|
|
| (183 intermediate revisions by 4 users not shown) |
| Line 1: |
Line 1: |
| | {{Paris_Bettencourt}} | | {{Paris_Bettencourt}} |
| | <html> | | <html> |
| − |
| |
| − |
| |
| | | | |
| | | | |
| Line 9: |
Line 7: |
| | <style type="text/css"> | | <style type="text/css"> |
| | | | |
| − | #header { | + | #header1 { |
| − | width : 100%;
| + | background-image : url("https://static.igem.org/mediawiki/2017/5/5e/Biomaterial_main.jpeg"); |
| − | height : 100vh;
| + | |
| − | background-image : url(""); | + | |
| − | background-attachment: fixed;
| + | |
| − | background-position : center;
| + | |
| − | background-repeat : no-repeat;
| + | |
| − | background-size : cover;
| + | |
| − | background-color : rgb(200,200,200);
| + | |
| − | text-align : center;
| + | |
| − | font-size : 50px;
| + | |
| − | display : block;
| + | |
| | } | | } |
| − | #side { | + | |
| − | height : 100%; | + | |
| − | width : 15%;
| + | #header2 { |
| − | position : fixed;
| + | background-image : url("https://static.igem.org/mediawiki/2017/b/b7/PHA2.png"); |
| − | right : 0;
| + | |
| | } | | } |
| − | #sidemenu { | + | #header3 { |
| − | text-align : left; | + | background-image : url("https://static.igem.org/mediawiki/2017/0/0c/CaCO3_Alizarin.png"); |
| − | right : 0px;
| + | |
| − | width : