which subsequently binds to pmrC promoter and induces expression of chemotaxis CheZ protein. In this part iron binding tag on the extracellular loop was exchanged with a lanthanide binding tag (LBT), to allow PmrA-PmrB two-component system to respond to lanthanide ions.</br><br />
If you wish to study PmrA-PmrB system more closely, we suggest reading following papers:</br><br />
[1] H. Liang, X. Deng, M. Bosscher, Q. Ji, M. P. Jensen, C. He, <i>Engineering Bacterial Two-Component System PmrA/PmrB to Sense<br />
Lanthanide Ions</i>, <i>J.Am.Chem.Soc.</i> 2013, 135, 2037−2039</br><br />
[2] M. Wonsten, L. Kox, S. Chamnogpol, F. Soncini, E. Groisman,<i>A Signal Transduction System that Responds to Extracellular Iron</i>,<i>Cell</i>, Vol. 103, 113–125, September 29, 2000</br><br />
SENT TO REGISTRY</br><br />
</p><br />
<br />
<br><br><br><br />
<h3>BBa_K1459005 - PmrA-PmrB(N-term)</h3></br><br />
<p align="justify"><br />
This is N-terminal part of PmrA-PmrB two-component system native to <i>Salmonella enterica</i>. PmrA-PmrB two-component system is native to Salmonella enterica and in its native state it is responsible for chemotaxis. PmrB is a transmembrane protein with iron binding peptide on its extracellular loop. When PmrB binds iron (III) iron, it's intracellular domain gains kinase activity and phosphorylates PmrA, which subsequently binds to pmrC promoter and induces expression of chemotaxis CheZ protein. In this part iron binding tag on the extracellular loop was exchanged with a lanthanide binding tag (LBT), to allow PmrA-PmrB two-component system to respond to lanthanide ions. In this part, PmrB protein is truncated just before iron binding tag, which enables one to put any desired tag between two parts of PmrB, to construct a detecting system based on PmrA-PmrB system.</br><br />
If you wish to study PmrA-PmrB system more closely, we suggest reading following papers:</br><br />
[1] H. Liang, X. Deng, M. Bosscher, Q. Ji, M. P. Jensen, C. He, <i>Engineering Bacterial Two-Component System PmrA/PmrB to Sense<br />
Lanthanide Ions</i>, <i>J.Am.Chem.Soc.</i> 2013, 135, 2037−2039</br><br />
[2] M. Wonsten, L. Kox, S. Chamnogpol, F. Soncini, E. Groisman,<i>A Signal Transduction System that Responds to Extracellular Iron</i>,<i>Cell</i>, Vol. 103, 113–125, September 29, 2000</br><br />
SENT TO REGISTRY</br><br />
</p><br />
<br />
<br><br><br><br />
<h3>BBa_K1459006 - pmrC</h3></br><br />
<p align="justify"><br />
This is pmrC promoter native to <i>Salmonella enterica</i>. PmrA-PmrB two-component system is native to <i>Salmonella enterica</i> and in its native state it is responsible for chemotaxis. PmrB is a transmembrane protein with iron binding peptide on its extracellular loop. When PmrB binds iron (III) iron, the intracellular domain gains kinase activity and phosphorylates PmrA, which subsequently binds to pmrC promoter and induces expression of chemotaxis CheZ protein. In this part iron binding tag on the extracellular loop was exchanged with a lanthanide binding tag (LBT), to allow PmrA-PmrB two-component system to respond to lanthanide ions.</br><br />
This part is extraordinary long for a promoter. It is designed this way because of the ca. 400 bp spacer at 5' end of the part - promoter is only 46 bp long, which would prove almost impossible to amplify through PCR or direct synthesis and ligation with pSB1C3. To counter this, we amplified pmrC with this spacer.</b><br />
This part contains also RBS from <i>Salmonella enterica</i>, just before the start of translation.</b><br />
If you wish to study PmrA-PmrB system more closely, we suggest reading following papers:</br><br />
[1] H. Liang, X. Deng, M. Bosscher, Q. Ji, M. P. Jensen, C. He, <i>Engineering Bacterial Two-Component System PmrA/PmrB to Sense<br />
Lanthanide Ions</i>, <i>J.Am.Chem.Soc.</i> 2013, 135, 2037−2039</br><br />
[2] M. Wonsten, L. Kox, S. Chamnogpol, F. Soncini, E. Groisman,<i>A Signal Transduction System that Responds to Extracellular Iron</i>,<i>Cell</i>, Vol. 103, 113–125, September 29, 2000</br><br />
</p><br />
<br />
<br><br><br><br />
<h3>BBa_K1459008 - pmrC-GFP-terminator</h3></br><br />
<p align="justify"><br />
This is pmrC promoter from <i>Salmonella enterica</i>, with subsequent GFP and BBa_B1006 terminator. This part is one part of PmrA-PmrB detecting system. Upon phosphorylation by PmrB, PmrA binds to pmrC and induces expression of GFP.</br><br />
This part is extraordinary long for a pmr<sup>C</sup>-GFP. It is designed this way because of the ca. 400 bp spacer at 5' end of the part - promoter is only 46 bp long, which would prove almost impossible to amplify through PCR or direct synthesis and ligation with pSB1C3. To counter this, we amplified pmrC with this spacer.</b><br />
This part contains also RBS from <i>Salmonella enterica</i>, just before the start of translation.</b><br />
If you wish to study PmrA-PmrB system more closely, we suggest reading following papers:</br><br />
[1] H. Liang, X. Deng, M. Bosscher, Q. Ji, M. P. Jensen, C. He, <i>Engineering Bacterial Two-Component System PmrA/PmrB to Sense<br />
Lanthanide Ions</i>, <i>J.Am.Chem.Soc.</i> 2013, 135, 2037−2039</br><br />
[2] M. Wonsten, L. Kox, S. Chamnogpol, F. Soncini, E. Groisman,<i>A Signal Transduction System that Responds to Extracellular Iron</i>,<i>Cell</i>, Vol. 103, 113–125, September 29, 2000</br><br />
SENT TO REGISTRY</br><br />
</p><br />
<br />
<br><br><br><br />
<h3>BBa_K1459010 - PmrB(LBT)</h3></br><br />
<b>Protein name: </b>PmrB</br><br />
<b>Other names: </b>basS, parB</br><br />
<b>Gene name: </b>basS</br><br />
<b>Source organism for the data: </b><i>Salmonella enterica</i> subsp. enterica serovar Typhimurium str. <i>strain LT2 / SGSC1412 / ATCC 700720</i></br><br />
<b>UniProtKB signature: </b>P36557/br><br />
<b>Gene sequence RefSeq accession number: </b>NC_003197.1</br><br />
<b>Protein sequence RefSeq accession number: </b>NP_463157.1</br><br />
<b>Length: </b>356 aa</br><br />
<b>Molecular mass: </b>40,262 Da</br><br />
<b>Cellular localization: </b>inner plasma membrane</br><br />
<b>Biological function: </b>Signal transduction via kinase acivities</br><br />
<p align="justify"><br />
PmrB(LBT) is a engineered PmrB gene, where PmrB is a sensor histidine kinase present in the inner cell membrane of many species of bacteria,<br />
including<br />
<i>E. coli</i> and <i>S. enterica</i>. With a 30 amino acid periplasmic loop, it is capable of binding iron<br />
(III) and aluminium ions. The binding event induces<br />
a conformational change of the protein, which<br />
leads to ATP phosphate-derived autophosphorylation<br />
of the C-terminal cytoplasmic domain,<br />
followed by transfer of the phosphate group onto the transcriptional regulator PmrA.<br />
As part of our project, the periplasmic iron/alumin<br />
ium-binding loop of the PmrB was substituted<br />
with a synthetic sequence - a lanthanide-binding ta<br />
g, intended to bind lanthanide ions, with terbium<br />
in particular. Such a binding event would then induce the aforementioned conformation change and<br />
phosphorylation of the PmrA, leading it to bind to<br />
the PmrC promoter, to allow for expression of the<br />
Green Fluorescent Protein - our reporter gene. </br><br />
If you wish to study PmrA-PmrB system more closely, we suggest reading following papers:</br><br />
[1] H. Liang, X. Deng, M. Bosscher, Q. Ji, M. P. Jensen, C. He, <i>Engineering Bacterial Two-Component System PmrA/PmrB to Sense<br />
Lanthanide Ions</i>, <i>J.Am.Chem.Soc.</i> 2013, 135, 2037−2039</br><br />
[2] M. Wonsten, L. Kox, S. Chamnogpol, F. Soncini, E. Groisman,<i>A Signal Transduction System that Responds to Extracellular Iron</i>,<i>Cell</i>, Vol. 103, 113–125, September 29, 2000</br><br />
SENT TO REGISTRY</br><br />
</p><br />
<br />
<br><br><br><br />
<h3>BBa_K1459011 - PmrB(N-term)</h3></br><br />
<p align="justify"><br />
PmrA-PmrB two-component system is native to <i>Salmonella enterica</i> and in its native state it is responsible for chemotaxis. PmrB is a transmembrane protein with iron binding peptide on its extracellular loop. When PmrB binds iron (III) iron, the intracellular domain gains kinase activity and phosphorylates PmrA, which subsequently binds to pmrC promoter and induces expression of chemotaxis CheZ protein.</br><br />
In this part iron binding tag on the extracellular loop was exchanged with a lanthanide binding tag (LBT), to allow PmrA-PmrB two-component system to respond to lanthanide ions. This part was truncated just before the iron binding tag, and PmrB(N-term) is functionally complementar to PmrB(C-term).</br><br />
If you wish to study PmrA-PmrB system more closely, we suggest reading following papers:</br><br />
[1] H. Liang, X. Deng, M. Bosscher, Q. Ji, M. P. Jensen, C. He, <i>Engineering Bacterial Two-Component System PmrA/PmrB to Sense<br />
Lanthanide Ions</i>, <i>J.Am.Chem.Soc.</i> 2013, 135, 2037−2039</br><br />
[2] M. Wonsten, L. Kox, S. Chamnogpol, F. Soncini, E. Groisman,<i>A Signal Transduction System that Responds to Extracellular Iron</i>,<i>Cell</i>, Vol. 103, 113–125, September 29, 2000</br><br />
SENT TO REGISTRY</br><br />
</p><br />
<br />
<br><br><br><br />
<h3>BBa_K1459012 - SENG lanthanide binding tag</h3></br><br />
<p align="justify"><br />
This is a DNA sequence coding lanthanide binding tag described in literature. Its literatural dissociation constants are as follows:</br><br />
K<sub>Tb<sup>3+</sup></sub>=18 nM</br><br />
This is the lowest known value of dissociation constant for a Tb<sup>3+</sup>, thus making the binding strenght highest amongst known LBTs.</br><br />
[1] J. M. Langdon, <i>Development of Lanthanide-Binding Tags (LBTs) as Powerful and Versatile PeptidesFor Use in Studies of Proteins and Protein Interactions</i>, © 2008 Massachusetts Institute of Technology<br />
All rights reserved</br><br />
</p><br />
<br />
<br><br><br><br />
<h3>BBa_K1459013 - wSE3 lanthanide binding tag</h3></br><br />
<p align="justify"><br />
This is sequence of DNA coding wSE3 lanthanide binding tag. It's dissociation constants are as follows:</br><br />
K<sub>Tb<sup>3+</sup></sub>=2000 nM</br><br />
[1] J. M. Langdon, <i>Development of Lanthanide-Binding Tags (LBTs) as Powerful and Versatile PeptidesFor Use in Studies of Proteins and Protein Interactions</i>, © 2008 Massachusetts Institute of Technology<br />
All rights reserved</br><br />
</p><br />
<br />
<br><br><br><br />
<h3>BBa_K1459014 - Lanthanide Binding Tag</h3></br><br />
<p align="justify"><br />
This is DNA sequence coding a lanthanide binding tag. This one is one of the best described LBTs in literature, with dissociation constants following:</br><br />
K<sub>La<sup>3+</sup></sub>= 3500 nM</br><br />
K<sub>Ce<sup>3+</sup></sub>= 950 nM</br><br />
K<sub>Nd<sup>3+</sup></sub>= 270 nM</br><br />
K<sub>Eu<sup>3+</sup></sub>= 62 nM</br><br />
K<sub>Gd<sup>3+</sup></sub>= 84 nM</br><br />
K<sub>Tb<sup>3+</sup></sub>= 57 nM</br><br />
K<sub>Dy<sup>3+</sup></sub>= 71 nM</br><br />
K<sub>Er<sup>3+</sup></sub>= 78 nM</br><br />
K<sub>Yb<sup>3+</sup></sub>= 100 nM</br><br />
K<sub>Lu<sup>3+</sup></sub>= 128 nM</br><br />
[1] M. Nitz, M. Sherawat, K. J. Franz, E. Peisach, K. N. Allen, B. Imperiali, <i>Structural Origin of the High Affinity of a Chemically Evolved Lanthanide-Binding Peptide</i><br />
, <i>Angew.Chem.Int.Ed.</i> 2004, 43, 3682–368</br><br />
</P<br />
<br />
<br><br><br><br />
<h3>BBa_K1459015 - 1L2Y short peptide</h3></br><br />
<p align="justify"><br />
This is DNA sequence coding short peptide (PDB 1L2Y) is highly structured in water and could provide a structural foundation for small binding tags, such as we were planning to use it.</br><br />
[1] Neidigh, J.W., Fesinmeyer, R.M., Andersen, N.H., <i>Designing a 20-residue protein</i>, <i>Nat.Struct.Biol.</i>, 2002 9: 425-430</br><br />
</p><br />
<br />
<br><br><br><br />
<h3>BBa_K1459016 - PmrB(WT)</h3></br><br />
<b>Protein name: </b>PmrB</br><br />
<b>Other names: </b>basS, parB</br><br />
<b>Gene name: </b>basS</br><br />
<b>Source organism for the data: </b><i>Salmonella enterica</i> subsp. enterica serovar Typhimurium str. <i>strain LT2 / SGSC1412 / ATCC 700720</i></br><br />
<b>UniProtKB signature: </b>P36557/br><br />
<b>Gene sequence RefSeq accession number: </b>NC_003197.1</br><br />
<b>Protein sequence RefSeq accession number: </b>NP_463157.1</br><br />
<b>Length: </b>356 aa</br><br />
<b>Molecular mass: </b>40,262 Da</br><br />
<b>Cellular localization: </b>inner plasma membrane</br><br />
<b>Biological function: </b>Signal transduction via kinase acivities</br><br />
<p align="justify"><br />
PmrB(LBT) is a engineered PmrB gene, where PmrB is a sensor histidine kinase present in the inner cell membrane of many species of bacteria,<br />
including<br />
<i>E. coli</i> and <i>S. enterica</i>. With a 30 amino acid periplasmic loop, it is capable of binding iron<br />
(III) and aluminium ions. The binding event induces<br />
a conformational change of the protein, which<br />
leads to ATP phosphate-derived autophosphorylation<br />
of the C-terminal cytoplasmic domain,<br />
followed by transfer of the phosphate group onto the transcriptional regulator PmrA.<br />
As part of our project, the periplasmic iron/alumin<br />
ium-binding loop of the PmrB was substituted<br />
with a synthetic sequence - a lanthanide-binding ta<br />
g, intended to bind lanthanide ions, with terbium<br />
in particular. Such a binding event would then induce the aforementioned conformation change and<br />
phosphorylation of the PmrA, leading it to bind to<br />
the PmrC promoter, to allow for expression of the<br />
Green Fluorescent Protein - our reporter gene. </br><br />
If you wish to study PmrA-PmrB system more closely, we suggest reading following papers:</br><br />
[1] H. Liang, X. Deng, M. Bosscher, Q. Ji, M. P. Jensen, C. He, <i>Engineering Bacterial Two-Component System PmrA/PmrB to Sense<br />
Lanthanide Ions</i>, <i>J.Am.Chem.Soc.</i> 2013, 135, 2037−2039</br><br />
[2] M. Wonsten, L. Kox, S. Chamnogpol, F. Soncini, E. Groisman,<i>A Signal Transduction System that Responds to Extracellular Iron</i>,<i>Cell</i>, Vol. 103, 113–125, September 29, 2000</br><br />
</p><br />
<br />
<br><br><br><br />
<h3>BBa_K1459017 - pmrC-GFP</h3></br><br />
<p align="justify"><br />
This is pmrC promoter from <i>Salmonella enterica</i>, with subsequent GFP. This part is one part of PmrA-PmrB detecting system. Upon phosphorylation by PmrB, PmrA binds to pmrC and induces expression of GFP.</br><br />
This part is extraordinary long for a pmr<sup>C</sup>-GFP. It is designed this way because of the ca. 400 bp spacer at 5' end of the part - promoter is only 46 bp long, which would prove almost impossible to amplify through PCR or direct synthesis and ligation with pSB1C3. To counter this, we amplified pmrC with this spacer.</b><br />
This part contains also RBS from <i>Salmonella enterica</i>, just before the start of translation.</b><br />
If you wish to study PmrA-PmrB system more closely, we suggest reading following papers:</br><br />
[1] H. Liang, X. Deng, M. Bosscher, Q. Ji, M. P. Jensen, C. He, <i>Engineering Bacterial Two-Component System PmrA/PmrB to Sense<br />
Lanthanide Ions</i>, <i>J.Am.Chem.Soc.</i> 2013, 135, 2037−2039</br><br />
[2] M. Wonsten, L. Kox, S. Chamnogpol, F. Soncini, E. Groisman,<i>A Signal Transduction System that Responds to Extracellular Iron</i>,<i>Cell</i>, Vol. 103, 113–125, September 29, 2000</br><br />
</p><br />
<br />
<br><br><br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<hr noshade="noshade" /><br />
<a name="results"><h2>Results</h2></a></br><br />
<h4>In what we succeded</h4><br />
We did succed in constructing the lanthanide sensor<br />
in BioBrick standard and cloning its parts into<br />
pSB1C3 and sending seven of them to the Registry.</br><br />
As for 17.10.2014, we are trying to measure pmr<sup>C</sup> not activated by PmrA and also we are trying to measure GFP expression both in presence and in absence of lanthanide ions in the environment.</br><br />
We also measured the relative strenght of pmr<sup>C</sup> promoter in the absence of lanthanides.</br><br />
<h4>What would we do (given more time)</h4><br />
Given more time, we would certainly try to test more lanthanide binding tags and to construct a<br />
system to effectively bind those ions, not only detect them. </br><br />
We would also try to quantify better our existing system. </br><br />
<br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<br />
<hr noshade="noshade" /><br />
<a name="cooperation"><h2>Cooperation with other iGEM Teams</h2></a></br><br />
<h5>During this year’s iGEM we have exchanged with the following teams:</h5><br />
<p align="justify"><li>Paris_Bettencourt – we participated in the iGEM newsletter, sending them information about our team, our project and trying to answer other teams questions from the previous newsletter</li><br />
<li>Toulouse – we sent them 4 of our BioBricks (BBa_K780003, BBa_K780002, BBa_K780001, BBa_K780000) </li><br />
<li>Groningen – we exchanged our official iGEM abstracts, translated their abstract into Polish and got our abstract translated into Dutch</li><br />
<li>Paris Saclay - we exchanged our official iGEM abstracts and we translated their into Polish and got our abstract translated into French</li><br />
<li>ETH Zurich – we filled in a survey about complexity in everyday life</li><br />
<li>Warwick - we filled in a survey about policy and practices</li><br />
<li>Valencia Biocampus - we filled in a survey</li><br />
<br><br />
<br />
<br />
<a name="medal_criteria"><h2>Medal Criteria</h2></a></br><br />
<hr noshade="noshade" /><br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a></div>
ASamsel
http://2014.igem.org/Team:Warsaw/Team
Team:Warsaw/Team
2014-10-17T23:42:48Z
<p>ASamsel: </p>
<hr />
<div><html lang="pl"><br />
<head><br />
<meta charset="utf-8"><br />
<br />
<title>iGEM Team Warsaw</title><br />
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<a href="https://2014.igem.org/Team:Warsaw">Project</a><br />
<ul><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#background">Background</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#proof_of_concept">Proof of concept</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#detailed_explanation">Detailed explanation</a></li> <br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#modelling">Modelling</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#weeestudy">WEEE study</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#safety">Safety</a></li> <br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#possibilities_of_development">Possibilities of development</a><br />
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<a href="/Team:Warsaw/Achievements">Achievements</a><br />
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<li><a href="https://2014.igem.org/Team:Warsaw/Achievements#parts">Parts</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Achievements#results">Results</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Achievements#cooperation">Cooperation</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Achievements#medal_criteria">Medal Criteria</a></li><br />
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<a href="/Team:Warsaw/HP">HP</a><br />
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<li><a href="https://2014.igem.org/Team:Warsaw/HP#overview">Overview</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/HP#lanthan_hospital">Lanthan Hospital</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/HP#science_festival">Science Festival</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/HP#media">Media</a></li><br />
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<a href="/Team:Warsaw/EXTRAS">EXTRAS</a><br />
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<li><a href="https://2014.igem.org/Team:Warsaw/EXTRAS#bioprocess">Bioprocess</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/EXTRAS#discussion">Discussion</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/EXTRAS#alternative_methods">Alternative methods</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Notebook">Notebook</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Protocoles">Protocoles</a></li><br />
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<a href="/Team:Warsaw/Team">Team</a><br />
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<li><a href="https://2014.igem.org/Team:Warsaw/Team#members">Members</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Team#advisors">Advisors</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Team#sponsors">Sponsors</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Team#acknowledgements">Acknowledgements</a></li><br />
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<h1>The Team</h1> </br><br />
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<td> <img src="https://static.igem.org/mediawiki/2014/0/05/Zdjecie_nurkowanie.JPG" width="200px"; alt="Aleksandra_Bartosik"></td> <td><h4>Aleksandra Bartosik</h4>I am studying biotechnology and chemistry at the College of Inter-faculty Individual Studies in Mathematics and Natural Science. I'm fascinated by biomimicry, immunobiology, mechanisms of bacterial multi-drug resistance and ABC-transporters. In my spare time I dive, travel across Northern Europe and talk about graphics with my best friend. I am the 2nd Team Leader and this year beside of working in the lab I take care of the Human Practice.</td><br />
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<td><img src="https://static.igem.org/mediawiki/2014/8/86/M_G%C3%B3rka.jpg" width="200px" ; alt="Magdalena_Górka"></td> <td><br><h4>Magdalena Górka</h4>I study biotechnology and chemistry at the College of Inter-faculty Individual Studies in Mathematics and Natural Science at University of Warsaw. <br>I am mostly interested in human genetics and medical biotechnology but I don't remain indifferent to other fields of science. <br>In my free time, I like reading books, swimming and traveling.<br><br><br><br><br><br></td><br />
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<td><img src="https://static.igem.org/mediawiki/2014/9/99/IMG_0616aa.jpg" width="200px"; alt="Ela_Gralińska"></td> <td><h4>Ela Gralińska</h4>Currently I study Biotechnology at TU Berlin. Besides biological engineering, I'm particularly fascinated with otters thanks to which my adventure with biology has begun. In my freetime I play badminton. <br><br><br><br><br><br><br><br></td><br />
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<td><img src="https://static.igem.org/mediawiki/2014/f/f4/Img2.jpg" width="200px" ; alt="Mieszko_Majka"></td> <td><br><h4>Mieszko Majka</h4>I'm a sophomore student of chemistry & biotechnology at the College of Interfaculty Studies in Mathematics and Natural Sciences (a.k.a. Kolegium MISMaP). My scientific interests entail biological chemistry (mostly chemistry of nucleic acids and proteins), bits of neurochemistry, molecular biology of nucleic acids and synthetic biology (mostly fantasizing how to combine fancy natural stuff as proteins, RNAs, etc. into systems that would serve whatever purpose I have on my mind at the given time). Outside the lab, I do enjoy a good bike trip, some ballroom dancing or teaching people... whatever they want to learn and I can provide, so it's usually English or chemistry. During this year's iGEM, I try to blow up the lab every other day (i.e. I'm a wetlab worker) but parallely, I help make sure that everything flows smoothly enough to prevent others from doing so (mostly by telling people when to come to the lab and when not & mundane stuff like this, really).</td><br />
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<td><img src="https://static.igem.org/mediawiki/2014/f/f3/Rafał_zdjęcie.jpg" width="200px" ; alt="Rafał_Meysztowicz"></td> <td><h4>Rafał Meysztowicz</h4> I'm studying Physics and Mathematics at College of Inter-Faculty Studies in Mathematics and Natural Sciences. I'm fascinated with their application on other fields like Biology, Economics and in everyday life. I'm also a music producer. In free time I like to play sports, read books and watch movies.<br><br><br><br><br><br><br></td><br />
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<td><img src="https://static.igem.org/mediawiki/2014/2/27/PaulinaOsiadacz.jpg" width="200px"; alt="Paulina_Osiadacz"></td> <td><h4>Paulina Osiadacz</h4>My name is Paulina Osiadacz. I study biotechnology in the second years in the Department in the Biology in the University of Warsaw. I always interesting how to work the all body, but in particular at the level of cell. In the free time I try to help people and participate in charitable actions. <br><br><br><br><br><br></td><br />
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<td><img src="https://static.igem.org/mediawiki/2014/7/7c/DSC02006.jpg" width="200px"; alt="Zosia_Rudnicka"></td> <td><h4>Zosia Rudnicka</h4>My name is Zosia Rudnicka. I’m a student of biology and physics at University of Warsaw. I’m interested in genetics and bioinformatics. In my free time I’m reading history and SF books. I love traveling and my dog Daisy. <br><br><br><br></td><br />
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<td><img src="https://static.igem.org/mediawiki/2014/c/c7/20130902_120836.jpg" width="200px"; alt="Anna_Saffray-Borowski"></td> <td><h4>Anna Saffray-Borowski</h4>I am a second year student at the Faculty of Biology of our university and I am studying biotechnology. I enjoy spending time in the laboratory learning new exciting approaches, methods and facts, that is why I am trying different projects. I have met the World of: plants, bacteria, yeasts, as well as the World of cells, I don't know yet what to choose but I guess at one point I will just know. Apart from my first hobby (science) I enjoy listening to classical music, especially Sergei Rachmaninoff and play the piano. </td><br />
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<td><img src="https://static.igem.org/mediawiki/2014/d/df/1407528791959.jpg" width="200px"; alt="Agnieszka_Samsel"></td> <td><h4>Agnieszka Samsel</h4>I am studying biotechnology and chemistry at the College of Inter-faculty Individual Studies In Mathematics and Natural Science. I’m fascinated by biotechnology in environmental protection, bioprocess engineering and genetics. In free time I like to travell, search for interesting places in Warsaw and delve into Kurpie’s history.<br></td><br />
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<td><img src="https://static.igem.org/mediawiki/2014/a/ac/20140719_143455.jpg" width="200px"; alt="Natalia_Szulc"></td> <td><h4>Natalia Szulc</h4>I study biotechnology and bioinformatics at College of Inter-Faculty Individual Studies in Mathematicsand Natural Sciences, University of Warsaw. My scientific interests include molecular mechanisms of disease, medicinal chemistry and computer-aided drug design. iGEM allows me to combine my scientific passion with Human Practice and popularization of science. In my free time I go with my friends to the cinema or theatre. I also love reading, travelling and visiting museums of natural history and galleries of fine arts.<br><br><br><br><br><br><br></td><br />
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<td><img src="https://static.igem.org/mediawiki/2014/3/3b/GŚ_zdjęcie.jpg" width="200px"; alt="Grzegorz_Ścibisz"></td> <td><h4>Grzegorz Ścibisz</h4>I'm studying chemistry and biotechnology at College of Inter-Faculty Studies in Mathematics and Natural Sciences. My scientific interests contain supramolecular chemistry, synthetic biology and biophysics and self-assembly of proteins. My "non-proffesional" interests are physics, astronomy and reading, not only sci-fi.<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br></td><br />
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<a href="https://2014.igem.org/Team:Warsaw/Team"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
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<a name="advisors"><h2>Advisors</h2></a></br><br />
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<td><img src="https://static.igem.org/mediawiki/2014/6/67/IMG_0826_%283%29.jpg" width="200px"; alt="Anna_Kornakiewicz"></td> <td><br><h4>Anna Kornakiewicz</h4>I am a young M.D. with interdisciplinary research background and want to contribute to open health and innovation in medicine. I am fascinated with mechanisms and machines - from watches and trains to elucidation of the mechanisms of action of drugs. I' ve discovered that IGEM reflects the way in which I see medicine – as ‘information science and art of healing’ and gives a unique opportunity to merge multiple interest in one clear idea. I want to act also in the area of mobile health, medical startups and healthcare markets design and contribute to gifted and STEM education.</td><br />
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<td><img src="https://static.igem.org/mediawiki/2014/c/c7/KamilKoziara.jpg" width="200px"; alt="Kamil_Koziara"></td> <td><br><h4>Kamil Koziara</h4>I have MSc in Computer Science and I am studying Biotechnology at the University of <br />
Warsaw. I am interested in bioinformatics, molecular biology and bioengineering. I am <br />
supporter of open source and in my free time I like to do some diy projects. I love to read.<br><br><br><br><br><br><br><br><br><br></td><br />
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<td><img src="https://static.igem.org/mediawiki/2014/d/d9/1618440_10151842531087126_414762827_n.jpg" width="200px"; alt="Masia_Maksymowicz"></td> <td><br><h4>Małgorzata Maksymowicz</h4>I am studying biology; last year I did my Bachelor thesis on circadian clock in different organisms. Now I'm doing my Masters in the Department of Plant Molecular Ecophysiology. I am mostly interested in plant physiology and molecular biology, though I really like being in the field and finding natural wonders.<br><br />
When I'm not doing biology, I sing, play a guitar and travel across Poland and around the world.<br><br><br><br><br><br><br></td><br />
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<td><img src="https://static.igem.org/mediawiki/2014/8/84/Zdjecie.jpg" width="200px"; alt="Piotr_Skłodowski"></td> <td><br><h4>Piotr Skłodowski</h4>I am student of third year Biology course at University of Warsaw. I am mostly interested in issues concerning molecular biology and genetic engineering. I got involved in iGEM project because it contains many fields of science, which is inseparable in development of modern knowledge. Additionally iGEM has interesting idea itself and aims to help to solve World-scale problems. After classes I like to climb and ride a bike.<br><br><br></td><br />
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<a href="https://2014.igem.org/Team:Warsaw/Team"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
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<a name="instructors"><h2>Instructors</h2></a></br><br />
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<td><img src="https://static.igem.org/mediawiki/2014/7/74/Prof._Bielecki.jpg" width="200px"; alt="prof.Jacek_Bielecki"></td> <td><br><h4>Prof. Jacek Bielecki</h4>Education: MSc, University of Warsaw, 1975; PhD, University of Warsaw, 1981; Associated professor, Warsaw University, 1995; Professor at University of Warsaw , 1996; Vice Dean of Faculty of Biology, 1996 - 1999, and 1999-2002 <br />
Research interests: Molecular mechanisms of virulence of bacteria Listeria monocytogenes, especially the role of a hemolysin, listeriolysin O (LLO). <br><br><br><br><br></td><br />
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<td><img src="https://static.igem.org/mediawiki/2014/3/33/LSB_1.jpg" width="200px"; alt="Łukasz_Borowski"></td> <td><br><h4><br>Dr. Łukasz Borowski</h4>Currently I am working as a postdoctoral fellow at The Laboratory of RNA Biology and Functional Genomics, led by Andrzej Dziembowski. My research is mainly focused on RNA biology. So far I have been involved in scientific projects concerning RNA decay in human <br />
mitochondria. We have discovered that human mitochondrial RNA degradation mediated by PNPase-hSuv3 complex takes place in distinct foci. Currently my main goal is to identify and characterise different protein complexes involved in RNA metabolism in human cells.<br><br><br><br><br></td><br />
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<td><img src="https://static.igem.org/mediawiki/2014/e/e3/Clipboard01.jpg" width="200px"; alt="Dr. Takao Ishikawa"></td> <td><br><h4><br>Dr. Takao Ishikawa</h4>Born in Tokyo, working and living in Warsaw, I am interested in protein-protein interactions. In my research, I employ in silico modeling of protein complexes and theirs verification by molecular biology methods. I am not only fascinated by life science, but also being in love with popularization of science. Giving lectures for students, teachers, and children give me a lot of fun!<br><br><br><br><br><br><br><br><br></td><br />
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<td><img src="https://static.igem.org/mediawiki/2014/5/51/Zdj%C4%99cie.jpg" width="200px"; alt="Dr. Radosław_Stachowiak"></td> <td><br><h4><br>Dr. Radosław Stachowiak</h4>My adventure with biology dates back to the previous century when I became a biotechnology student at the University of Warsaw. Currently I am a postdoc at the Department of Applied Microbiology. My research concerns molecular mechanisms of bacterial pathogenesis and possible applications of bacterial toxins in biotechnology. Apart from research activity I take care of students and guide them through experimental work. Additionally, I am a caretaker of Synthetic Biology Students’ Association and enjoy working with enthusiastic students and being involved in their daring projects.<br><br></td><br />
</tr><br />
</table><br />
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<table><br />
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<td><img src="https://static.igem.org/mediawiki/2014/1/1a/Marcin_Ziemniak.jpg" width="200px"; alt="Marcin_Ziemniak"></td> <td><br><h4><br>Marcin Ziemniak</h4>I have graduated from the University of Warsaw (M.S in bioorganic chemistry and B.S in molecular biology). Currently, I am a PhD student at the Faculty of Physics, University of Warsaw . The scientific project in which I am involved is interdisciplinary, hence in my research I employ not only chemical synthesis of modified nucleotides but also a variety of biochemical and biophysical techniques. I am also quite interested in structural biology and nanotechnology. Apart from academia I enjoy travelling, taking photos and sport. I am also an avid fan of science fiction and fantasy. <br><br><br><br></td><br />
</tr><br />
</table><br />
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<br />
<a href="https://2014.igem.org/Team:Warsaw/Team"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<hr noshade="noshade" /><br />
<br />
<a name="acknowledgements"><h2>Acknowledgements</h2></a></br><br />
Special thanks to <b>prof. Paweł Golik</b> and <b>dr. Katarzyna Tońska</b> from Institute of Genetics and Biotechnology, The University of Warsaw for allowing us working in their lab.<br><br><br />
<br />
We would like to thank <b>prof. Chuan He</b> and his team from Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago for inspiration and sending us plasmids with lanthanide-responsive system created by them.<br><br><br />
<br />
We would also like to thank <b>Anna Olchowik</b> and her company <b>123code.me</b> for incredible support in creating iGEM Team Warsaw computer game.<br><br><br />
<br />
All people that helped us in every possible way and to whom we are grateful are listed here (in alphabetic order): <br><br><br />
<br />
<b>Prof. Dariusz Bartosik</b> - Institute of Microbiology<br><br />
<b>Prof. Jacek Bielecki</b> - Department of Applied Microbiology<br><br />
<b>Dr. Łukasz Borowski</b> - Instructor, the Institute of Genetics and Biotechnology<br><br />
<b>Dr. Piotr Borsuk</b> - Associate Dean of Academic Affairs University of Warsaw<br><br />
<b>Dr. Łukasz Drewniak</b> - Laboratory of Environmental Pollution Analysis<br><br />
<b>Dr. Maciej Garstka</b> - Associate Dean for Financial Affairs University of Warsaw<br><br />
<b>Prof. Anna Giza-Poleszczuk</b> - Vice-Rector for Development and Financial Policy University of Warsaw<br><br />
<b>Prof. Paweł Golik</b> - Director of the Institute of Genetics and Biotechnology<br><br />
<b>Dr. Takao Ishikawa</b> - Institute of Biochemistry<br> <br />
<b>Prof. Jacek Jemielity</b> - Centre of New Technologies, University of Warsaw<br> <br />
<b>Prof. Agnieszka Mostowska</b> - Dean of The Faculty of Biology University of Warsaw<br> <br />
<b>Prof. Marcin Pałys</b> - Rector of University of Warsaw<br> <br />
<b>MA Jakub Piątkowski</b> - Institute of Genetics and Biotechnology<br> <br />
<b>Prof. Aleksandra Danuta Skłodowska</b> - Laboratory of Environmental Pollution Analysis<br><br />
<b>Dr. Radosław Stachowiak</b> - Instructor, Institute of Microbiology<br><br />
<b>Dr. Magdalena Szuplewska</b> -Institute of Microbiology<br><br />
<b>Dr. Katarzyna Tońska</b> - Deputy Director of the Institute of Genetics and Biotechnology<br><br />
<b>Prof. Andrzej Twardowski</b> - Director of the College of Inter-Faculty Individual Studies in Mathematics and Natural Sciences, University of Warsaw<br><br><br />
<a href="https://2014.igem.org/Team:Warsaw/Team"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
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<a name="sponsors"><h2>Sponsors</h2></a><br />
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<a href="http://www.uw.edu.pl/"><img src='https://static.igem.org/mediawiki/2014/0/08/UW.gif' width="200px" ></a><br />
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<div style='position:absolute; left:41%'><br />
<a href="http://www.biol.uw.edu.pl/"><img src='https://static.igem.org/mediawiki/2014/7/73/WB.png' width="300px"></a><br />
</div><br />
<div style='position:absolute; left:63%'><br />
<a href="http://www.mismap.uw.edu.pl/"><img src='https://static.igem.org/mediawiki/2014/c/c7/MISMaP.png' width="170px"></a><br />
</div><br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<div style='float:left'><br />
<a href="http://www.fuw.pl/"><img src='https://static.igem.org/mediawiki/2014/c/c2/FUW.jpg' width="180px" ></a><br />
</div><br />
<div style='position:absolute; left:41%'><br />
<a href="http://www.aabiot.com/"><img src='https://static.igem.org/mediawiki/2014/4/45/Fit_company_profile_LOGO-Mono-Blue12-zmniejszone.jpg' width="300px"></a><br />
</div><br />
<div style='position:absolute; left:63%'><br />
<a href="http://www.cft.edu.pl/ "><img src='https://static.igem.org/mediawiki/2014/6/60/Cft.jpg' width="170px"></a><br />
</div><br />
<br><br><br><br><br><br><br><br><br><br><br><br />
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<div style='float:left'><br />
<a href="http://www.genomed.pl/"><img src='https://static.igem.org/mediawiki/2014/9/92/Genomed.png' width="400px" ></a><br />
</div><br />
<br><br />
<div style='position:absolute; left:50%'><br />
<a href="http://www.eppendorf.com/ "><img src='https://static.igem.org/mediawiki/2014/3/38/Eppendorf-logo.jpg' width="400px"></a><br />
</div><br />
<br />
<br><br><br><br><br><br />
<br />
<div style='position:absolute; left:45%'><br />
<a href="http://www.serwis.zwik.szczecin.pl/"><img src='https://static.igem.org/mediawiki/2014/e/ed/Logo_zwik.jpg' width="130px" ></a><br />
</div><br />
<br />
<br />
<div style='float:left'><br />
<a href="http://samorzad.uw.edu.pl/ "><img src='https://static.igem.org/mediawiki/2014/c/c8/Logotyp-samorzad-rgb.jpg' width="300px" ></a><br />
</div><br />
<br />
<div style='position:absolute; left:55%'><br />
<a href="http://www.igib.uw.edu.pl/ "><img src='https://static.igem.org/mediawiki/2014/5/50/IGiB.jpg' width="320px" ></a><br />
</div><br />
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<br><br><br><br><br><br><br><br><br><br><br><br><br />
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<hr noshade="noshade" /><br />
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<a name="Patrons/Partners"><h2>Patrons/Partners</h2></a><br />
<br />
<br />
<br><br><br />
<br />
<div style='position:absolute; left:28%'><br />
<a href="http://www.naszaziemia.pl/ "><img src='https://static.igem.org/mediawiki/2014/2/2c/Nasza_Ziemia.png' width="300px" ></a><br />
</div><br />
<br />
<div style='position:absolute; left:50%'><br />
<a href="http://www.123code.me/ "><img src='https://static.igem.org/mediawiki/2014/b/bf/Logo_123codeme.jpg' width="300px" ></a><br />
</div><br />
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<br><br><br><br><br><br><br><br><br><br><br><br><br />
<a href="https://2014.igem.org/Team:Warsaw/Team"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<hr noshade="noshade" /></div>
ASamsel
http://2014.igem.org/Team:Warsaw/EXTRAS
Team:Warsaw/EXTRAS
2014-10-17T23:40:14Z
<p>ASamsel: </p>
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<li><br />
<a href="https://2014.igem.org/Team:Warsaw">Project</a><br />
<ul><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#background">Background</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#proof_of_concept">Proof of concept</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#detailed_explanation">Detailed explanation</a></li> <br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#modelling">Modelling</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#weeestudy">WEEE study</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#safety">Safety</a></li> <br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#possibilities_of_development">Possibilities of development</a><br />
</ul><br />
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<a href="/Team:Warsaw/Achievements">Achievements</a><br />
<ul><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Achievements#parts">Parts</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Achievements#results">Results</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Achievements#cooperation">Cooperation</a></li><br />
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<br />
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<a href="/Team:Warsaw/HP">HP</a><br />
<ul><br />
<li><a href="https://2014.igem.org/Team:Warsaw/HP#overview">Overview</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/HP#lanthan_hospital">Lanthan Hospital</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/HP#science_festival">Science Festival</a></li><br />
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</ul><br />
</li><br />
<li><br />
<a href="/Team:Warsaw/EXTRAS">EXTRAS</a><br />
<ul><br />
<li><a href="https://2014.igem.org/Team:Warsaw/EXTRAS#bioprocess">Bioprocess</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/EXTRAS#discussion">Discussion</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/EXTRAS#alternative_methods">Alternative methods</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Notebook">Notebook</a></li><br />
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<br />
<br />
</ul><br />
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<li><br />
<a href="/Team:Warsaw/Team">Team</a><br />
<ul><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Team#members">Members</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Team#advisors">Advisors</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Team#sponsors">Sponsors</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Team#acknowledgements">Acknowledgements</a></li><br />
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<a href="https://2014.igem.org/"></a><br />
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<div class="main-content"><br />
<br />
<h1>Technology</h1> </br><br />
<hr noshade="noshade" /><br />
<br />
<br />
<a name="bioprocess"><h2>Bioprocess</h2></a></br><br />
<p align="justify">Electronic equipment is the fastest growing waste category of waste in many developed countries. Amount of electronic waste grows rapidly because markets in which electronic is produced cross the other side of the ‘Digital Divide’. We stand in front of the following problem: what we are we going to do with all those computers, smartphones which we buy? All these products become obsolete or just unwanted within 1-3 years of purchase. Where will we find a new source of metals necessary to fabricate electronic equipment?</br><br />
Using old and broken WEEE in our project not only gives us a source of metals to produce new smartphones or computers, but also solves problem of storing WEEE.</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/6/6e/Recycling.jpg" alt="recyclin_itw" /></center><br />
<p align="justify">This figure shows a view on the context of re-use of EEE or its components. At the end of the use EEE returns to producers or to companies, which specialize in reuse of e-waste. <br />
The next step is preparation for re-use. On this step wasted electronic equipment is deconstructed. Not only computer memories or other parts which contains valuable metals are re-use. We also want to recover such materials as plastic or glass. That step requires labor work or special machines which will prepare WEE for another point of the plan. <br />
Some parts of the TVs or computers may not be useful in another manufactory. Sometimes we need to remarked parts which we need and work in place with special equipment. <br />
In the future we can use our bacteria with lanthanides binding sequences to re-use lanthanides.</br><br />
1) Removing from WEEE plastic and stirring parts which are abundant of rare earth metals such as lanthanides, especially computer memories. Stirring increase availability WEEE with acid which is produced by <i>Thiobacillus ferrooxidans</i>. It display terbium from alloy.</br><br />
2) Effluent from our bioreactor has low pH (about 2,5) what is a lethal for <i>E.coli</i>. This is the reason why we add Ca(OH)<sub>2</sub> to effluent. It increases our pH to a level which is optimal to <i>E.coli</i> (about 7). Ca(OH)<sub>2</sub> is also cheap and it is not increasing cost of the process very much.</br> <br />
3) Effluent goes to packed column bioreactor in which <i>E.coli</i> is immobilized. It gets across whole column. Ions of terbium binds and senses trough periplasmic domain.</br><br />
4) Column (with no initial effluent) is flushed by NaCl. PmrB can easily denaturate in NaCl solution losing its conformation. Ions of terbium do not precipitate with NaCl aq and do not make insoluble components with it.</br><br />
5) Ions of terbium are recovered from solution by electrolysis <br />
<a href="https://2014.igem.org/Team:Warsaw/EXTRAS"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<hr noshade="noshade" /><br />
<br />
<a name="discussion"><h2>Discussion</h2></a></br><br />
Our process finds not only a way to recovery the lanthanides, but also other metals such as copper ( it takes place at first point of it) and other rare metals.We not only gain rare metals, but also solve the problem of WEEE. There are alternative ways to recycle metals such as cooper or iron by using bacteria, but lanthanides are usually extraced from motherlode, which nowdays is really costly.</br><br />
Our way of recovery lanthanides does not produce pollution but it also is a safe way of managing with WEEE. Other methods of gaining lanthanides use mix of concentrated acids such as HCl or HNO<sub>3</sub>. The most common problems with these methods are difficult to neutralise byproducts. Byproducts, especially Ca(NO<sub>3</sub>)<sub>2</sub> are environmental-threatening. Their storing is very difficult and costly.Mining of lanthanides by old methods need to be placed on big areas, but our process takes less place. Area which will be taken to run it strictly depends on scale (in contrast to minors which always will take more space).</br><br />
Method which we presented is eco-friendly and solves the problem of full landfills with WEEE.</br> In our opinion using microorganisms to recycle e-waste will be a standard way of raising lanthanides.<br />
<a href="https://2014.igem.org/Team:Warsaw/EXTRAS"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<hr noshade="noshade" /><br />
<br />
<a name="alternative_methods"><h2>Alternative methods</h2></a></br><br />
<p align="justify">Our final system was of course not the only possibility. There were some points where we had to decide...<br />
<h4>Reporter protein</h4><br />
Finally we decided for GFP protein because of its prevalence, simplicity and fact that GFP does not require any additional reagents (eg. IPTG).</br><br />
We could have used other fluorescent proteins, for instance superfolder fluorescent proteins constructed by iGEM Warsaw 2013 Team, but regular GFP was the simpliest choice.</br><br />
<h4>Binding agent</h4><br />
Although we were unable to implement lanthanide binding system because of lack of time, we had several ideas how to accomplish this goal.</br><br />
<u>Poly-LBT peptide</u></br><br />
First was to construct a poly-LBT peptide which would be then anchored in the outer membrane of <i>E. coli</i> or transported to periplasm. We discarded this idea due to problems with modelling of behaviour of this polymer and problems with wet-lab design.</br><br />
<u>PmrB over-expression</u></br><br />
Another idea consisted of PmrB dependent over-expression of PmrB(LBT) protein. In such system we would have pmr<sup>C</sup> promoter - some logical device to boost the signal - PmrB(LBT), so in presence of lanthanides amount of PmrB(LBT) protein per cell would rise sharply, which should allow effective binding of lanthanides.</br><br />
<u>Small peptide fused with LBT</u></br><br />
Our final and probably best idea was to create a construct peptide of such composition:</br><br />
BBa_J32015 (<i>E. coli</i> periplasm signal peptide)-structure peptide (ubiquitin or 1L2Y [BBa_K1459015]) - lanthanide binding tag.</br><br />
The plan was to create a small, 'rubbish' protein which would only bind lanthanides without having any physiological function in cell (since we were afraid whether over-expression of PmrB would be cytotoxic).</br><br />
<a href="https://2014.igem.org/Team:Warsaw/EXTRAS"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<hr noshade="noshade" /></div>
ASamsel
http://2014.igem.org/Team:Warsaw/Project
Team:Warsaw/Project
2014-10-17T22:24:44Z
<p>ASamsel: </p>
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<a href="https://2014.igem.org/Team:Warsaw">Project</a><br />
<ul><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#background">Background</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#proof_of_concept">Proof of concept</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#detailed_explanation">Detailed explanation</a></li> <br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#modelling">Modelling</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#weeestudy">WEEE study</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#safety">Safety</a></li> <br />
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<li><a href="https://2014.igem.org/Team:Warsaw/EXTRAS#discussion">Discussion</a></li><br />
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<br />
<br />
<h1>The Project</h1></br><br />
<hr noshade="noshade" /><br />
<br />
<a name="parts"><h2>Background</h2></a></br><br />
<p align="justify"><br />
Lanthanides are a series of fifteen chemical elements with atomic numbers 57 through 71, from lanthanum to lutetium. All but one of the lanthanides are f-block elements, corresponding to the filling of the 4f electron shell (lutetium is a d-block element). The presence of f orbitals is responsible for their unique properties such as strong paramagnetism.</br><br />
<img src="https://static.igem.org/mediawiki/2014/8/8f/600px-Lanthanides1.jpg" alt="Something went straight to Hell" width="600" height="169" /></br><br />
They are required in a variety of modern technologies, such as electronics, aviation (eg. jet engines) and superconductors</br><br />
In spite of their name 'rare earths metals' their deposits on Earth are not scarce but very dispersed. Hence, there are only a few places (eg. China) where their concentration is high enough to be exploited on a commercial scale. Due to that situation the lanthanides market is not getting any better, especially taking into account that demand on these metals is still on increase.</br><br />
<img src="https://static.igem.org/mediawiki/2014/6/63/REE_world_deposits_map.jpeg" alt="Something went straight to Hell" width="751" height="307" /></br><br />
1. Worldwide deposits of rare earths elements [1].</br><br />
<img src="https://static.igem.org/mediawiki/2014/3/37/World_deposits_of_REE_graph.png" alt="Something went straight to Hell" width="601" height="401" /></br><br />
2. Total deposits of rare earth metals [1].</br><br />
Other problems with lanthanides avaibility are their difficult extraction from the ore. Most of lanthanides appear together and very similar physical properties make extraction and purification difficult. Despite heavy costs and technical difficulties lanthanides are usually purified by ion-exchange chromatography.</br><br />
[1] Chapter 6, Kołodyńska D., Hubicki Z., <i>Investigation of Sorption and Separation of Lanthanides on the Ion Exchangers of Various Types</i>, <i>Ion exchange technologies</i>, edited by Ayben Kilislioğlu, Published: November 7, 2012 under CC BY 3.0 license</br><br />
</p><br />
<hr noshade="noshade" /><br />
<br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<br />
<a name="proof_of_concept"><h2>Proof of concept</h2></a></br><br />
<p align="justify"><br />
In 2013 group of prof. He from the University of Chicago published paper in <i>Journal of American Chemical Society</i> (<i>J. Am. Chem. Soc.</i> 2013 Feb 13;135(6):2037-9) in which they described thr devised lanthanide detecting system.<br />
</br><br />
To accomplish this, they engineered two-component system from <i>Salmonella enterica</i> creating the first bacteria capable of detecting lanthanides.<br />
These findings inspired us to create our bioremediating system.</br><br />
<img src="https://static.igem.org/mediawiki/2014/thumb/2/2e/ZDJ%C4%98CIE_WIKI_PMRA-PMRB.jpg/702px-ZDJ%C4%98CIE_WIKI_PMRA-PMRB.jpg" alt="Something went straight to Hell" width="350" height="300" /><br />
A general scheme of PmrA-PmrB system.<br />
</p><br />
<hr noshade="noshade" /><br />
<br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<br />
<a name="detailed_explanation"><h2>Detailed explanation</h2></a></br><br />
<p align="justify"><br />
Initially, our project was intended to have two different parts. First being a lanthanide detecting system in BioBrick standard, much like the one constructed by group of prof. He<br />
and the second being lanthanide binding/recovery system, which would bind lanthanides much more effectively than the detecting system.</br><br />
Both of these systems were based on PmrA-PmrB two-component system, native to <i>Salmonella enterica</i>. This system consists of two proteins, PmrA and PmrB. PmrB is a transmembrane kinase with iron (III) binding motif on its extracellular loop.<br />
When iron (III) is bound to this tag, PmrB gains kinase activity and phosphorylates PmrA. PmrA is a transctriptoral factor and, upon activation, binds to pmr<sup>C</sup> promoter and induces expression of CheZ, a chemotaxis protein.</br><br />
So much for native systems.</br><br />
</p><br />
<br />
<h4>Design</h4><br />
<p align="justify"><br />
<b>Detecting system</b></br><br />
Our detecting system is planned as follows:</br><br />
Iron binding tag would be replaced with lanthanide binding tag (of which a various collection can be find in literature) and a reporter protein would be inserted downstream of pmr<sup>C</sup>. Thus, in the presence of lanthanides, fluorescence of GFP should be observed.</br><br />
<b>Binding system</b></br><br />
Binding system has more complicated design. PmrA-PmrB is not changed significantly, the only modification was introduction of LBT (lanthanide binding tag) instead of iron binding motif. The difference is downstream the pmrC promoter. First of all, we need to introduce some sort of binding agent, presumably a small protein.<br />
We decided to use ubiquitin or an artificial structurised peptide and combine it with a LBT to create synthetic protein capable of binding lanthanide ions. Since lanthanide cations are not transported to the bacterium cell the binding agent need to be secreted outside the cytoplasm. Hence, we planned to add a signal peptide to the N or C terminus of the protein. Such modification could allow the protein to be located in the bacterial periplasmic space.</br><br />
Another possible problem is connected with pmr<sup>C</sup>, which is a very weak promoter (even if induced by PmrA). So, even in the presence of lanthanides, expression of a binding agent could be inefficient. To overcome that, we planned to use some activating sequences to boost the expression from upon the pmr<sup>C</sup>. Our first idea was to put two subsequent inverters (based on different proteins, eg. tetR and lacI), which should alleviate the problem. Expression of binding agent is expected to be high in the presence of lanthanides and low in their absence.</br><br />
</p><br />
<table border="1"><br />
<tr><br />
<td></td><br />
<td colspan="3">Binding agent expression</td><br />
</tr><br />
<tr><br />
<td>Lanthanide presence</td><br />
<td>pmr<sup>C</sup></td><br />
<td>pmr<sup>C</sup>-inverter1</td><br />
<td>pmr<sup>C</sup>-inverter1-inverter2</td><br />
</tr><br />
<tr><br />
<td>none</td><br />
<td>zero (very low)</td><br />
<td>high</td><br />
<td>low</td><br />
</tr><br />
<tr><br />
<td>present</td><br />
<td>low</td><br />
<td>low</td><br />
<td>high</td><br />
</tr><br />
</table><br />
</br><br />
<p align="justify"><br />
This may seem like an excessive mean, but we could not have invented anything subtler.</br><br />
</p><br />
<h4>Project goals</h4><br />
<ol><br />
<li>Construction of a lanthanide sensor in the BioBrick standard</li><br />
<li>Cloning of PmrA/PmrB parts into pSB1C3 in the BioBrick standard</li><br />
<li>Construction of a lanthanide sensoring system with<br />
other LBT described in the literature </li><br />
<li>Construction of a lanthanide binding system</li><br />
</ol><br />
<hr noshade="noshade" /><br />
<br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<br />
<a name="modelling"><h2>Modelling</h2></a></br><br />
<br />
<p> Two-component systems (TCSs) are the most prevalent mechanism of transmembrane signal transduction. They control gene expression thus make bacteria respond to environmental changes and drive pathogen-host interactions. A typical TCS consists of a membrane-bound histidine kinase and<br />
a partner response regulator protein. The pmrA/pmrB system, which our team used in the project, also belongs to this class. pmrB is a histidine kinase and pmrA is a response regulator which strongly enhances expression upon binding to Pmr<sup>C</sup>. In order to understand better the mechanism of the system and to prevent any problems before starting the experiments in the wetlab we decided to create a simple model of this signaling pathway. Some other TCSs were successfully modeled before, but not the pmrA/pmrB.<br />
</p><br />
<br />
<b>The model</b><br />
<br />
<p> When designing our model we assumed the following pathway:<br />
<ol><br />
<li> lanthanide ion binds to the pmrB protein which leads to its autophosphorylation, </li><br />
<li> phosphorylated pmrB transfers the phosphate group onto pmrA </li><br />
<li> phosphorylated pmrA binds to pmrC and initiate expression of the reporter GFP protein </li><br />
<li> dephosphorylated pmrB induces pmrA dephoshporylation </li><br />
Additionally for model to work properly feedback loop in which phoshporylated pmrA induces pmrA expression is needed.<br />
</p><br />
<p><br />
The model diagram looks as follows:<br />
<br />
<img src="https://static.igem.org/mediawiki/2014/b/b4/Warsaw_pathway.png" width=780px alt="Signaling pathway" /><br />
<br />
</ol><br />
</p><br />
<br />
<p> We concluded that quantities of observed species change according to these equations:<br><br><br><br />
<img src="https://static.igem.org/mediawiki/2014/3/34/Warsaw_dpmrnaprmb.gif" alt="Equation 1" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/1/1f/Mrnapmra2.gif" alt="Equation 2" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/3/33/Warsaw_eq3.gif" alt="Equation 3" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/5/52/Warsaw_eq4.gif" alt="Equation 4" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/2/20/Warsaw_eq5.gif" alt="Equation 5" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/4/48/Warsaw_eq6.gif" alt="Equation 6" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/3/3b/Warsaw_eq7.gif" alt="Equation 7" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/c/c1/Mrnarp2.gif" alt="Equation 8" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/c/c3/Warsaw_eq9.gif" alt="Equation 9" /><br><br><br></br><br />
<br />
<br />
where:<br />
<ul><br />
<li> <i>mRNApmrB</i> is concentration of pmrB mRNA, the same goes for <i>mRNApmrA</i> and <i>mRNARP</i>, </li><br />
<li> <i>L</i> is lanthanide concentration, </li><br />
<li> <i>RP</i> is reporter protein concentration, </li><br />
<li> <i>pmrB.bound</i> is <i>pmrB</i> with lanthanide ion bound, </li><br />
<li> <i>prmB.bound.ph</i> is phosphorylated <i>pmrB</i> with lanthanide ion bound, </li><br />
<li> <i>pmrA.ph</i> is phosphorylated pmrA, </li><br />
<li> <i>ABComplex</i> is complex of <i>pmrA</i> and <i>pmrB.bound.ph</i> during <i>pmrA</i> phosphorylation, </li><br />
<li> <i>AComplex</i>, <i>RPComplex</i> are <i>pmrA.ph</i> inductors bound to respective promoters, </li><br />
<li> <i>ABRevComplex</i> is complex of <i>pmrA.ph</i> and <i>pmrB</i> during <i>pmrA</i> dephosphorylation </li><br />
</ul><br />
</p><br />
<br><br><br />
<br />
<br />
<b> The parameters </b><br />
<p><br />
Initial parameters were found in literature as we did not make independent component measures.<br />
</p><br />
<b> Simulation and results </b><br />
<p><br />
Deterministic simulations were performed using TinkerCell software. There are few bugs in it, but it allows for fast model building and makes changes to the model quite easy. <br />
Simulation showed that signal greatly enhances GFP expression, the its growth is exponential and correlate positively with increased concentration of lanthanide ions.<br />
</p><p><br />
GFP level when there is no lanthanide ions:<br />
<img src="https://static.igem.org/mediawiki/2014/f/f8/Warsaw_noL.png" width=780px alt="Chart" /><br />
</p><br />
<p><br />
GFP levels with 100 um of ions:<br />
<img src="https://static.igem.org/mediawiki/2014/c/c0/Warsaw_yesL.png" width=780px alt="Chart" /><br />
</p><br />
</br><br />
</br><br />
<b> References </b><br />
<p><br />
<i> Kierzek AM, Zhou L, Wanner BL. Stochastic kinetic model of two compo-<br />
nent system signalling reveals all-or-none, graded and mixed mode stochastic switching responses. Mol Biosyst. 2010;6(3):531-42<br />
</i><br />
</p><br />
<hr noshade="noshade" /><br />
<br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<br />
<a name="weeestudy"><h2>WEEE study</h2></a></br><br />
WEEE stands for ‘Waste Electrical and Electronic Equipment’ such as computers, mobile phones, TV-sets and fridges.</br> <br />
Modern electronic products contain up to 60 elements, many of them are very valuable. The most complex of it is usually presented in printed wiring boards. Metals represent on average 23% of weight of the phone, in majority copper. Single mobile phone can contain up to 9 g Cu, 250 mg Ag, 24mg Au and 0,5 mg Tb. <br />
<center><img src="https://static.igem.org/mediawiki/2014/b/b9/Periodic_table.jpg" alt="periodic_table_itw"width="751"height="350" /></center></br></br><br />
<i>Material content mobile phone [Umicore 2008]</i></br> <br />
It seems to be not too much, but we have to remember how much WEEE average European citizen products.</br> <br />
<center><img src="https://static.igem.org/mediawiki/2014/7/71/Weee_collection-page-001.jpg" alt="weee_collection"width="550"height="500" /></center></br><br />
By 2014 number of active cell phones will reach 7.3 billion. It gives 375 000 tons of Tb. All of it can be stored on landfill or recycled. Many of e-waste is transported to China, Ghana or Pakistan where fly dumpling is cheaper and the law is not as strict as European or American one.</br> <br />
The developing countries become toxic yard for e-waste. Heavy metals and toxins leak trough landfills into waterways, poisoing local people.<br />
<hr noshade="noshade" /><br />
<br />
<br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<br />
<a name="safety"><h2>Safety</h2></a></br><br />
<h3>1. Introduction</h3><br />
<p align="justify"><br />
We at Team Warsaw understand the need for good safety training and biosafe conduct in the lab.<br />
In the following sections, we will show you how we went about making sure we didn't put anyone at an unnecessary risk either at our faculty or in the outside world.<br />
</p><br />
<h3>2. (Bio)safe conduct</h3><br />
<p align="justify"><br />
Before summer, our work in the lab began with a safety training provided by our instructors. We were trained <br />
in accordance with the biosafety guidelines of our institution, focusing on lab-practical aspects of biosafety,<br />
i.e. where to work with bacteria, to always do it in the same place, to account for where the bacteria-containing material is being put, to always disinfect the immediate vicinity of your workbench once the work is finished, etc. We were also taught to properly store biological material, such as bacterial broths imbued with colonies, waste agar plates, or pipette tips and plastic tubes, and handle them in a manner suitable for preventing the spread of bacteria. Whenever some biological material-containing glass, as flasks or tubes, was broken into pieces, the adjacent area was mopped dry and disinfected with ethanol,<br />
and broken glass was stored in a separate container for glass, but only after possible remains of liquids have been removed by mopping and the pieces were disinfected by spraying with 70% ethanol.<br />
</p><br />
<p align="justify"><br />
Usually, we worked with DNA constructs so it was a matter of keeping everything else out the workplace (i.e. every type of contamination). Therefore, we had a set place for bacterial work on a bench, which was cleaned after each use, always worked with bacteria under conditions of closed windows and burner turned on, always worked in non-reusable gloves, which were disinfected with 70% ethanol at the start of work, proceeded to disinfect them regularly, stored contaminated plastic and liquids in separate containers suitable for autoclaving under standard conditions (which is taken care of by our Institute) and removed contaminated materials from our lab on a weekly basis. Whenever working with bacteria, we also refrained from touching objects outside the workbench (to prevent the possible spread of bacteria) and disinfected the working place using 70% ethanol.Of course, since our DNA constructs often carried antibiotic resistance gene, we took particular care to make sure all of our liquids remained in their respective tubes and cleaned the leakage places with 70% ethanol whenever these happened. In terms of work with hazardous substances, the only one we encountered was the ethidium bromide (used when working with agarose gels): to avoid any skin contact we always used gloves and we worked on a separate bench, devoted to ethidium bromide. Gels after visualization were stored in a separate container to be destroyed.<br />
</p><br />
<h3>3. Biosafety level</h3><br />
<p align="justify"><br />
In this year's project, we used the K-12 <i>E. coli</i> bacteria as chassis for our proteins: which are PmrA, PmrB and GFP. All the proteins we worked on, as well as with <i>Escherichia</i> chassis are harmless (non-pathogenic)<br />
to humans, which allowed for classification of our work at biosafety level 1 according to<br />
<a href="http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf?ua=1">WHO</a>.<br />
We therefore worked with our bacteria and constructs on ordinary open top benches, using Bunsen burners for sterility whenever aliquoting media, imbuing overnight cultures or sowing onto agar plates.<br />
We used <i>Salmonella enterica</i> subsp. <i>enterica</i> ser. Typhimurium, which is technically a biosafety level 2 organism, as source of parts isolated by PCR. However, we worked with a non-pathogenic, attenuated<br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/15063560">&#967;3987</a> <i>Salmonella enterica</i> subsp. <i>enterica</i> ser. Typhimurium carrying an <i>asd</i> (aspartate dehydrogenase) gene from <i>E.coli</i> on the p3342 plasmid, as derived from the <i>Salmonella Typhimurium</i> <a href="http://www.ncbi.nlm.nih.gov/pubmed/21622747">UK-1 strain</a>. This strain is non-virulent (#916;crp, #916;cya), hence provides no risk to personal or community health. Even though the genomic DNA isolation was a one-time operation, we still performed all manual activities under a suitable biological safety cabinet.</p><p align="justify"><br />
As regarding the parts isolated, i.e. proteins coded by the BasR (PmrA) and BasS (PmrB) genes, they are said to partake in the <i>Salmonella</i> virulence. Nonetheless, these two proteins merely regulate expression of the genes, whose products (usually LPS-modifyingenzymes) take part in virulence processes and so, "our" proteins are not involved in these processes themselves. We also used a PmrC/GFP construct-carrying plasmid (which we were glad to have received from<br />
<a href="http://he-group.uchicago.edu/">Prof. Chuan He's group</a> at the University of Chicago), whereby GFP was expressed from a PmrA-induced PmrC promoter. The PmrC gene codes for the phosphoethanolaminetransferase enzyme, which is required for Salmonella resistance to polymyxin. However, the plasmid we used was not carrying PmrC gene, but only the mentioned promoter which [the promoter] does not constitute a biosafety risk and neither does the GFP protein.<br />
</p><br />
<h3>4. Safety forms</h3><br />
<p align="justify"><br />
We submitted our <a href="https://igem.org/Safety/About_Our_Lab?team_id=1459">About Our Lab</a> form as well as the<br />
<a href="https://igem.org/Safety/Safety_Form?team_id=1459">Safety Form</a>, which can be found by clicking<br />
the hyperlinks on their respective names. We did not, however, need to fill out any Check-Ins, as neither of our parts nor the chassis fell under the required categories. The White List of parts<br />
and organisms and their Check-In necessity status can be found on the website of the<br />
<a href="https://2014.igem.org/Safety/White_List"> Safety Hub</a>.<br />
</p><br />
<h3>5. Environmental concerns</h3><br />
<p align="justify"><br />
The question about potential environmental concerns of our project was central to our attempts. However, due to the nature of the proteins expressed, our chassis bacteria have not acquired any characteristics that would enable them to compromise human immune system/other systems<br />
or evade detection and destruction by the former or facilitate spread between people/animals, which makes them harmless from both a personal and public health point of view.<br />
At the same time, neither the proteins encoded themselves, nor the functionality of the lanthanide detecting/binding system as a whole, imbue the bacteria with characteristics that would convey<br />
an evolutionary advantage against other organisms in the environment, both microorganisms and plants or animals, or act as toxins against the aforementioned, making the bacteria modified with the PmrA/PmrB system environmentally biosafe with no risk or them dominating any ecological niche. Our modified bacteria have, however, survival capabilities comparable to the wildtype ones.<br />
There must be the point stressed, however, that since the transformed bacteria carry<br />
a chloramphenicol resistance-encoding plasmid, the actual biosafety of the detection/binding system (i.e. prevention of HGT of the antibiotic resistance between the modified and wildtype bacteria) and so - the potential impact on the environment - depends greatly on the design of the bioreactor and the technological process, to minimize, or best prevent, the influx and efflux<br />
of non-transformed bacteria, and microorganisms in general, into the reactor.<br />
To sum up, our bacteria are not toxic towards either humans, plants, animals or other microorganisms, making them both biosafe both environmentally and health-wise. However, they can survive in the environment just as well as the wildtype bacteria, therefore the potential technological process of lanthanide detection and recycling must be optimized (esp. in the terms of preventing GMO bacteria efflux from the bioreactor) to prevent HGT and efflux of the acceptor bacteria back into the environment.<br />
</p><br />
<br />
<hr noshade="noshade" /><br />
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<br />
<a name="possibilities_of_development"><h2>Possibilities of development</h2></a></br><br />
<p align="justify"><br />
We envisage two opportunities allowing our project to be improved. First, to test more LBTs described in literature (or even design new ones) and second to create more effective binding systems.</br><br />
Furthermore, we considered utilising some kind of sulphur bacterias instead of <i>E. coli</i>. Their sulphur-based metabolism and ability to survive in low pH (in which metal leaching is more efficient) makes them excellent candidates for industrial application of our project.</br><br />
Another thing which is worth investigating: our system should not be present in bacterias as plasmids. It could be interesting to integrate it with bacteria genome, so it would be more stable within bacteria. We briefly investigated applying pMAT plasmid (known for it's remarkable stability in bacterial cells) in our project to fix the problem of 'deevolution' and eradication of our construct plasmids from bacterias. We also planned to construct a BioBrick compatible version of pMAT, but had to scratch the idea because of lack of time.</br><br />
</p><br />
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2014-10-17T22:15:48Z
<p>ASamsel: </p>
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<a href="https://2014.igem.org/Team:Warsaw">Project</a><br />
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<li><a href="https://2014.igem.org/Team:Warsaw/Project#background">Background</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#proof_of_concept">Proof of concept</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#detailed_explanation">Detailed explanation</a></li> <br />
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<li><a href="https://2014.igem.org/Team:Warsaw/Team#members">Members</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Team#advisors">Advisors</a></li><br />
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<li><a href="https://2014.igem.org/Team:Warsaw/Team#acknowledgements">Acknowledgements</a></li><br />
</ul><br />
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<div class="main-content"><br />
<br />
<br />
<h1>The Project</h1></br><br />
<hr noshade="noshade" /><br />
<br />
<a name="parts"><h2>Background</h2></a></br><br />
<p align="justify"><br />
Lanthanides are a series of fifteen chemical elements with atomic numbers 57 through 71, from lanthanum to lutetium. All but one of the lanthanides are f-block elements, corresponding to the filling of the 4f electron shell (lutetium is a d-block element). The presence of f orbitals is responsible for their unique properties such as strong paramagnetism.</br><br />
<img src="https://static.igem.org/mediawiki/2014/8/8f/600px-Lanthanides1.jpg" alt="Something went straight to Hell" width="600" height="169" /></br><br />
They are required in a variety of modern technologies, such as electronics, aviation (eg. jet engines) and superconductors</br><br />
In spite of their name 'rare earths metals' their deposits on Earth are not scarce but very dispersed. Hence, there are only a few places (eg. China) where their concentration is high enough to be exploited on a commercial scale. Due to that situation the lanthanides market is not getting any better, especially taking into account that demand on these metals is still on increase.</br><br />
<img src="https://static.igem.org/mediawiki/2014/6/63/REE_world_deposits_map.jpeg" alt="Something went straight to Hell" width="751" height="307" /></br><br />
1. Worldwide deposits of rare earths elements [1].</br><br />
<img src="https://static.igem.org/mediawiki/2014/3/37/World_deposits_of_REE_graph.png" alt="Something went straight to Hell" width="601" height="401" /></br><br />
2. Total deposits of rare earth metals [1].</br><br />
Other problems with lanthanides avaibility are their difficult extraction from the ore. Most of lanthanides appear together and very similar physical properties make extraction and purification difficult. Despite heavy costs and technical difficulties lanthanides are usually purified by ion-exchange chromatography.</br><br />
[1] Chapter 6, Kołodyńska D., Hubicki Z., <i>Investigation of Sorption and Separation of Lanthanides on the Ion Exchangers of Various Types</i>, <i>Ion exchange technologies</i>, edited by Ayben Kilislioğlu, Published: November 7, 2012 under CC BY 3.0 license</br><br />
</p><br />
<hr noshade="noshade" /><br />
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<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
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<a name="proof_of_concept"><h2>Proof of concept</h2></a></br><br />
<p align="justify"><br />
In 2013 group of prof. He from the University of Chicago published paper in <i>Journal of American Chemical Society</i> (<i>J. Am. Chem. Soc.</i> 2013 Feb 13;135(6):2037-9) in which they described thr devised lanthanide detecting system.<br />
</br><br />
To accomplish this, they engineered two-component system from <i>Salmonella enterica</i> creating the first bacteria capable of detecting lanthanides.<br />
These findings inspired us to create our bioremediating system.</br><br />
<img src="https://static.igem.org/mediawiki/2014/thumb/2/2e/ZDJ%C4%98CIE_WIKI_PMRA-PMRB.jpg/702px-ZDJ%C4%98CIE_WIKI_PMRA-PMRB.jpg" alt="Something went straight to Hell" width="350" height="300" /><br />
A general scheme of PmrA-PmrB system.<br />
</p><br />
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<a name="detailed_explanation"><h2>Detailed explanation</h2></a></br><br />
<p align="justify"><br />
Initially, our project was intended to have two different parts. First being a lanthanide detecting system in BioBrick standard, much like the one constructed by group of prof. He<br />
and the second being lanthanide binding/recovery system, which would bind lanthanides much more effectively than the detecting system.</br><br />
Both of these systems were based on PmrA-PmrB two-component system, native to <i>Salmonella enterica</i>. This system consists of two proteins, PmrA and PmrB. PmrB is a transmembrane kinase with iron (III) binding motif on its extracellular loop.<br />
When iron (III) is bound to this tag, PmrB gains kinase activity and phosphorylates PmrA. PmrA is a transctriptoral factor and, upon activation, binds to pmr<sup>C</sup> promoter and induces expression of CheZ, a chemotaxis protein.</br><br />
So much for native systems.</br><br />
</p><br />
<br />
<h4>Design</h4><br />
<p align="justify"><br />
<b>Detecting system</b></br><br />
Our detecting system is planned as follows:</br><br />
Iron binding tag would be replaced with lanthanide binding tag (of which a various collection can be find in literature) and a reporter protein would be inserted downstream of pmr<sup>C</sup>. Thus, in the presence of lanthanides, fluorescence of GFP should be observed.</br><br />
<b>Binding system</b></br><br />
Binding system has more complicated design. PmrA-PmrB is not changed significantly, the only modification was introduction of LBT (lanthanide binding tag) instead of iron binding motif. The difference is downstream the pmrC promoter. First of all, we need to introduce some sort of binding agent, presumably a small protein.<br />
We decided to use ubiquitin or an artificial structurised peptide and combine it with a LBT to create synthetic protein capable of binding lanthanide ions. Since lanthanide cations are not transported to the bacterium cell the binding agent need to be secreted outside the cytoplasm. Hence, we planned to add a signal peptide to the N or C terminus of the protein. Such modification could allow the protein to be located in the bacterial periplasmic space.</br><br />
Another possible problem is connected with pmr<sup>C</sup>, which is a very weak promoter (even if induced by PmrA). So, even in the presence of lanthanides, expression of a binding agent could be inefficient. To overcome that, we planned to use some activating sequences to boost the expression from upon the pmr<sup>C</sup>. Our first idea was to put two subsequent inverters (based on different proteins, eg. tetR and lacI), which should alleviate the problem. Expression of binding agent is expected to be high in the presence of lanthanides and low in their absence.</br><br />
</p><br />
<table border="1"><br />
<tr><br />
<td></td><br />
<td colspan="3">Binding agent expression</td><br />
</tr><br />
<tr><br />
<td>Lanthanide presence</td><br />
<td>pmr<sup>C</sup></td><br />
<td>pmr<sup>C</sup>-inverter1</td><br />
<td>pmr<sup>C</sup>-inverter1-inverter2</td><br />
</tr><br />
<tr><br />
<td>none</td><br />
<td>zero (very low)</td><br />
<td>high</td><br />
<td>low</td><br />
</tr><br />
<tr><br />
<td>present</td><br />
<td>low</td><br />
<td>low</td><br />
<td>high</td><br />
</tr><br />
</table><br />
</br><br />
<p align="justify"><br />
This may seem like an excessive mean, but we could not have invented anything subtler.</br><br />
</p><br />
<h4>Project goals</h4><br />
<ol><br />
<li>Construction of a lanthanide sensor in the BioBrick standard</li><br />
<li>Cloning of PmrA/PmrB parts into pSB1C3 in the BioBrick standard</li><br />
<li>Construction of a lanthanide sensoring system with<br />
other LBT described in the literature </li><br />
<li>Construction of a lanthanide binding system</li><br />
</ol><br />
<hr noshade="noshade" /><br />
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<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
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<a name="modelling"><h2>Modelling</h2></a></br><br />
<br />
<p> Two-component systems (TCSs) are the most prevalent mechanism of transmembrane signal transduction. They control gene expression thus make bacteria respond to environmental changes and drive pathogen-host interactions. A typical TCS consists of a membrane-bound histidine kinase and<br />
a partner response regulator protein. The pmrA/pmrB system, which our team used in the project, also belongs to this class. pmrB is a histidine kinase and pmrA is a response regulator which strongly enhances expression upon binding to Pmr<sup>C</sup>. In order to understand better the mechanism of the system and to prevent any problems before starting the experiments in the wetlab we decided to create a simple model of this signaling pathway. Some other TCSs were successfully modeled before, but not the pmrA/pmrB.<br />
</p><br />
<br />
<b>The model</b><br />
<br />
<p> When designing our model we assumed the following pathway:<br />
<ol><br />
<li> lanthanide ion binds to the pmrB protein which leads to its autophosphorylation, </li><br />
<li> phosphorylated pmrB transfers the phosphate group onto pmrA </li><br />
<li> phosphorylated pmrA binds to pmrC and initiate expression of the reporter GFP protein </li><br />
<li> dephosphorylated pmrB induces pmrA dephoshporylation </li><br />
Additionally for model to work properly feedback loop in which phoshporylated pmrA induces pmrA expression is needed.<br />
</p><br />
<p><br />
The model diagram looks as follows:<br />
<br />
<img src="https://static.igem.org/mediawiki/2014/b/b4/Warsaw_pathway.png" width=780px alt="Signaling pathway" /><br />
<br />
</ol><br />
</p><br />
<br />
<p> We concluded that quantities of observed species change according to these equations:<br><br><br><br />
<img src="https://static.igem.org/mediawiki/2014/3/34/Warsaw_dpmrnaprmb.gif" alt="Equation 1" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/1/1f/Mrnapmra2.gif" alt="Equation 2" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/3/33/Warsaw_eq3.gif" alt="Equation 3" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/5/52/Warsaw_eq4.gif" alt="Equation 4" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/2/20/Warsaw_eq5.gif" alt="Equation 5" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/4/48/Warsaw_eq6.gif" alt="Equation 6" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/3/3b/Warsaw_eq7.gif" alt="Equation 7" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/c/c1/Mrnarp2.gif" alt="Equation 8" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/c/c3/Warsaw_eq9.gif" alt="Equation 9" /><br><br><br></br><br />
<br />
<br />
where:<br />
<ul><br />
<li> <i>mRNApmrB</i> is concentration of pmrB mRNA, the same goes for <i>mRNApmrA</i> and <i>mRNARP</i>, </li><br />
<li> <i>L</i> is lanthanide concentration, </li><br />
<li> <i>RP</i> is reporter protein concentration, </li><br />
<li> <i>pmrB.bound</i> is <i>pmrB</i> with lanthanide ion bound, </li><br />
<li> <i>prmB.bound.ph</i> is phosphorylated <i>pmrB</i> with lanthanide ion bound, </li><br />
<li> <i>pmrA.ph</i> is phosphorylated pmrA, </li><br />
<li> <i>ABComplex</i> is complex of <i>pmrA</i> and <i>pmrB.bound.ph</i> during <i>pmrA</i> phosphorylation, </li><br />
<li> <i>AComplex</i>, <i>RPComplex</i> are <i>pmrA.ph</i> inductors bound to respective promoters, </li><br />
<li> <i>ABRevComplex</i> is complex of <i>pmrA.ph</i> and <i>pmrB</i> during <i>pmrA</i> dephosphorylation </li><br />
</ul><br />
</p><br />
<br><br><br />
<br />
<br />
<b> The parameters </b><br />
<p><br />
Initial parameters were found in literature as we did not make independent component measures.<br />
</p><br />
<b> Simulation and results </b><br />
<p><br />
Deterministic simulations were performed using TinkerCell software. There are few bugs in it, but it allows for fast model building and makes changes to the model quite easy. <br />
Simulation showed that signal greatly enhances GFP expression, the its growth is exponential and correlate positively with increased concentration of lanthanide ions.<br />
</p><p><br />
GFP level when there is no lanthanide ions:<br />
<img src="https://static.igem.org/mediawiki/2014/f/f8/Warsaw_noL.png" width=780px alt="Chart" /><br />
</p><br />
<p><br />
GFP levels with 100 um of ions:<br />
<img src="https://static.igem.org/mediawiki/2014/c/c0/Warsaw_yesL.png" width=780px alt="Chart" /><br />
</p><br />
</br><br />
</br><br />
<b> References </b><br />
<p><br />
<i> Kierzek AM, Zhou L, Wanner BL. Stochastic kinetic model of two compo-<br />
nent system signalling reveals all-or-none, graded and mixed mode stochastic switching responses. Mol Biosyst. 2010;6(3):531-42<br />
</i><br />
</p><br />
<hr noshade="noshade" /><br />
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<br />
<a name="weeestudy"><h2>WEEE study</h2></a></br><br />
WEEE stands for ‘Waste Electrical and Electronic Equipment’ such as computers, mobile phones, TV-sets and fridges.</br> <br />
Modern electronic products contain up to 60 elements, many of them are very valuable. The most complex of it is usually presented in printed wiring boards. Metals represent on average 23% of weight of the phone, in majority copper. Single mobile phone can contain up to 9 g Cu, 250 mg Ag, 24mg Au and 0,5 mg Tb. <br />
<center><img src="https://static.igem.org/mediawiki/2014/b/b9/Periodic_table.jpg" alt="periodic_table_itw"width="751"height="350" /></center></br></br><br />
Material content mobile phone [Umicore 2008]</br> <br />
It seems to be not too much, but we have to remember how much WEEE average European citizen products.</br> <br />
<center><img src="https://static.igem.org/mediawiki/2014/7/71/Weee_collection-page-001.jpg" alt="weee_collection"width="550"height="500" /></center></br><br />
By 2014 number of active cell phones will reach 7.3 billion. It gives 375 000 tons of Tb. All of it can be stored on landfill or recycled. Many of e-waste is transported to China, Ghana or Pakistan where fly dumpling is cheaper and the law is not as strict as European or American one.</br> <br />
The developing countries become toxic yard for e-waste. Heavy metals and toxins leak trough landfills into waterways, poisoing local people.<br />
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<a name="safety"><h2>Safety</h2></a></br><br />
<h3>1. Introduction</h3><br />
<p align="justify"><br />
We at Team Warsaw understand the need for good safety training and biosafe conduct in the lab.<br />
In the following sections, we will show you how we went about making sure we didn't put anyone at an unnecessary risk either at our faculty or in the outside world.<br />
</p><br />
<h3>2. (Bio)safe conduct</h3><br />
<p align="justify"><br />
Before summer, our work in the lab began with a safety training provided by our instructors. We were trained <br />
in accordance with the biosafety guidelines of our institution, focusing on lab-practical aspects of biosafety,<br />
i.e. where to work with bacteria, to always do it in the same place, to account for where the bacteria-containing material is being put, to always disinfect the immediate vicinity of your workbench once the work is finished, etc. We were also taught to properly store biological material, such as bacterial broths imbued with colonies, waste agar plates, or pipette tips and plastic tubes, and handle them in a manner suitable for preventing the spread of bacteria. Whenever some biological material-containing glass, as flasks or tubes, was broken into pieces, the adjacent area was mopped dry and disinfected with ethanol,<br />
and broken glass was stored in a separate container for glass, but only after possible remains of liquids have been removed by mopping and the pieces were disinfected by spraying with 70% ethanol.<br />
</p><br />
<p align="justify"><br />
Usually, we worked with DNA constructs so it was a matter of keeping everything else out the workplace (i.e. every type of contamination). Therefore, we had a set place for bacterial work on a bench, which was cleaned after each use, always worked with bacteria under conditions of closed windows and burner turned on, always worked in non-reusable gloves, which were disinfected with 70% ethanol at the start of work, proceeded to disinfect them regularly, stored contaminated plastic and liquids in separate containers suitable for autoclaving under standard conditions (which is taken care of by our Institute) and removed contaminated materials from our lab on a weekly basis. Whenever working with bacteria, we also refrained from touching objects outside the workbench (to prevent the possible spread of bacteria) and disinfected the working place using 70% ethanol.Of course, since our DNA constructs often carried antibiotic resistance gene, we took particular care to make sure all of our liquids remained in their respective tubes and cleaned the leakage places with 70% ethanol whenever these happened. In terms of work with hazardous substances, the only one we encountered was the ethidium bromide (used when working with agarose gels): to avoid any skin contact we always used gloves and we worked on a separate bench, devoted to ethidium bromide. Gels after visualization were stored in a separate container to be destroyed.<br />
</p><br />
<h3>3. Biosafety level</h3><br />
<p align="justify"><br />
In this year's project, we used the K-12 <i>E. coli</i> bacteria as chassis for our proteins: which are PmrA, PmrB and GFP. All the proteins we worked on, as well as with <i>Escherichia</i> chassis are harmless (non-pathogenic)<br />
to humans, which allowed for classification of our work at biosafety level 1 according to<br />
<a href="http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf?ua=1">WHO</a>.<br />
We therefore worked with our bacteria and constructs on ordinary open top benches, using Bunsen burners for sterility whenever aliquoting media, imbuing overnight cultures or sowing onto agar plates.<br />
We used <i>Salmonella enterica</i> subsp. <i>enterica</i> ser. Typhimurium, which is technically a biosafety level 2 organism, as source of parts isolated by PCR. However, we worked with a non-pathogenic, attenuated<br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/15063560">&#967;3987</a> <i>Salmonella enterica</i> subsp. <i>enterica</i> ser. Typhimurium carrying an <i>asd</i> (aspartate dehydrogenase) gene from <i>E.coli</i> on the p3342 plasmid, as derived from the <i>Salmonella Typhimurium</i> <a href="http://www.ncbi.nlm.nih.gov/pubmed/21622747">UK-1 strain</a>. This strain is non-virulent (#916;crp, #916;cya), hence provides no risk to personal or community health. Even though the genomic DNA isolation was a one-time operation, we still performed all manual activities under a suitable biological safety cabinet.</p><p align="justify"><br />
As regarding the parts isolated, i.e. proteins coded by the BasR (PmrA) and BasS (PmrB) genes, they are said to partake in the <i>Salmonella</i> virulence. Nonetheless, these two proteins merely regulate expression of the genes, whose products (usually LPS-modifyingenzymes) take part in virulence processes and so, "our" proteins are not involved in these processes themselves. We also used a PmrC/GFP construct-carrying plasmid (which we were glad to have received from<br />
<a href="http://he-group.uchicago.edu/">Prof. Chuan He's group</a> at the University of Chicago), whereby GFP was expressed from a PmrA-induced PmrC promoter. The PmrC gene codes for the phosphoethanolaminetransferase enzyme, which is required for Salmonella resistance to polymyxin. However, the plasmid we used was not carrying PmrC gene, but only the mentioned promoter which [the promoter] does not constitute a biosafety risk and neither does the GFP protein.<br />
</p><br />
<h3>4. Safety forms</h3><br />
<p align="justify"><br />
We submitted our <a href="https://igem.org/Safety/About_Our_Lab?team_id=1459">About Our Lab</a> form as well as the<br />
<a href="https://igem.org/Safety/Safety_Form?team_id=1459">Safety Form</a>, which can be found by clicking<br />
the hyperlinks on their respective names. We did not, however, need to fill out any Check-Ins, as neither of our parts nor the chassis fell under the required categories. The White List of parts<br />
and organisms and their Check-In necessity status can be found on the website of the<br />
<a href="https://2014.igem.org/Safety/White_List"> Safety Hub</a>.<br />
</p><br />
<h3>5. Environmental concerns</h3><br />
<p align="justify"><br />
The question about potential environmental concerns of our project was central to our attempts. However, due to the nature of the proteins expressed, our chassis bacteria have not acquired any characteristics that would enable them to compromise human immune system/other systems<br />
or evade detection and destruction by the former or facilitate spread between people/animals, which makes them harmless from both a personal and public health point of view.<br />
At the same time, neither the proteins encoded themselves, nor the functionality of the lanthanide detecting/binding system as a whole, imbue the bacteria with characteristics that would convey<br />
an evolutionary advantage against other organisms in the environment, both microorganisms and plants or animals, or act as toxins against the aforementioned, making the bacteria modified with the PmrA/PmrB system environmentally biosafe with no risk or them dominating any ecological niche. Our modified bacteria have, however, survival capabilities comparable to the wildtype ones.<br />
There must be the point stressed, however, that since the transformed bacteria carry<br />
a chloramphenicol resistance-encoding plasmid, the actual biosafety of the detection/binding system (i.e. prevention of HGT of the antibiotic resistance between the modified and wildtype bacteria) and so - the potential impact on the environment - depends greatly on the design of the bioreactor and the technological process, to minimize, or best prevent, the influx and efflux<br />
of non-transformed bacteria, and microorganisms in general, into the reactor.<br />
To sum up, our bacteria are not toxic towards either humans, plants, animals or other microorganisms, making them both biosafe both environmentally and health-wise. However, they can survive in the environment just as well as the wildtype bacteria, therefore the potential technological process of lanthanide detection and recycling must be optimized (esp. in the terms of preventing GMO bacteria efflux from the bioreactor) to prevent HGT and efflux of the acceptor bacteria back into the environment.<br />
</p><br />
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<a name="possibilities_of_development"><h2>Possibilities of development</h2></a></br><br />
<p align="justify"><br />
We envisage two opportunities allowing our project to be improved. First, to test more LBTs described in literature (or even design new ones) and second to create more effective binding systems.</br><br />
Furthermore, we considered utilising some kind of sulphur bacterias instead of <i>E. coli</i>. Their sulphur-based metabolism and ability to survive in low pH (in which metal leaching is more efficient) makes them excellent candidates for industrial application of our project.</br><br />
Another thing which is worth investigating: our system should not be present in bacterias as plasmids. It could be interesting to integrate it with bacteria genome, so it would be more stable within bacteria. We briefly investigated applying pMAT plasmid (known for it's remarkable stability in bacterial cells) in our project to fix the problem of 'deevolution' and eradication of our construct plasmids from bacterias. We also planned to construct a BioBrick compatible version of pMAT, but had to scratch the idea because of lack of time.</br><br />
</p><br />
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ASamsel
http://2014.igem.org/File:Weee_collection-page-001.jpg
File:Weee collection-page-001.jpg
2014-10-17T22:00:21Z
<p>ASamsel: weee_collection</p>
<hr />
<div>weee_collection</div>
ASamsel
http://2014.igem.org/Team:Warsaw/Project
Team:Warsaw/Project
2014-10-17T21:59:34Z
<p>ASamsel: </p>
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<ul><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#background">Background</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#proof_of_concept">Proof of concept</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#detailed_explanation">Detailed explanation</a></li> <br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#modelling">Modelling</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#weeestudy">WEEE study</a></li><br />
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<br />
<br />
<h1>The Project</h1></br><br />
<hr noshade="noshade" /><br />
<br />
<a name="parts"><h2>Background</h2></a></br><br />
<p align="justify"><br />
Lanthanides are a series of fifteen chemical elements with atomic numbers 57 through 71, from lanthanum to lutetium. All but one of the lanthanides are f-block elements, corresponding to the filling of the 4f electron shell (lutetium is a d-block element). The presence of f orbitals is responsible for their unique properties such as strong paramagnetism.</br><br />
<img src="https://static.igem.org/mediawiki/2014/8/8f/600px-Lanthanides1.jpg" alt="Something went straight to Hell" width="600" height="169" /></br><br />
They are required in a variety of modern technologies, such as electronics, aviation (eg. jet engines) and superconductors</br><br />
In spite of their name 'rare earths metals' their deposits on Earth are not scarce but very dispersed. Hence, there are only a few places (eg. China) where their concentration is high enough to be exploited on a commercial scale. Due to that situation the lanthanides market is not getting any better, especially taking into account that demand on these metals is still on increase.</br><br />
<img src="https://static.igem.org/mediawiki/2014/6/63/REE_world_deposits_map.jpeg" alt="Something went straight to Hell" width="751" height="307" /></br><br />
1. Worldwide deposits of rare earths elements [1].</br><br />
<img src="https://static.igem.org/mediawiki/2014/3/37/World_deposits_of_REE_graph.png" alt="Something went straight to Hell" width="601" height="401" /></br><br />
2. Total deposits of rare earth metals [1].</br><br />
Other problems with lanthanides avaibility are their difficult extraction from the ore. Most of lanthanides appear together and very similar physical properties make extraction and purification difficult. Despite heavy costs and technical difficulties lanthanides are usually purified by ion-exchange chromatography.</br><br />
[1] Chapter 6, Kołodyńska D., Hubicki Z., <i>Investigation of Sorption and Separation of Lanthanides on the Ion Exchangers of Various Types</i>, <i>Ion exchange technologies</i>, edited by Ayben Kilislioğlu, Published: November 7, 2012 under CC BY 3.0 license</br><br />
</p><br />
<hr noshade="noshade" /><br />
<br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<br />
<a name="proof_of_concept"><h2>Proof of concept</h2></a></br><br />
<p align="justify"><br />
In 2013 group of prof. He from the University of Chicago published paper in <i>Journal of American Chemical Society</i> (<i>J. Am. Chem. Soc.</i> 2013 Feb 13;135(6):2037-9) in which they described thr devised lanthanide detecting system.<br />
</br><br />
To accomplish this, they engineered two-component system from <i>Salmonella enterica</i> creating the first bacteria capable of detecting lanthanides.<br />
These findings inspired us to create our bioremediating system.</br><br />
<img src="https://static.igem.org/mediawiki/2014/thumb/2/2e/ZDJ%C4%98CIE_WIKI_PMRA-PMRB.jpg/702px-ZDJ%C4%98CIE_WIKI_PMRA-PMRB.jpg" alt="Something went straight to Hell" width="350" height="300" /><br />
A general scheme of PmrA-PmrB system.<br />
</p><br />
<hr noshade="noshade" /><br />
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<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<br />
<a name="detailed_explanation"><h2>Detailed explanation</h2></a></br><br />
<p align="justify"><br />
Initially, our project was intended to have two different parts. First being a lanthanide detecting system in BioBrick standard, much like the one constructed by group of prof. He<br />
and the second being lanthanide binding/recovery system, which would bind lanthanides much more effectively than the detecting system.</br><br />
Both of these systems were based on PmrA-PmrB two-component system, native to <i>Salmonella enterica</i>. This system consists of two proteins, PmrA and PmrB. PmrB is a transmembrane kinase with iron (III) binding motif on its extracellular loop.<br />
When iron (III) is bound to this tag, PmrB gains kinase activity and phosphorylates PmrA. PmrA is a transctriptoral factor and, upon activation, binds to pmr<sup>C</sup> promoter and induces expression of CheZ, a chemotaxis protein.</br><br />
So much for native systems.</br><br />
</p><br />
<br />
<h4>Design</h4><br />
<p align="justify"><br />
<b>Detecting system</b></br><br />
Our detecting system is planned as follows:</br><br />
Iron binding tag would be replaced with lanthanide binding tag (of which a various collection can be find in literature) and a reporter protein would be inserted downstream of pmr<sup>C</sup>. Thus, in the presence of lanthanides, fluorescence of GFP should be observed.</br><br />
<b>Binding system</b></br><br />
Binding system has more complicated design. PmrA-PmrB is not changed significantly, the only modification was introduction of LBT (lanthanide binding tag) instead of iron binding motif. The difference is downstream the pmrC promoter. First of all, we need to introduce some sort of binding agent, presumably a small protein.<br />
We decided to use ubiquitin or an artificial structurised peptide and combine it with a LBT to create synthetic protein capable of binding lanthanide ions. Since lanthanide cations are not transported to the bacterium cell the binding agent need to be secreted outside the cytoplasm. Hence, we planned to add a signal peptide to the N or C terminus of the protein. Such modification could allow the protein to be located in the bacterial periplasmic space.</br><br />
Another possible problem is connected with pmr<sup>C</sup>, which is a very weak promoter (even if induced by PmrA). So, even in the presence of lanthanides, expression of a binding agent could be inefficient. To overcome that, we planned to use some activating sequences to boost the expression from upon the pmr<sup>C</sup>. Our first idea was to put two subsequent inverters (based on different proteins, eg. tetR and lacI), which should alleviate the problem. Expression of binding agent is expected to be high in the presence of lanthanides and low in their absence.</br><br />
</p><br />
<table border="1"><br />
<tr><br />
<td></td><br />
<td colspan="3">Binding agent expression</td><br />
</tr><br />
<tr><br />
<td>Lanthanide presence</td><br />
<td>pmr<sup>C</sup></td><br />
<td>pmr<sup>C</sup>-inverter1</td><br />
<td>pmr<sup>C</sup>-inverter1-inverter2</td><br />
</tr><br />
<tr><br />
<td>none</td><br />
<td>zero (very low)</td><br />
<td>high</td><br />
<td>low</td><br />
</tr><br />
<tr><br />
<td>present</td><br />
<td>low</td><br />
<td>low</td><br />
<td>high</td><br />
</tr><br />
</table><br />
</br><br />
<p align="justify"><br />
This may seem like an excessive mean, but we could not have invented anything subtler.</br><br />
</p><br />
<h4>Project goals</h4><br />
<ol><br />
<li>Construction of a lanthanide sensor in the BioBrick standard</li><br />
<li>Cloning of PmrA/PmrB parts into pSB1C3 in the BioBrick standard</li><br />
<li>Construction of a lanthanide sensoring system with<br />
other LBT described in the literature </li><br />
<li>Construction of a lanthanide binding system</li><br />
</ol><br />
<hr noshade="noshade" /><br />
<br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<br />
<a name="modelling"><h2>Modelling</h2></a></br><br />
<br />
<p> Two-component systems (TCSs) are the most prevalent mechanism of transmembrane signal transduction. They control gene expression thus make bacteria respond to environmental changes and drive pathogen-host interactions. A typical TCS consists of a membrane-bound histidine kinase and<br />
a partner response regulator protein. The pmrA/pmrB system, which our team used in the project, also belongs to this class. pmrB is a histidine kinase and pmrA is a response regulator which strongly enhances expression upon binding to Pmr<sup>C</sup>. In order to understand better the mechanism of the system and to prevent any problems before starting the experiments in the wetlab we decided to create a simple model of this signaling pathway. Some other TCSs were successfully modeled before, but not the pmrA/pmrB.<br />
</p><br />
<br />
<b>The model</b><br />
<br />
<p> When designing our model we assumed the following pathway:<br />
<ol><br />
<li> lanthanide ion binds to the pmrB protein which leads to its autophosphorylation, </li><br />
<li> phosphorylated pmrB transfers the phosphate group onto pmrA </li><br />
<li> phosphorylated pmrA binds to pmrC and initiate expression of the reporter GFP protein </li><br />
<li> dephosphorylated pmrB induces pmrA dephoshporylation </li><br />
Additionally for model to work properly feedback loop in which phoshporylated pmrA induces pmrA expression is needed.<br />
</p><br />
<p><br />
The model diagram looks as follows:<br />
<br />
<img src="https://static.igem.org/mediawiki/2014/b/b4/Warsaw_pathway.png" width=780px alt="Signaling pathway" /><br />
<br />
</ol><br />
</p><br />
<br />
<p> We concluded that quantities of observed species change according to these equations:<br><br><br><br />
<img src="https://static.igem.org/mediawiki/2014/3/34/Warsaw_dpmrnaprmb.gif" alt="Equation 1" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/1/1f/Mrnapmra2.gif" alt="Equation 2" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/3/33/Warsaw_eq3.gif" alt="Equation 3" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/5/52/Warsaw_eq4.gif" alt="Equation 4" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/2/20/Warsaw_eq5.gif" alt="Equation 5" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/4/48/Warsaw_eq6.gif" alt="Equation 6" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/3/3b/Warsaw_eq7.gif" alt="Equation 7" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/c/c1/Mrnarp2.gif" alt="Equation 8" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/c/c3/Warsaw_eq9.gif" alt="Equation 9" /><br><br><br></br><br />
<br />
<br />
where:<br />
<ul><br />
<li> <i>mRNApmrB</i> is concentration of pmrB mRNA, the same goes for <i>mRNApmrA</i> and <i>mRNARP</i>, </li><br />
<li> <i>L</i> is lanthanide concentration, </li><br />
<li> <i>RP</i> is reporter protein concentration, </li><br />
<li> <i>pmrB.bound</i> is <i>pmrB</i> with lanthanide ion bound, </li><br />
<li> <i>prmB.bound.ph</i> is phosphorylated <i>pmrB</i> with lanthanide ion bound, </li><br />
<li> <i>pmrA.ph</i> is phosphorylated pmrA, </li><br />
<li> <i>ABComplex</i> is complex of <i>pmrA</i> and <i>pmrB.bound.ph</i> during <i>pmrA</i> phosphorylation, </li><br />
<li> <i>AComplex</i>, <i>RPComplex</i> are <i>pmrA.ph</i> inductors bound to respective promoters, </li><br />
<li> <i>ABRevComplex</i> is complex of <i>pmrA.ph</i> and <i>pmrB</i> during <i>pmrA</i> dephosphorylation </li><br />
</ul><br />
</p><br />
<br><br><br />
<br />
<br />
<b> The parameters </b><br />
<p><br />
Initial parameters were found in literature as we did not make independent component measures.<br />
</p><br />
<b> Simulation and results </b><br />
<p><br />
Deterministic simulations were performed using TinkerCell software. There are few bugs in it, but it allows for fast model building and makes changes to the model quite easy. <br />
Simulation showed that signal greatly enhances GFP expression, the its growth is exponential and correlate positively with increased concentration of lanthanide ions.<br />
</p><p><br />
GFP level when there is no lanthanide ions:<br />
<img src="https://static.igem.org/mediawiki/2014/f/f8/Warsaw_noL.png" width=780px alt="Chart" /><br />
</p><br />
<p><br />
GFP levels with 100 um of ions:<br />
<img src="https://static.igem.org/mediawiki/2014/c/c0/Warsaw_yesL.png" width=780px alt="Chart" /><br />
</p><br />
</br><br />
</br><br />
<b> References </b><br />
<p><br />
<i> Kierzek AM, Zhou L, Wanner BL. Stochastic kinetic model of two compo-<br />
nent system signalling reveals all-or-none, graded and mixed mode stochastic switching responses. Mol Biosyst. 2010;6(3):531-42<br />
</i><br />
</p><br />
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<a name="weeestudy"><h2>WEEE study</h2></a></br><br />
WEEE stands for ‘Waste Electrical and Electronic Equipment’ such as computers, mobile phones, TV-sets and fridges.</br> <br />
Modern electronic products contain up to 60 elements, many of them are very valuable. The most complex of it is usually presented in printed wiring boards. Metals represent on average 23% of weight of the phone, in majority copper. Single mobile phone can contain up to 9 g Cu, 250 mg Ag, 24mg Au and 0,5 mg Tb. <br />
<center><img src="https://static.igem.org/mediawiki/2014/b/b9/Periodic_table.jpg" alt="periodic_table_itw"width="751"height="350" /></center></br> <br />
Material content mobile phone [Umicore 2008]</br> <br />
It seems to be not too much, but we have to remember how much WEEE average European citizen products.</br> <br />
<br />
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<a name="safety"><h2>Safety</h2></a></br><br />
<h3>1. Introduction</h3><br />
<p align="justify"><br />
We at Team Warsaw understand the need for good safety training and biosafe conduct in the lab.<br />
In the following sections, we will show you how we went about making sure we didn't put anyone at an unnecessary risk either at our faculty or in the outside world.<br />
</p><br />
<h3>2. (Bio)safe conduct</h3><br />
<p align="justify"><br />
Before summer, our work in the lab began with a safety training provided by our instructors. We were trained <br />
in accordance with the biosafety guidelines of our institution, focusing on lab-practical aspects of biosafety,<br />
i.e. where to work with bacteria, to always do it in the same place, to account for where the bacteria-containing material is being put, to always disinfect the immediate vicinity of your workbench once the work is finished, etc. We were also taught to properly store biological material, such as bacterial broths imbued with colonies, waste agar plates, or pipette tips and plastic tubes, and handle them in a manner suitable for preventing the spread of bacteria. Whenever some biological material-containing glass, as flasks or tubes, was broken into pieces, the adjacent area was mopped dry and disinfected with ethanol,<br />
and broken glass was stored in a separate container for glass, but only after possible remains of liquids have been removed by mopping and the pieces were disinfected by spraying with 70% ethanol.<br />
</p><br />
<p align="justify"><br />
Usually, we worked with DNA constructs so it was a matter of keeping everything else out the workplace (i.e. every type of contamination). Therefore, we had a set place for bacterial work on a bench, which was cleaned after each use, always worked with bacteria under conditions of closed windows and burner turned on, always worked in non-reusable gloves, which were disinfected with 70% ethanol at the start of work, proceeded to disinfect them regularly, stored contaminated plastic and liquids in separate containers suitable for autoclaving under standard conditions (which is taken care of by our Institute) and removed contaminated materials from our lab on a weekly basis. Whenever working with bacteria, we also refrained from touching objects outside the workbench (to prevent the possible spread of bacteria) and disinfected the working place using 70% ethanol.Of course, since our DNA constructs often carried antibiotic resistance gene, we took particular care to make sure all of our liquids remained in their respective tubes and cleaned the leakage places with 70% ethanol whenever these happened. In terms of work with hazardous substances, the only one we encountered was the ethidium bromide (used when working with agarose gels): to avoid any skin contact we always used gloves and we worked on a separate bench, devoted to ethidium bromide. Gels after visualization were stored in a separate container to be destroyed.<br />
</p><br />
<h3>3. Biosafety level</h3><br />
<p align="justify"><br />
In this year's project, we used the K-12 <i>E. coli</i> bacteria as chassis for our proteins: which are PmrA, PmrB and GFP. All the proteins we worked on, as well as with <i>Escherichia</i> chassis are harmless (non-pathogenic)<br />
to humans, which allowed for classification of our work at biosafety level 1 according to<br />
<a href="http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf?ua=1">WHO</a>.<br />
We therefore worked with our bacteria and constructs on ordinary open top benches, using Bunsen burners for sterility whenever aliquoting media, imbuing overnight cultures or sowing onto agar plates.<br />
We used <i>Salmonella enterica</i> subsp. <i>enterica</i> ser. Typhimurium, which is technically a biosafety level 2 organism, as source of parts isolated by PCR. However, we worked with a non-pathogenic, attenuated<br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/15063560">&#967;3987</a> <i>Salmonella enterica</i> subsp. <i>enterica</i> ser. Typhimurium carrying an <i>asd</i> (aspartate dehydrogenase) gene from <i>E.coli</i> on the p3342 plasmid, as derived from the <i>Salmonella Typhimurium</i> <a href="http://www.ncbi.nlm.nih.gov/pubmed/21622747">UK-1 strain</a>. This strain is non-virulent (#916;crp, #916;cya), hence provides no risk to personal or community health. Even though the genomic DNA isolation was a one-time operation, we still performed all manual activities under a suitable biological safety cabinet.</p><p align="justify"><br />
As regarding the parts isolated, i.e. proteins coded by the BasR (PmrA) and BasS (PmrB) genes, they are said to partake in the <i>Salmonella</i> virulence. Nonetheless, these two proteins merely regulate expression of the genes, whose products (usually LPS-modifyingenzymes) take part in virulence processes and so, "our" proteins are not involved in these processes themselves. We also used a PmrC/GFP construct-carrying plasmid (which we were glad to have received from<br />
<a href="http://he-group.uchicago.edu/">Prof. Chuan He's group</a> at the University of Chicago), whereby GFP was expressed from a PmrA-induced PmrC promoter. The PmrC gene codes for the phosphoethanolaminetransferase enzyme, which is required for Salmonella resistance to polymyxin. However, the plasmid we used was not carrying PmrC gene, but only the mentioned promoter which [the promoter] does not constitute a biosafety risk and neither does the GFP protein.<br />
</p><br />
<h3>4. Safety forms</h3><br />
<p align="justify"><br />
We submitted our <a href="https://igem.org/Safety/About_Our_Lab?team_id=1459">About Our Lab</a> form as well as the<br />
<a href="https://igem.org/Safety/Safety_Form?team_id=1459">Safety Form</a>, which can be found by clicking<br />
the hyperlinks on their respective names. We did not, however, need to fill out any Check-Ins, as neither of our parts nor the chassis fell under the required categories. The White List of parts<br />
and organisms and their Check-In necessity status can be found on the website of the<br />
<a href="https://2014.igem.org/Safety/White_List"> Safety Hub</a>.<br />
</p><br />
<h3>5. Environmental concerns</h3><br />
<p align="justify"><br />
The question about potential environmental concerns of our project was central to our attempts. However, due to the nature of the proteins expressed, our chassis bacteria have not acquired any characteristics that would enable them to compromise human immune system/other systems<br />
or evade detection and destruction by the former or facilitate spread between people/animals, which makes them harmless from both a personal and public health point of view.<br />
At the same time, neither the proteins encoded themselves, nor the functionality of the lanthanide detecting/binding system as a whole, imbue the bacteria with characteristics that would convey<br />
an evolutionary advantage against other organisms in the environment, both microorganisms and plants or animals, or act as toxins against the aforementioned, making the bacteria modified with the PmrA/PmrB system environmentally biosafe with no risk or them dominating any ecological niche. Our modified bacteria have, however, survival capabilities comparable to the wildtype ones.<br />
There must be the point stressed, however, that since the transformed bacteria carry<br />
a chloramphenicol resistance-encoding plasmid, the actual biosafety of the detection/binding system (i.e. prevention of HGT of the antibiotic resistance between the modified and wildtype bacteria) and so - the potential impact on the environment - depends greatly on the design of the bioreactor and the technological process, to minimize, or best prevent, the influx and efflux<br />
of non-transformed bacteria, and microorganisms in general, into the reactor.<br />
To sum up, our bacteria are not toxic towards either humans, plants, animals or other microorganisms, making them both biosafe both environmentally and health-wise. However, they can survive in the environment just as well as the wildtype bacteria, therefore the potential technological process of lanthanide detection and recycling must be optimized (esp. in the terms of preventing GMO bacteria efflux from the bioreactor) to prevent HGT and efflux of the acceptor bacteria back into the environment.<br />
</p><br />
<br />
<hr noshade="noshade" /><br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<br />
<a name="possibilities_of_development"><h2>Possibilities of development</h2></a></br><br />
<p align="justify"><br />
We envisage two opportunities allowing our project to be improved. First, to test more LBTs described in literature (or even design new ones) and second to create more effective binding systems.</br><br />
Furthermore, we considered utilising some kind of sulphur bacterias instead of <i>E. coli</i>. Their sulphur-based metabolism and ability to survive in low pH (in which metal leaching is more efficient) makes them excellent candidates for industrial application of our project.</br><br />
Another thing which is worth investigating: our system should not be present in bacterias as plasmids. It could be interesting to integrate it with bacteria genome, so it would be more stable within bacteria. We briefly investigated applying pMAT plasmid (known for it's remarkable stability in bacterial cells) in our project to fix the problem of 'deevolution' and eradication of our construct plasmids from bacterias. We also planned to construct a BioBrick compatible version of pMAT, but had to scratch the idea because of lack of time.</br><br />
</p><br />
<hr noshade="noshade" /><br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a></div>
ASamsel
http://2014.igem.org/File:Periodic_table.jpg
File:Periodic table.jpg
2014-10-17T21:45:03Z
<p>ASamsel: periodic_table_itw</p>
<hr />
<div>periodic_table_itw</div>
ASamsel
http://2014.igem.org/Team:Warsaw/Project
Team:Warsaw/Project
2014-10-17T21:44:01Z
<p>ASamsel: </p>
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<li><a href="https://2014.igem.org/Team:Warsaw/Project#background">Background</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#proof_of_concept">Proof of concept</a></li><br />
<li><a href="https://2014.igem.org/Team:Warsaw/Project#detailed_explanation">Detailed explanation</a></li> <br />
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<br />
<br />
<h1>The Project</h1></br><br />
<hr noshade="noshade" /><br />
<br />
<a name="parts"><h2>Background</h2></a></br><br />
<p align="justify"><br />
Lanthanides are a series of fifteen chemical elements with atomic numbers 57 through 71, from lanthanum to lutetium. All but one of the lanthanides are f-block elements, corresponding to the filling of the 4f electron shell (lutetium is a d-block element). The presence of f orbitals is responsible for their unique properties such as strong paramagnetism.</br><br />
<img src="https://static.igem.org/mediawiki/2014/8/8f/600px-Lanthanides1.jpg" alt="Something went straight to Hell" width="600" height="169" /></br><br />
They are required in a variety of modern technologies, such as electronics, aviation (eg. jet engines) and superconductors</br><br />
In spite of their name 'rare earths metals' their deposits on Earth are not scarce but very dispersed. Hence, there are only a few places (eg. China) where their concentration is high enough to be exploited on a commercial scale. Due to that situation the lanthanides market is not getting any better, especially taking into account that demand on these metals is still on increase.</br><br />
<img src="https://static.igem.org/mediawiki/2014/6/63/REE_world_deposits_map.jpeg" alt="Something went straight to Hell" width="751" height="307" /></br><br />
1. Worldwide deposits of rare earths elements [1].</br><br />
<img src="https://static.igem.org/mediawiki/2014/3/37/World_deposits_of_REE_graph.png" alt="Something went straight to Hell" width="601" height="401" /></br><br />
2. Total deposits of rare earth metals [1].</br><br />
Other problems with lanthanides avaibility are their difficult extraction from the ore. Most of lanthanides appear together and very similar physical properties make extraction and purification difficult. Despite heavy costs and technical difficulties lanthanides are usually purified by ion-exchange chromatography.</br><br />
[1] Chapter 6, Kołodyńska D., Hubicki Z., <i>Investigation of Sorption and Separation of Lanthanides on the Ion Exchangers of Various Types</i>, <i>Ion exchange technologies</i>, edited by Ayben Kilislioğlu, Published: November 7, 2012 under CC BY 3.0 license</br><br />
</p><br />
<hr noshade="noshade" /><br />
<br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<br />
<a name="proof_of_concept"><h2>Proof of concept</h2></a></br><br />
<p align="justify"><br />
In 2013 group of prof. He from the University of Chicago published paper in <i>Journal of American Chemical Society</i> (<i>J. Am. Chem. Soc.</i> 2013 Feb 13;135(6):2037-9) in which they described thr devised lanthanide detecting system.<br />
</br><br />
To accomplish this, they engineered two-component system from <i>Salmonella enterica</i> creating the first bacteria capable of detecting lanthanides.<br />
These findings inspired us to create our bioremediating system.</br><br />
<img src="https://static.igem.org/mediawiki/2014/thumb/2/2e/ZDJ%C4%98CIE_WIKI_PMRA-PMRB.jpg/702px-ZDJ%C4%98CIE_WIKI_PMRA-PMRB.jpg" alt="Something went straight to Hell" width="350" height="300" /><br />
A general scheme of PmrA-PmrB system.<br />
</p><br />
<hr noshade="noshade" /><br />
<br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<br />
<a name="detailed_explanation"><h2>Detailed explanation</h2></a></br><br />
<p align="justify"><br />
Initially, our project was intended to have two different parts. First being a lanthanide detecting system in BioBrick standard, much like the one constructed by group of prof. He<br />
and the second being lanthanide binding/recovery system, which would bind lanthanides much more effectively than the detecting system.</br><br />
Both of these systems were based on PmrA-PmrB two-component system, native to <i>Salmonella enterica</i>. This system consists of two proteins, PmrA and PmrB. PmrB is a transmembrane kinase with iron (III) binding motif on its extracellular loop.<br />
When iron (III) is bound to this tag, PmrB gains kinase activity and phosphorylates PmrA. PmrA is a transctriptoral factor and, upon activation, binds to pmr<sup>C</sup> promoter and induces expression of CheZ, a chemotaxis protein.</br><br />
So much for native systems.</br><br />
</p><br />
<br />
<h4>Design</h4><br />
<p align="justify"><br />
<b>Detecting system</b></br><br />
Our detecting system is planned as follows:</br><br />
Iron binding tag would be replaced with lanthanide binding tag (of which a various collection can be find in literature) and a reporter protein would be inserted downstream of pmr<sup>C</sup>. Thus, in the presence of lanthanides, fluorescence of GFP should be observed.</br><br />
<b>Binding system</b></br><br />
Binding system has more complicated design. PmrA-PmrB is not changed significantly, the only modification was introduction of LBT (lanthanide binding tag) instead of iron binding motif. The difference is downstream the pmrC promoter. First of all, we need to introduce some sort of binding agent, presumably a small protein.<br />
We decided to use ubiquitin or an artificial structurised peptide and combine it with a LBT to create synthetic protein capable of binding lanthanide ions. Since lanthanide cations are not transported to the bacterium cell the binding agent need to be secreted outside the cytoplasm. Hence, we planned to add a signal peptide to the N or C terminus of the protein. Such modification could allow the protein to be located in the bacterial periplasmic space.</br><br />
Another possible problem is connected with pmr<sup>C</sup>, which is a very weak promoter (even if induced by PmrA). So, even in the presence of lanthanides, expression of a binding agent could be inefficient. To overcome that, we planned to use some activating sequences to boost the expression from upon the pmr<sup>C</sup>. Our first idea was to put two subsequent inverters (based on different proteins, eg. tetR and lacI), which should alleviate the problem. Expression of binding agent is expected to be high in the presence of lanthanides and low in their absence.</br><br />
</p><br />
<table border="1"><br />
<tr><br />
<td></td><br />
<td colspan="3">Binding agent expression</td><br />
</tr><br />
<tr><br />
<td>Lanthanide presence</td><br />
<td>pmr<sup>C</sup></td><br />
<td>pmr<sup>C</sup>-inverter1</td><br />
<td>pmr<sup>C</sup>-inverter1-inverter2</td><br />
</tr><br />
<tr><br />
<td>none</td><br />
<td>zero (very low)</td><br />
<td>high</td><br />
<td>low</td><br />
</tr><br />
<tr><br />
<td>present</td><br />
<td>low</td><br />
<td>low</td><br />
<td>high</td><br />
</tr><br />
</table><br />
</br><br />
<p align="justify"><br />
This may seem like an excessive mean, but we could not have invented anything subtler.</br><br />
</p><br />
<h4>Project goals</h4><br />
<ol><br />
<li>Construction of a lanthanide sensor in the BioBrick standard</li><br />
<li>Cloning of PmrA/PmrB parts into pSB1C3 in the BioBrick standard</li><br />
<li>Construction of a lanthanide sensoring system with<br />
other LBT described in the literature </li><br />
<li>Construction of a lanthanide binding system</li><br />
</ol><br />
<hr noshade="noshade" /><br />
<br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<br />
<a name="modelling"><h2>Modelling</h2></a></br><br />
<br />
<p> Two-component systems (TCSs) are the most prevalent mechanism of transmembrane signal transduction. They control gene expression thus make bacteria respond to environmental changes and drive pathogen-host interactions. A typical TCS consists of a membrane-bound histidine kinase and<br />
a partner response regulator protein. The pmrA/pmrB system, which our team used in the project, also belongs to this class. pmrB is a histidine kinase and pmrA is a response regulator which strongly enhances expression upon binding to Pmr<sup>C</sup>. In order to understand better the mechanism of the system and to prevent any problems before starting the experiments in the wetlab we decided to create a simple model of this signaling pathway. Some other TCSs were successfully modeled before, but not the pmrA/pmrB.<br />
</p><br />
<br />
<b>The model</b><br />
<br />
<p> When designing our model we assumed the following pathway:<br />
<ol><br />
<li> lanthanide ion binds to the pmrB protein which leads to its autophosphorylation, </li><br />
<li> phosphorylated pmrB transfers the phosphate group onto pmrA </li><br />
<li> phosphorylated pmrA binds to pmrC and initiate expression of the reporter GFP protein </li><br />
<li> dephosphorylated pmrB induces pmrA dephoshporylation </li><br />
Additionally for model to work properly feedback loop in which phoshporylated pmrA induces pmrA expression is needed.<br />
</p><br />
<p><br />
The model diagram looks as follows:<br />
<br />
<img src="https://static.igem.org/mediawiki/2014/b/b4/Warsaw_pathway.png" width=780px alt="Signaling pathway" /><br />
<br />
</ol><br />
</p><br />
<br />
<p> We concluded that quantities of observed species change according to these equations:<br><br><br><br />
<img src="https://static.igem.org/mediawiki/2014/3/34/Warsaw_dpmrnaprmb.gif" alt="Equation 1" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/1/1f/Mrnapmra2.gif" alt="Equation 2" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/3/33/Warsaw_eq3.gif" alt="Equation 3" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/5/52/Warsaw_eq4.gif" alt="Equation 4" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/2/20/Warsaw_eq5.gif" alt="Equation 5" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/4/48/Warsaw_eq6.gif" alt="Equation 6" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/3/3b/Warsaw_eq7.gif" alt="Equation 7" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/c/c1/Mrnarp2.gif" alt="Equation 8" /><br><br><br></br><br />
<img src="https://static.igem.org/mediawiki/2014/c/c3/Warsaw_eq9.gif" alt="Equation 9" /><br><br><br></br><br />
<br />
<br />
where:<br />
<ul><br />
<li> <i>mRNApmrB</i> is concentration of pmrB mRNA, the same goes for <i>mRNApmrA</i> and <i>mRNARP</i>, </li><br />
<li> <i>L</i> is lanthanide concentration, </li><br />
<li> <i>RP</i> is reporter protein concentration, </li><br />
<li> <i>pmrB.bound</i> is <i>pmrB</i> with lanthanide ion bound, </li><br />
<li> <i>prmB.bound.ph</i> is phosphorylated <i>pmrB</i> with lanthanide ion bound, </li><br />
<li> <i>pmrA.ph</i> is phosphorylated pmrA, </li><br />
<li> <i>ABComplex</i> is complex of <i>pmrA</i> and <i>pmrB.bound.ph</i> during <i>pmrA</i> phosphorylation, </li><br />
<li> <i>AComplex</i>, <i>RPComplex</i> are <i>pmrA.ph</i> inductors bound to respective promoters, </li><br />
<li> <i>ABRevComplex</i> is complex of <i>pmrA.ph</i> and <i>pmrB</i> during <i>pmrA</i> dephosphorylation </li><br />
</ul><br />
</p><br />
<br><br><br />
<br />
<br />
<b> The parameters </b><br />
<p><br />
Initial parameters were found in literature as we did not make independent component measures.<br />
</p><br />
<b> Simulation and results </b><br />
<p><br />
Deterministic simulations were performed using TinkerCell software. There are few bugs in it, but it allows for fast model building and makes changes to the model quite easy. <br />
Simulation showed that signal greatly enhances GFP expression, the its growth is exponential and correlate positively with increased concentration of lanthanide ions.<br />
</p><p><br />
GFP level when there is no lanthanide ions:<br />
<img src="https://static.igem.org/mediawiki/2014/f/f8/Warsaw_noL.png" width=780px alt="Chart" /><br />
</p><br />
<p><br />
GFP levels with 100 um of ions:<br />
<img src="https://static.igem.org/mediawiki/2014/c/c0/Warsaw_yesL.png" width=780px alt="Chart" /><br />
</p><br />
</br><br />
</br><br />
<b> References </b><br />
<p><br />
<i> Kierzek AM, Zhou L, Wanner BL. Stochastic kinetic model of two compo-<br />
nent system signalling reveals all-or-none, graded and mixed mode stochastic switching responses. Mol Biosyst. 2010;6(3):531-42<br />
</i><br />
</p><br />
<hr noshade="noshade" /><br />
<br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<br />
<a name="weeestudy"><h2>WEEE study</h2></a></br><br />
WEEE stands for ‘Waste Electrical and Electronic Equipment’ such as computers, mobile phones, TV-sets and fridges.</br> <br />
Modern electronic products contain up to 60 elements, many of them are very valuable. The most complex of it is usually presented in printed wiring boards. Metals represent on average 23% of weight of the phone, in majority copper. Single mobile phone can contain up to 9 g Cu, 250 mg Ag, 24mg Au and 0,5 mg Tb. <br />
<br />
<hr noshade="noshade" /><br />
<br />
<br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<br />
<a name="safety"><h2>Safety</h2></a></br><br />
<h3>1. Introduction</h3><br />
<p align="justify"><br />
We at Team Warsaw understand the need for good safety training and biosafe conduct in the lab.<br />
In the following sections, we will show you how we went about making sure we didn't put anyone at an unnecessary risk either at our faculty or in the outside world.<br />
</p><br />
<h3>2. (Bio)safe conduct</h3><br />
<p align="justify"><br />
Before summer, our work in the lab began with a safety training provided by our instructors. We were trained <br />
in accordance with the biosafety guidelines of our institution, focusing on lab-practical aspects of biosafety,<br />
i.e. where to work with bacteria, to always do it in the same place, to account for where the bacteria-containing material is being put, to always disinfect the immediate vicinity of your workbench once the work is finished, etc. We were also taught to properly store biological material, such as bacterial broths imbued with colonies, waste agar plates, or pipette tips and plastic tubes, and handle them in a manner suitable for preventing the spread of bacteria. Whenever some biological material-containing glass, as flasks or tubes, was broken into pieces, the adjacent area was mopped dry and disinfected with ethanol,<br />
and broken glass was stored in a separate container for glass, but only after possible remains of liquids have been removed by mopping and the pieces were disinfected by spraying with 70% ethanol.<br />
</p><br />
<p align="justify"><br />
Usually, we worked with DNA constructs so it was a matter of keeping everything else out the workplace (i.e. every type of contamination). Therefore, we had a set place for bacterial work on a bench, which was cleaned after each use, always worked with bacteria under conditions of closed windows and burner turned on, always worked in non-reusable gloves, which were disinfected with 70% ethanol at the start of work, proceeded to disinfect them regularly, stored contaminated plastic and liquids in separate containers suitable for autoclaving under standard conditions (which is taken care of by our Institute) and removed contaminated materials from our lab on a weekly basis. Whenever working with bacteria, we also refrained from touching objects outside the workbench (to prevent the possible spread of bacteria) and disinfected the working place using 70% ethanol.Of course, since our DNA constructs often carried antibiotic resistance gene, we took particular care to make sure all of our liquids remained in their respective tubes and cleaned the leakage places with 70% ethanol whenever these happened. In terms of work with hazardous substances, the only one we encountered was the ethidium bromide (used when working with agarose gels): to avoid any skin contact we always used gloves and we worked on a separate bench, devoted to ethidium bromide. Gels after visualization were stored in a separate container to be destroyed.<br />
</p><br />
<h3>3. Biosafety level</h3><br />
<p align="justify"><br />
In this year's project, we used the K-12 <i>E. coli</i> bacteria as chassis for our proteins: which are PmrA, PmrB and GFP. All the proteins we worked on, as well as with <i>Escherichia</i> chassis are harmless (non-pathogenic)<br />
to humans, which allowed for classification of our work at biosafety level 1 according to<br />
<a href="http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf?ua=1">WHO</a>.<br />
We therefore worked with our bacteria and constructs on ordinary open top benches, using Bunsen burners for sterility whenever aliquoting media, imbuing overnight cultures or sowing onto agar plates.<br />
We used <i>Salmonella enterica</i> subsp. <i>enterica</i> ser. Typhimurium, which is technically a biosafety level 2 organism, as source of parts isolated by PCR. However, we worked with a non-pathogenic, attenuated<br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/15063560">&#967;3987</a> <i>Salmonella enterica</i> subsp. <i>enterica</i> ser. Typhimurium carrying an <i>asd</i> (aspartate dehydrogenase) gene from <i>E.coli</i> on the p3342 plasmid, as derived from the <i>Salmonella Typhimurium</i> <a href="http://www.ncbi.nlm.nih.gov/pubmed/21622747">UK-1 strain</a>. This strain is non-virulent (#916;crp, #916;cya), hence provides no risk to personal or community health. Even though the genomic DNA isolation was a one-time operation, we still performed all manual activities under a suitable biological safety cabinet.</p><p align="justify"><br />
As regarding the parts isolated, i.e. proteins coded by the BasR (PmrA) and BasS (PmrB) genes, they are said to partake in the <i>Salmonella</i> virulence. Nonetheless, these two proteins merely regulate expression of the genes, whose products (usually LPS-modifyingenzymes) take part in virulence processes and so, "our" proteins are not involved in these processes themselves. We also used a PmrC/GFP construct-carrying plasmid (which we were glad to have received from<br />
<a href="http://he-group.uchicago.edu/">Prof. Chuan He's group</a> at the University of Chicago), whereby GFP was expressed from a PmrA-induced PmrC promoter. The PmrC gene codes for the phosphoethanolaminetransferase enzyme, which is required for Salmonella resistance to polymyxin. However, the plasmid we used was not carrying PmrC gene, but only the mentioned promoter which [the promoter] does not constitute a biosafety risk and neither does the GFP protein.<br />
</p><br />
<h3>4. Safety forms</h3><br />
<p align="justify"><br />
We submitted our <a href="https://igem.org/Safety/About_Our_Lab?team_id=1459">About Our Lab</a> form as well as the<br />
<a href="https://igem.org/Safety/Safety_Form?team_id=1459">Safety Form</a>, which can be found by clicking<br />
the hyperlinks on their respective names. We did not, however, need to fill out any Check-Ins, as neither of our parts nor the chassis fell under the required categories. The White List of parts<br />
and organisms and their Check-In necessity status can be found on the website of the<br />
<a href="https://2014.igem.org/Safety/White_List"> Safety Hub</a>.<br />
</p><br />
<h3>5. Environmental concerns</h3><br />
<p align="justify"><br />
The question about potential environmental concerns of our project was central to our attempts. However, due to the nature of the proteins expressed, our chassis bacteria have not acquired any characteristics that would enable them to compromise human immune system/other systems<br />
or evade detection and destruction by the former or facilitate spread between people/animals, which makes them harmless from both a personal and public health point of view.<br />
At the same time, neither the proteins encoded themselves, nor the functionality of the lanthanide detecting/binding system as a whole, imbue the bacteria with characteristics that would convey<br />
an evolutionary advantage against other organisms in the environment, both microorganisms and plants or animals, or act as toxins against the aforementioned, making the bacteria modified with the PmrA/PmrB system environmentally biosafe with no risk or them dominating any ecological niche. Our modified bacteria have, however, survival capabilities comparable to the wildtype ones.<br />
There must be the point stressed, however, that since the transformed bacteria carry<br />
a chloramphenicol resistance-encoding plasmid, the actual biosafety of the detection/binding system (i.e. prevention of HGT of the antibiotic resistance between the modified and wildtype bacteria) and so - the potential impact on the environment - depends greatly on the design of the bioreactor and the technological process, to minimize, or best prevent, the influx and efflux<br />
of non-transformed bacteria, and microorganisms in general, into the reactor.<br />
To sum up, our bacteria are not toxic towards either humans, plants, animals or other microorganisms, making them both biosafe both environmentally and health-wise. However, they can survive in the environment just as well as the wildtype bacteria, therefore the potential technological process of lanthanide detection and recycling must be optimized (esp. in the terms of preventing GMO bacteria efflux from the bioreactor) to prevent HGT and efflux of the acceptor bacteria back into the environment.<br />
</p><br />
<br />
<hr noshade="noshade" /><br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a><br />
<br />
<a name="possibilities_of_development"><h2>Possibilities of development</h2></a></br><br />
<p align="justify"><br />
We envisage two opportunities allowing our project to be improved. First, to test more LBTs described in literature (or even design new ones) and second to create more effective binding systems.</br><br />
Furthermore, we considered utilising some kind of sulphur bacterias instead of <i>E. coli</i>. Their sulphur-based metabolism and ability to survive in low pH (in which metal leaching is more efficient) makes them excellent candidates for industrial application of our project.</br><br />
Another thing which is worth investigating: our system should not be present in bacterias as plasmids. It could be interesting to integrate it with bacteria genome, so it would be more stable within bacteria. We briefly investigated applying pMAT plasmid (known for it's remarkable stability in bacterial cells) in our project to fix the problem of 'deevolution' and eradication of our construct plasmids from bacterias. We also planned to construct a BioBrick compatible version of pMAT, but had to scratch the idea because of lack of time.</br><br />
</p><br />
<hr noshade="noshade" /><br />
<a href="https://2014.igem.org/Team:Warsaw/Project"><p style="text-align:right;"><h6>Up↑</h6></p></a></div>
ASamsel
http://2014.igem.org/Team:Warsaw/EXTRAS
Team:Warsaw/EXTRAS
2014-10-17T19:21:54Z
<p>ASamsel: </p>
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<h1>Technology</h1> </br><br />
<hr noshade="noshade" /><br />
<br />
<br />
<a name="bioprocess"><h2>Bioprocess</h2></a></br><br />
<p align="justify">Electronic equipment is the fastest growing waste category of waste in many developed countries. Amount of electronic waste grows rapidly because markets in which electronic is produced cross the other side of the ‘Digital Divide’. We stand in front of the following problem: what we are we going to do with all those computers, smartphones which we buy? All these products become obsolete or just unwanted within 1-3 years of purchase. Where will we find a new source of metals necessary to fabricate electronic equipment?</br><br />
Using old and broken WEEE in our project not only gives us a source of metals to produce new smartphones or computers, but also solves problem of storing WEEE.</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/6/6e/Recycling.jpg" alt="recyclin_itw" /></center><br />
<p align="justify">This figure shows a view on the context of re-use of EEE or its components. At the end of the use EEE returns to producers or to companies, which specialize in reuse of e-waste. <br />
The next step is preparation for re-use. On this step wasted electronic equipment is deconstructed. Not only computer memories or other parts which contains valuable metals are re-use. We also want to recover such materials as plastic or glass. That step requires labor work or special machines which will prepare WEE for another point of the plan. <br />
Some parts of the TVs or computers may not be useful in another manufactory. Sometimes we need to remarked parts which we need and work in place with special equipment. <br />
In the future we can use our bacteria with lanthanides binding sequences to re-use lanthanides.</br><br />
1) Removing from WEEE plastic and stirring parts which are abundant of rare earth metals such as lanthanides, especially computer memories. Stirring increase availability WEEE with acid which is produced by <i>Thiobacillus ferrooxidans</i>. It display terbium from alloy.</br><br />
2) Effluent from our bioreactor has low pH (about 2,5) what is a lethal for <i>E.coli</i>. This is the reason why we add Ca(OH)<sub>2</sub> to effluent. It increases our pH to a level which is optimal to <i>E.coli</i> (about 7). Ca(OH)<sub>2</sub> is also cheap and it is not increasing cost of the process very much.</br> <br />
3) Effluent goes to packed column bioreactor in which <i>E.coli</i> is immobilized. It gets across whole column. Ions of terbium binds and senses trough periplasmic domain.</br><br />
4) Column (with no initial effluent) is flushed by NaCl. PmrB can easily denaturate in NaCl solution losing its conformation. Ions of terbium do not precipitate with NaCl aq and do not make insoluble components with it.</br><br />
5) Ions of terbium are recovered from solution by electrolysis <br />
<hr noshade="noshade" /><br />
<br />
<a name="discussion"><h2>Discussion</h2></a></br><br />
Our process finds not only a way to recovery the lanthanides, but also other metals such as copper ( it takes place at first point of it) and other rare metals.We not only gain rare metals, but also solve the problem of WEEE. There are alternative ways to recycle metals such as cooper or iron by using bacteria, but lanthanides are usually extraced from motherlode, which nowdays is really costly.</br><br />
Our way of recovery lanthanides does not produce pollution but it also is a safe way of managing with WEEE. Other methods of gaining lanthanides use mix of concentrated acids such as HCl or HNO<sub>3</sub>. The most common problems with these methods are difficult to neutralise byproducts. Byproducts, especially Ca(NO<sub>3</sub>)<sub>2</sub> are environmental-threatening. Their storing is very difficult and costly.Mining of lanthanides by old methods need to be placed on big areas, but our process takes less place. Area which will be taken to run it strictly depends on scale (in contrast to minors which always will take more space).</br><br />
Method which we presented is eco-friendly and solves the problem of full landfills with WEEE.</br> In our opinion using microorganisms to recycle e-waste will be a standard way of raising lanthanides.<br />
<hr noshade="noshade" /><br />
<br />
<a name="challenges"><h2>Challenges</h2></a></br><br />
<hr noshade="noshade" /><br />
<br />
<a name="alternative_methods"><h2>Alternative methods</h2></a></br><br />
<p align="justify">Our final system was of course not the only possibility. There were some points where we had to decide...<br />
<h4>Reporter protein</h4><br />
Finally we decided for GFP protein because of its prevalence, simplicity and fact that GFP does not require any additional reagents (eg. IPTG).</br><br />
We could have used other fluorescent proteins, for instance superfolder fluorescent proteins constructed by iGEM Warsaw 2013 Team, but regular GFP was the simpliest choice.</br><br />
<h4>Binding agent</h4><br />
Although we were unable to implement lanthanide binding system because of lack of time, we had several ideas how to accomplish this goal.</br><br />
<u>Poly-LBT peptide</u></br><br />
First was to construct a poly-LBT peptide which would be then anchored in the outer membrane of <i>E. coli</i> or transported to periplasm. We discarded this idea due to problems with modelling of behaviour of this polymer and problems with wet-lab design.</br><br />
<u>PmrB over-expression</u></br><br />
Another idea consisted of PmrB dependent over-expression of PmrB(LBT) protein. In such system we would have pmr<sup>C</sup> promoter - some logical device to boost the signal - PmrB(LBT), so in presence of lanthanides amount of PmrB(LBT) protein per cell would rise sharply, which should allow effective binding of lanthanides.</br><br />
<u>Small peptide fused with LBT</u></br><br />
Our final and probably best idea was to create a construct peptide of such composition:</br><br />
BBa_J32015 (<i>E. coli</i> periplasm signal peptide)-structure peptide (ubiquitin or 1L2Y [BBa_K1459015]) - lanthanide binding tag.</br><br />
The plan was to create a small, 'rubbish' protein which would only bind lanthanides without having any physiological function in cell (since we were afraid whether over-expression of PmrB would be cytotoxic).</br><br />
<hr noshade="noshade" /></div>
ASamsel
http://2014.igem.org/Team:Warsaw/EXTRAS
Team:Warsaw/EXTRAS
2014-10-17T18:18:24Z
<p>ASamsel: </p>
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<h1>Technology</h1> </br><br />
<hr noshade="noshade" /><br />
<br />
<br />
<a name="bioprocess"><h2>Bioprocess</h2></a></br><br />
Electronic equipment is the fastest growing waste stream in many countries. E waste grows rapidly because markets in which these products are produced cross the other side of the ‘Digital Divide’. We stand in front of the following problem: what we are we going to do with all those computers, smartphones which we buy? All these products become obsolete or unwanted within 1-3 years of purchase. Where will we find a new source of metals necessary to fabricate electronic equipment?</br><br />
Using old and broken WEEE in our project not only gives us a source of metals to produce new smartphones or computers, but also solves problem of storing WEEE.</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/6/6e/Recycling.jpg" alt="recyclin_itw" /></center><br />
This figure shows a view on the context of re-use of EEE or its components. At the end of the use EEE returns to producers or to companies, which specialize in reuse of e-waste. <br />
The next step is preparation for re-use. On this step waste electronic equipment is deconstructed. Not only computer memories or other parts which contains valuable metals are re-use. We also want to recover such materials as plastic or glass. That step requires labor work or special machines which will prepare WEE for another point of the plan. <br />
Some parts of the TVs or computers may not be useful in another manufactory. Sometimes we need to remarked parts which we need and work in place with specialist equipment. <br />
Later we can use our bacteria with lanthanides binding sequences to re-use lanthanides.</br><br />
1) Removing from WEEE plastic and stirring parts which are abundant of rare earth metals such as lanthanides, especially computer memories. Stirring increase availability WEEE with acid which is produced by Thiobacillus ferrooxidans. It display terbium from alloy.</br><br />
2) Effluent from our bioreactor has low pH (about 2,5) what is a lethal for E.coli. That is why we add Ca(OH)<sub>2</sub> to effluent. It increases our pH to a level which is optimal to E.coli (about 7). Ca(OH)<sub>2</sub> is also cheap and it is not increasing cost of the process very much.</br> <br />
3) Effluent goes to packed column bioreactor in which E.coli is immobilized. It gets across whole column. Ions of terbium binds and senses trough periplasmic domain.</br><br />
4) Column (with no initial effluent) is flushed by NaCl. PmrB can easily denaturate in NaCl aq , losing shape. Ions of terbium do not precipitate with NaCl aq and do not make insoluble components with it.</br><br />
5) Ions of terbium are recovered from solution by electrolysis <br />
<hr noshade="noshade" /><br />
<br />
<a name="discussion"><h2>Discussion</h2></a></br><br />
Our process finds not only a way to recovery the lanthanides, but also other metals such as copper ( it takes place at first point of it and other rare metals.</br><br />
Our way of recovery lanthanides does not produce pollution but it also is a safe way of managing with WEE.<br />
<hr noshade="noshade" /><br />
<br />
<a name="challenges"><h2>Challenges</h2></a></br><br />
<hr noshade="noshade" /><br />
<br />
<a name="alternative_methods"><h2>Alternative methods</h2></a></br><br />
Our final system was of course not the only possibility. There were some points where we had to decide...<br />
<h4>Reporter protein</h4><br />
Finally we decided for GFP protein because of it's versality, simplicity and fact that GFP does not require any additional reagents (eg. IPTG).</br><br />
We could have used other fluorescent proteins, for instance superfolder fluorescent proteins constructed by iGEM Warsaw 2013 Team, but regular GFP was the simpliest choice.</br><br />
<h4>Binding agent</h4><br />
Although, unfortunately, we could not have implemented lanthanide binding system because of lack of time, we had several ideas how to accomplish this.</br><br />
<u>Poly-LBT peptide</u></br><br />
First was to construct a poly-LBT peptide which would be then anchored in the outer membrane of <i>E. coli</i> or transported to periplasm. We discarded this idea due to problems with modelling of behaviour of this polymer and problems with wet-lab design.</br><br />
<u>PmrB over-expression</u></br><br />
Another idea consisted of PmrB dependent over-expression of PmrB(LBT) protein. In such system we would have pmr<sup>C</sup> promoter - some logical device to boost the signal - PmrB(LBT), so in presence of lanthanides amount of PmrB(LBT) protein per cell would rise sharply, which should allow effective binding of lanthanides.</br><br />
<u>Small peptide fused with LBT</u></br><br />
Our final and probably best idea was to create a construct peptide of such composition:</br><br />
BBa_J32015(<i>E. coli</i> periplasm signal peptide)-structure peptide(ubiquitin or 1L2Y [BBa_K1459015]) - lanthanide binding tag.</br><br />
The plan was to create a small, 'rubbish' protein which would only bind lanthanides without having any physiological function in cell (since we were afraid whether over-expression of PmrB would be cytotoxic).</br><br />
<hr noshade="noshade" /></div>
ASamsel
http://2014.igem.org/Team:Warsaw/EXTRAS
Team:Warsaw/EXTRAS
2014-10-17T18:04:37Z
<p>ASamsel: </p>
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<a href="/Team:Warsaw/EXTRAS">EXTRAS</a><br />
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<h1>Technology</h1> </br><br />
<hr noshade="noshade" /><br />
<br />
<br />
<a name="bioprocess"><h2>Bioprocess</h2></a></br><br />
Electronic equipment is the fastest growing waste stream in many countries. E waste grows rapidly because markets in which these products are produced cross the other side of the ‘Digital Divide’. We stand in front of the following problem: what we are we going to do with all those computers, smartphones which we buy? All these products become obsolete or unwanted within 1-3 years of purchase. Where will we find a new source of metals necessary to fabricate electronic equipment?</br><br />
Using old and broken WEEE in our project not only gives us a source of metals to produce new smartphones or computers, but also solves problem of storing WEEE.</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/6/6e/Recycling.jpg" alt="recyclin_itw" /></center><br />
This figure shows a view on the context of re-use of EEE or its components. At the end of the use EEE returns to producers or to companies, which specialize in reuse of e-waste. <br />
The next step is preparation for re-use. On this step waste electronic equipment is deconstructed. Not only computer memories or other parts which contains valuable metals are re-use. We also want to recover such materials as plastic or glass. That step requires labor work or special machines which will prepare WEE for another point of the plan. <br />
Some parts of the TVs or computers may not be useful in another manufactory. Sometimes we need to remarked parts which we need and work in place with specialist equipment. <br />
Later we can use our bacteria with lanthanides binding sequences to re-use lanthanides.</br><br />
1) Removing from WEEE plastic and stirring parts which are abundant of rare earth metals such as lanthanides, especially computer memories. Stirring increase availability WEEE with acid which is produced by Thiobacillus ferrooxidans. It display terbium from alloy.</br><br />
2) Effluent from our bioreactor has low pH (about 2,5) what is a lethal for E.coli. That is why we add Ca(OH)2 to effluent. It increases our pH to a level which is optimal to E.coli (about 7). Ca(OH)2 is also cheap and it is not increasing cost of the process very much.</br> <br />
3) Effluent goes to packed column bioreactor in which E.coli is immobilized. It gets across whole column. Ions of terbium binds and senses trough periplasmic domain.</br><br />
4) Column (with no initial effluent) is flushed by NaCl. PmrB can easily denaturate in NaCl aq , losing shape. Ions of terbium do not precipitate with NaCl aq and do not make insoluble components with it.</br><br />
5) Ions of terbium are recovered from solution by electrolysis <br />
<hr noshade="noshade" /><br />
<br />
<a name="discussion"><h2>Discussion</h2></a></br><br />
Our process finds not only a way to recovery the lanthanides, but also other metals such as copper ( it takes place at first point of it and other rare metals.</br><br />
Our way of recovery lanthanides does not produce pollution but it also is a safe way of managing with WEE.<br />
<hr noshade="noshade" /><br />
<br />
<a name="challenges"><h2>Challenges</h2></a></br><br />
<hr noshade="noshade" /><br />
<br />
<a name="alternative_methods"><h2>Alternative methods</h2></a></br><br />
Our final system was of course not the only possibility. There were some points where we had to decide...<br />
<h4>Reporter protein</h4><br />
Finally we decided for GFP protein because of it's versality, simplicity and fact that GFP does not require any additional reagents (eg. IPTG).</br><br />
We could have used other fluorescent proteins, for instance superfolder fluorescent proteins constructed by iGEM Warsaw 2013 Team, but regular GFP was the simpliest choice.</br><br />
<h4>Binding agent</h4><br />
Although, unfortunately, we could not have implemented lanthanide binding system because of lack of time, we had several ideas how to accomplish this.</br><br />
<u>Poly-LBT peptide</u></br><br />
First was to construct a poly-LBT peptide which would be then anchored in the outer membrane of <i>E. coli</i> or transported to periplasm. We discarded this idea due to problems with modelling of behaviour of this polymer and problems with wet-lab design.</br><br />
<u>PmrB over-expression</u></br><br />
Another idea consisted of PmrB dependent over-expression of PmrB(LBT) protein. In such system we would have pmr<sup>C</sup> promoter - some logical device to boost the signal - PmrB(LBT), so in presence of lanthanides amount of PmrB(LBT) protein per cell would rise sharply, which should allow effective binding of lanthanides.</br><br />
<u>Small peptide fused with LBT</u></br><br />
Our final and probably best idea was to create a construct peptide of such composition:</br><br />
BBa_J32015(<i>E. coli</i> periplasm signal peptide)-structure peptide(ubiquitin or 1L2Y [BBa_K1459015]) - lanthanide binding tag.</br><br />
The plan was to create a small, 'rubbish' protein which would only bind lanthanides without having any physiological function in cell (since we were afraid whether over-expression of PmrB would be cytotoxic).</br><br />
<hr noshade="noshade" /></div>
ASamsel
http://2014.igem.org/Team:Warsaw/Team
Team:Warsaw/Team
2014-10-17T17:51:04Z
<p>ASamsel: </p>
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<div class="main-content"><br />
<br />
<h1>The Team</h1> </br><br />
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<a name="members"><h2>Members</h2></a></br><br />
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<td> <img src="https://static.igem.org/mediawiki/2014/0/05/Zdjecie_nurkowanie.JPG" width="200px"; alt="Aleksandra_Bartosik"></td> <td><h4>Aleksandra Bartosik</h4>I am studying biotechnology and chemistry at the College of Inter-faculty Individual Studies in Mathematics and Natural Science. I'm fascinated by biomimicry, immunobiology, mechanisms of bacterial multi-drug resistance and ABC-transporters. In my spare time I dive, travel across Northern Europe and talk about graphics with my best friend. I am the 2nd Team Leader and this year beside of working in the lab I take care of the Human Practice.</td><br />
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<td><img src="https://static.igem.org/mediawiki/2014/8/86/M_G%C3%B3rka.jpg" width="200px" ; alt="Magdalena_Górka"></td> <td><br><h4>Magdalena Górka</h4>I study biotechnology and chemistry at the College of Inter-faculty Individual Studies in Mathematics and Natural Science at University of Warsaw. <br>I am mostly interested in human genetics and medical biotechnology but I don't remain indifferent to other fields of science. <br>In my free time, I like reading books, swimming and traveling.<br><br><br><br><br><br></td><br />
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<td><img src="https://static.igem.org/mediawiki/2014/9/99/IMG_0616aa.jpg" width="200px"; alt="Ela_Gralińska"></td> <td><h4>Ela Gralińska</h4>Currently I study Biotechnology at TU Berlin. Besides biological engineering, I'm particularly fascinated with otters thanks to which my adventure with biology has begun. In my freetime I play badminton. <br><br><br><br><br><br><br><br></td><br />
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<td><img src="https://static.igem.org/mediawiki/2014/f/f4/Img2.jpg" width="200px" ; alt="Mieszko_Majka"></td> <td><br><h4>Mieszko Majka</h4>I'm a sophomore student of chemistry & biotechnology at the College of Interfaculty Studies in Mathematics and Natural Sciences (a.k.a. Kolegium MISMaP). My scientific interests entail biological chemistry (mostly chemistry of nucleic acids and proteins), bits of neurochemistry, molecular biology of nucleic acids and synthetic biology (mostly fantasizing how to combine fancy natural stuff as proteins, RNAs, etc. into systems that would serve whatever purpose I have on my mind at the given time). Outside the lab, I do enjoy a good bike trip, some ballroom dancing or teaching people... whatever they want to learn and I can provide, so it's usually English or chemistry. During this year's iGEM, I try to blow up the lab every other day (i.e. I'm a wetlab worker) but parallely, I help make sure that everything flows smoothly enough to prevent others from doing so (mostly by telling people when to come to the lab and when not & mundane stuff like this, really).</td><br />
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<td><img src="https://static.igem.org/mediawiki/2014/f/f3/Rafał_zdjęcie.jpg" width="200px" ; alt="Rafał_Meysztowicz"></td> <td><h4>Rafał Meysztowicz</h4> I'm studying Physics and Mathematics at College of Inter-Faculty Studies in Mathematics and Natural Sciences. I'm fascinated with their application on other fields like Biology, Economics and in everyday life. I'm also a music producer. In free time I like to play sports, read books and watch movies.<br><br><br><br><br><br><br></td><br />
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<td><img src="https://static.igem.org/mediawiki/2014/2/27/PaulinaOsiadacz.jpg" width="200px"; alt="Paulina_Osiadacz"></td> <td><h4>Paulina Osiadacz</h4>My name is Paulina Osiadacz. I study biotechnology in the second years in the Department in the Biology in the University of Warsaw. I always interesting how to work the all body, but in particular at the level of cell. In the free time I try to help people and participate in charitable actions. <br><br><br><br><br><br></td><br />
</tr><br />
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<td><img src="https://static.igem.org/mediawiki/2014/7/7c/DSC02006.jpg" width="200px"; alt="Zosia_Rudnicka"></td> <td><h4>Zosia Rudnicka</h4>My name is Zosia Rudnicka. I’m a student of biology and physics at University of Warsaw. I’m interested in genetics and bioinformatics. In my free time I’m reading history and SF books. I love traveling and my dog Daisy. <br><br><br><br></td><br />
</tr><br />
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<td><img src="https://static.igem.org/mediawiki/2014/c/c7/20130902_120836.jpg" width="200px"; alt="Anna_Saffray-Borowski"></td> <td><h4>Anna Saffray-Borowski</h4>I am a second year student at the Faculty of Biology of our university and I am studying biotechnology. I enjoy spending time in the laboratory learning new exciting approaches, methods and facts, that is why I am trying different projects. I have met the World of: plants, bacteria, yeasts, as well as the World of cells, I don't know yet what to choose but I guess at one point I will just know. Apart from my first hobby (science) I enjoy listening to classical music, especially Sergei Rachmaninoff and play the piano. </td><br />
</tr><br />
</table><br />
<br><br />
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<table><br />
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<td><img src="https://static.igem.org/mediawiki/2014/d/df/1407528791959.jpg" width="200px"; alt="Agnieszka_Samsel"></td> <td><h4>Agnieszka Samsel</h4>I am studying biotechnology and chemistry at the College of Inter-faculty Individual Studies In Mathematics and Natural Science. I’m fascinated by biotechnology in environmental protection, bioprocess engineering and genetics. In free time I like to travell, search for interesting places in Warsaw and delve into Kurpie’s history.<br></td><br />
</tr><br />
</table><br />
<br><br />
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<td><img src="https://static.igem.org/mediawiki/2014/a/ac/20140719_143455.jpg" width="200px"; alt="Natalia_Szulc"></td> <td><h4>Natalia Szulc</h4>I study biotechnology and bioinformatics at College of Inter-Faculty Individual Studies in Mathematicsand Natural Sciences, University of Warsaw. My scientific interests include molecular mechanisms of disease, medicinal chemistry and computer-aided drug design. iGEM allows me to combine my scientific passion with Human Practice and popularization of science. In my free time I go with my friends to the cinema or theatre. I also love reading, travelling and visiting museums of natural history and galleries of fine arts.<br><br><br><br><br><br><br></td><br />
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<td><img src="https://static.igem.org/mediawiki/2014/3/3b/GŚ_zdjęcie.jpg" width="200px"; alt="Grzegorz_Ścibisz"></td> <td><h4>Grzegorz Ścibisz</h4>I'm studying chemistry and biotechnology at College of Inter-Faculty Studies in Mathematics and Natural Sciences. My scientific interests contain supramolecular chemistry, synthetic biology and biophysics and self-assembly of proteins. My "non-proffesional" interests are physics, astronomy and reading, not only sci-fi.<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br></td><br />
</tr><br />
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<a name="advisors"><h2>Advisors</h2></a></br><br />
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<td><img src="https://static.igem.org/mediawiki/2014/6/67/IMG_0826_%283%29.jpg" width="200px"; alt="Anna_Kornakiewicz"></td> <td><br><h4>Anna Kornakiewicz</h4>I am a young M.D. with interdisciplinary research background and want to contribute to open health and innovation in medicine. I am fascinated with mechanisms and machines - from watches and trains to elucidation of the mechanisms of action of drugs. I' ve discovered that IGEM reflects the way in which I see medicine – as ‘information science and art of healing’ and gives a unique opportunity to merge multiple interest in one clear idea. I want to act also in the area of mobile health, medical startups and healthcare markets design and contribute to gifted and STEM education.</td><br />
</tr><br />
</table><br />
<br><br />
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<td><img src="https://static.igem.org/mediawiki/2014/c/c7/KamilKoziara.jpg" width="200px"; alt="Kamil_Koziara"></td> <td><br><h4>Kamil Koziara</h4>I have MSc in Computer Science and I am studying Biotechnology at the University of <br />
Warsaw. I am interested in bioinformatics, molecular biology and bioengineering. I am <br />
supporter of open source and in my free time I like to do some diy projects. I love to read.<br><br><br><br><br><br><br><br><br><br></td><br />
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<td><img src="https://static.igem.org/mediawiki/2014/d/d9/1618440_10151842531087126_414762827_n.jpg" width="200px"; alt="Masia_Maksymowicz"></td> <td><br><h4>Małgorzata Maksymowicz</h4>I am studying biology; last year I did my Bachelor thesis on circadian clock in different organisms. Now I'm doing my Masters in the Department of Plant Molecular Ecophysiology. I am mostly interested in plant physiology and molecular biology, though I really like being in the field and finding natural wonders.<br><br />
When I'm not doing biology, I sing, play a guitar and travel across Poland and around the world.<br><br><br><br><br><br><br></td><br />
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</table><br />
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<td><img src="https://static.igem.org/mediawiki/2014/8/84/Zdjecie.jpg" width="200px"; alt="Piotr_Skłodowski"></td> <td><br><h4>Piotr Skłodowski</h4>I am student of third year Biology course at University of Warsaw. I am mostly interested in issues concerning molecular biology and genetic engineering. I got involved in iGEM project because it contains many fields of science, which is inseparable in development of modern knowledge. Additionally iGEM has interesting idea itself and aims to help to solve World-scale problems. After classes I like to climb and ride a bike.<br><br><br></td><br />
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</table><br />
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<a name="instructors"><h2>Instructors</h2></a></br><br />
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<td><img src="https://static.igem.org/mediawiki/2014/7/74/Prof._Bielecki.jpg" width="200px"; alt="prof.Jacek_Bielecki"></td> <td><br><h4>Prof. Jacek Bielecki</h4>Education: MSc, University of Warsaw, 1975; PhD, University of Warsaw, 1981; Associated professor, Warsaw University, 1995; Professor at University of Warsaw , 1996; Vice Dean of Faculty of Biology, 1996 - 1999, and 1999-2002 <br />
Research interests: Molecular mechanisms of virulence of bacteria Listeria monocytogenes, especially the role of a hemolysin, listeriolysin O (LLO). <br><br><br><br><br></td><br />
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<td><img src="https://static.igem.org/mediawiki/2014/3/33/LSB_1.jpg" width="200px"; alt="Łukasz_Borowski"></td> <td><br><h4><br>Dr. Łukasz Borowski</h4>Currently I am working as a postdoctoral fellow at The Laboratory of RNA Biology and Functional Genomics, led by Andrzej Dziembowski. My research is mainly focused on RNA biology. So far I have been involved in scientific projects concerning RNA decay in human <br />
mitochondria. We have discovered that human mitochondrial RNA degradation mediated by PNPase-hSuv3 complex takes place in distinct foci. Currently my main goal is to identify and characterise different protein complexes involved in RNA metabolism in human cells.<br><br><br><br><br></td><br />
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<td><img src="https://static.igem.org/mediawiki/2014/e/e3/Clipboard01.jpg" width="200px"; alt="Dr. Takao Ishikawa"></td> <td><br><h4><br>Dr. Takao Ishikawa</h4>Born in Tokyo, working and living in Warsaw, I am interested in protein-protein interactions. In my research, I employ in silico modeling of protein complexes and theirs verification by molecular biology methods. I am not only fascinated by life science, but also being in love with popularization of science. Giving lectures for students, teachers, and children give me a lot of fun!<br><br><br><br><br><br><br><br><br></td><br />
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</table><br />
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<td><img src="https://static.igem.org/mediawiki/2014/5/51/Zdj%C4%99cie.jpg" width="200px"; alt="Dr. Radosław_Stachowiak"></td> <td><br><h4><br>Dr. Radosław Stachowiak</h4>My adventure with biology dates back to the previous century when I became a biotechnology student at the University of Warsaw. Currently I am a postdoc at the Department of Applied Microbiology. My research concerns molecular mechanisms of bacterial pathogenesis and possible applications of bacterial toxins in biotechnology. Apart from research activity I take care of students and guide them through experimental work. Additionally, I am a caretaker of Synthetic Biology Students’ Association and enjoy working with enthusiastic students and being involved in their daring projects.<br><br></td><br />
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<td><img src="https://static.igem.org/mediawiki/2014/1/1a/Marcin_Ziemniak.jpg" width="200px"; alt="Marcin_Ziemniak"></td> <td><br><h4><br>Marcin Ziemniak</h4>I have graduated from the University of Warsaw (M.S in bioorganic chemistry and B.S in molecular biology). Currently, I am a PhD student at the Faculty of Physics, University of Warsaw . The scientific project in which I am involved is interdisciplinary, hence in my research I employ not only chemical synthesis of modified nucleotides but also a variety of biochemical and biophysical techniques. I am also quite interested in structural biology and nanotechnology. Apart from academia I enjoy travelling, taking photos and sport. I am also an avid fan of science fiction and fantasy. <br><br><br><br></td><br />
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<a name="acknowledgements"><h2>Acknowledgements</h2></a></br><br />
Special thanks to <b>prof. Paweł Golik</b> and <b>dr. Katarzyna Tońska</b> from Institute of Genetics and Biotechnology, The University of Warsaw for allowing us working in their lab.<br><br><br />
<br />
We would also like to thank <b>prof. Chuan He</b> and his team from Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago for inspiration and sending us plasmids with lanthanide-responsive system created by them.<br><br><br />
<br />
All people that helped us in every possible way and to whom we are grateful are listed here (in alphabetic order): <br><br><br />
<br />
<b>Prof. Dariusz Bartosik</b> - Institute of Microbiology<br><br />
<b>Prof. Jacek Bielecki</b> - Department of Applied Microbiology<br><br />
<b>Dr. Łukasz Borowski</b> - Instructor, the Institute of Genetics and Biotechnology<br><br />
<b>Dr. Piotr Borsuk</b> - Associate Dean of Academic Affairs University of Warsaw<br><br />
<b>Dr. Łukasz Drewniak</b> - Laboratory of Environmental Pollution Analysis<br><br />
<b>Dr. Maciej Garstka</b> - Associate Dean for Financial Affairs University of Warsaw<br><br />
<b>Prof. Anna Giza-Poleszczuk</b> - Vice-Rector for Development and Financial Policy University of Warsaw<br><br />
<b>Prof. Paweł Golik</b> - Director of the Institute of Genetics and Biotechnology<br><br />
<b>Dr. Takao Ishikawa</b> - Institute of Biochemistry<br> <br />
<b>Prof. Jacek Jemielity</b> - Centre of New Technologies and Division of Biophysics, Faculty of Physics, University of Warsaw<br> <br />
<b>Prof. Agnieszka Mostowska</b> - Dean of The Faculty of Biology University of Warsaw<br> <br />
<b>Prof. Marcin Pałys</b> - Rector of University of Warsaw<br> <br />
<b>Prof. Aleksandra Danuta Skłodowska</b> - Laboratory of Environmental Pollution Analysis<br><br />
<b>Dr. Radosław Stachowiak</b> - Instructor, Institute of Microbiology<br><br />
<b>Dr. Magdalena Szuplewska</b> -Institute of Microbiology<br><br />
<b>Dr. Katarzyna Tońska</b> - Deputy Director of the Institute of Genetics and Biotechnology<br><br />
<b>Prof. Andrzej Twardowski</b> - Director of the College of Inter-Faculty Individual Studies in Mathematics and Natural Sciences, University of Warsaw<br><br><br />
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<hr noshade="noshade" /><br />
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<a name="sponsors"><h2>Sponsors</h2></a><br />
<br />
<div style='float:left'><br />
<a href="http://www.uw.edu.pl/"><img src='https://static.igem.org/mediawiki/2014/0/08/UW.gif' width="200px" ></a><br />
</div><br />
<div style='position:absolute; left:41%'><br />
<a href="http://www.biol.uw.edu.pl/"><img src='https://static.igem.org/mediawiki/2014/7/73/WB.png' width="300px"></a><br />
</div><br />
<div style='position:absolute; left:63%'><br />
<a href="http://www.mismap.uw.edu.pl/"><img src='https://static.igem.org/mediawiki/2014/c/c7/MISMaP.png' width="170px"></a><br />
</div><br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<div style='float:left'><br />
<a href="http://www.fuw.pl/"><img src='https://static.igem.org/mediawiki/2014/c/c2/FUW.jpg' width="180px" ></a><br />
</div><br />
<div style='position:absolute; left:41%'><br />
<a href="http://www.aabiot.com/"><img src='https://static.igem.org/mediawiki/2014/4/45/Fit_company_profile_LOGO-Mono-Blue12-zmniejszone.jpg' width="300px"></a><br />
</div><br />
<div style='position:absolute; left:63%'><br />
<a href="http://www.cft.edu.pl/ "><img src='https://static.igem.org/mediawiki/2014/6/60/Cft.jpg' width="170px"></a><br />
</div><br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<div style='float:left'><br />
<a href="http://www.genomed.pl/ "><img src='https://static.igem.org/mediawiki/2014/9/92/Genomed.png' width="400px" ></a><br />
</div><br />
<br><br />
<div style='position:absolute; left:50%'><br />
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ASamsel
http://2014.igem.org/Team:Warsaw/EXTRAS
Team:Warsaw/EXTRAS
2014-10-17T17:44:29Z
<p>ASamsel: </p>
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<li><a href="https://2014.igem.org/Team:Warsaw/HP#lanthan_hospital">Lanthan Hospital</a></li><br />
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<a href="/Team:Warsaw/EXTRAS">EXTRAS</a><br />
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<h1>Technology</h1> </br><br />
<hr noshade="noshade" /><br />
<br />
<br />
<a name="bioprocess"><h2>Bioprocess</h2></a></br><br />
Electronic equipment is the fastest growing waste stream in many countries. E waste grows rapidly because markets in which these products are produced cross the other side of the ‘Digital Divide’. We stand in front of the following problem: what we are we going to do with all those computers, smartphones which we buy? All these products become obsolete or unwanted within 1-3 years of purchase. Where will we find a new source of metals necessary to fabricate electronic equipment?</br><br />
Using old and broken WEEE in our project not only gives us a source of metals to produce new smartphones or computers, but also solves problem of storing WEEE.</br><br />
This figure shows a view on the context of re-use of EEE or its components. At the end of the use EEE returns to producers or to companies, which specialize in reuse of e-waste. <br />
The next step is preparation for re-use. On this step waste electronic equipment is deconstructed. Not only computer memories or other parts which contains valuable metals are re-use. We also want to recover such materials as plastic or glass. That step requires labor work or special machines which will prepare WEE for another point of the plan. <br />
Some parts of the TVs or computers may not be useful in another manufactory. Sometimes we need to remarked parts which we need and work in place with specialist equipment. <br />
Later we can use our bacteria with lanthanides binding sequences to re-use lanthanides.</br><br />
1) Removing from WEEE plastic and stirring parts which are abundant of rare earth metals such as lanthanides, especially computer memories. Stirring increase availability WEEE with acid which is produced by Thiobacillus ferrooxidans. It display terbium from alloy.</br><br />
2) Effluent from our bioreactor has low pH (about 2,5) what is a lethal for E.coli. That is why we add Ca(OH)2 to effluent. It increases our pH to a level which is optimal to E.coli (about 7). Ca(OH)2 is also cheap and it is not increasing cost of the process very much.</br> <br />
3) Effluent goes to packed column bioreactor in which E.coli is immobilized. It gets across whole column. Ions of terbium binds and senses trough periplasmic domain.</br><br />
4) Column (with no initial effluent) is flushed by NaCl. PmrB can easily denaturate in NaCl aq , losing shape. Ions of terbium do not precipitate with NaCl aq and do not make insoluble components with it.</br><br />
5) Ions of terbium are recovered from solution by electrolysis <br />
<hr noshade="noshade" /><br />
<br />
<a name="discussion"><h2>Discussion</h2></a></br><br />
Our process finds not only a way to recovery the lanthanides, but also other metals such as copper ( it takes place at first point of it and other rare metals.</br><br />
Our way of recovery lanthanides does not produce pollution but it also is a safe way of managing with WEE.<br />
<hr noshade="noshade" /><br />
<br />
<a name="challenges"><h2>Challenges</h2></a></br><br />
<hr noshade="noshade" /><br />
<br />
<a name="alternative_methods"><h2>Alternative methods</h2></a></br><br />
Our final system was of course not the only possibility. There were some points where we had to decide...<br />
<h4>Reporter protein</h4><br />
Finally we decided for GFP protein because of it's versality, simplicity and fact that GFP does not require any additional reagents (eg. IPTG).</br><br />
We could have used other fluorescent proteins, for instance superfolder fluorescent proteins constructed by iGEM Warsaw 2013 Team, but regular GFP was the simpliest choice.</br><br />
<h4>Binding agent</h4><br />
Although, unfortunately, we could not have implemented lanthanide binding system because of lack of time, we had several ideas how to accomplish this.</br><br />
<u>Poly-LBT peptide</u></br><br />
First was to construct a poly-LBT peptide which would be then anchored in the outer membrane of <i>E. coli</i> or transported to periplasm. We discarded this idea due to problems with modelling of behaviour of this polymer and problems with wet-lab design.</br><br />
<u>PmrB over-expression</u></br><br />
Another idea consisted of PmrB dependent over-expression of PmrB(LBT) protein. In such system we would have pmr<sup>C</sup> promoter - some logical device to boost the signal - PmrB(LBT), so in presence of lanthanides amount of PmrB(LBT) protein per cell would rise sharply, which should allow effective binding of lanthanides.</br><br />
<u>Small peptide fused with LBT</u></br><br />
Our final and probably best idea was to create a construct peptide of such composition:</br><br />
BBa_J32015(<i>E. coli</i> periplasm signal peptide)-structure peptide(ubiquitin or 1L2Y [BBa_K1459015]) - lanthanide binding tag.</br><br />
The plan was to create a small, 'rubbish' protein which would only bind lanthanides without having any physiological function in cell (since we were afraid whether over-expression of PmrB would be cytotoxic).</br><br />
<hr noshade="noshade" /></div>
ASamsel
http://2014.igem.org/File:Recycling.jpg
File:Recycling.jpg
2014-10-17T09:29:17Z
<p>ASamsel: Recycling_process_itw</p>
<hr />
<div>Recycling_process_itw</div>
ASamsel
http://2014.igem.org/Team:Warsaw/EXTRAS
Team:Warsaw/EXTRAS
2014-10-17T09:28:09Z
<p>ASamsel: </p>
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<div class="main-content"><br />
<h1>Technology</h1> </br><br />
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<a name="bioprocess"><h2>Bioprocess</h2></a></br><br />
Electronic equipment is the fastest growing waste stream in many countries. E waste grows rapidly because markets in which these products are produced cross the other side of the ‘Digital Divide’. We stand in front of the following problem: what we are we going to do with all those computers, smartphones which we buy? All these products become obsolete or unwanted within 1-3 years of purchase. Where will we find a new source of metals necessary to fabricate electronic equipment?</br><br />
Using old and broken WEEE in our project not only gives us a source of metals to produce new smartphones or computers, but also solves problem of storing WEEE.</br><br />
This figure shows a view on the context of re-use of EEE or its components. At the end of the use EEE returns to producers or to companies, which specialize in reuse of e-waste. <br />
The next step is preparation for re-use. On this step waste electronic equipment is deconstructed. Not only computer memories or other parts which contains valuable metals are re-use. We also want to recover such materials as plastic or glass. That step requires labor work or special machines which will prepare WEE for another point of the plan. <br />
Some parts of the TVs or computers may not be useful in another manufactory. Sometimes we need to remarked parts which we need and work in place with specialist equipment. <br />
Later we can use our bacteria with lanthanides binding sequences to re-use lanthanides. <br />
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<a name="discussion"><h2>Discussion</h2></a></br><br />
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<a name="challenges"><h2>Challenges</h2></a></br><br />
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<a name="alternative_methods"><h2>Alternative methods</h2></a></br><br />
Our final system was of course not the only possibility. There were some points where we had to decide...<br />
<h4>Reporter protein</h4><br />
Finally we decided for GFP protein because of it's versality, simplicity and fact that GFP does not require any additional reagents (eg. IPTG).</br><br />
We could have used other fluorescent proteins, for instance superfolder fluorescent proteins constructed by iGEM Warsaw 2013 Team, but regular GFP was the simpliest choice.</br><br />
<h4>Binding agent</h4><br />
Although, unfortunately, we could not have implemented lanthanide binding system because of lack of time, we had several ideas how to accomplish this.</br><br />
<u>Poly-LBT peptide</u></br><br />
First was to construct a poly-LBT peptide which would be then anchored in the outer membrane of <i>E. coli</i> or transported to periplasm. We discarded this idea due to problems with modelling of behaviour of this polymer and problems with wet-lab design.</br><br />
<u>PmrB over-expression</u></br><br />
Another idea consisted of PmrB dependent over-expression of PmrB(LBT) protein. In such system we would have pmr<sup>C</sup> promoter - some logical device to boost the signal - PmrB(LBT), so in presence of lanthanides amount of PmrB(LBT) protein per cell would rise sharply, which should allow effective binding of lanthanides.</br><br />
<u>Small peptide fused with LBT</u></br><br />
Our final and probably best idea was to create a construct peptide of such composition:</br><br />
BBa_J32015(<i>E. coli</i> periplasm signal peptide)-structure peptide(ubiquitin or 1L2Y [BBa_K1459015]) - lanthanide binding tag.</br><br />
The plan was to create a small, 'rubbish' protein which would only bind lanthanides without having any physiological function in cell (since we were afraid whether over-expression of PmrB would be cytotoxic).</br><br />
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ASamsel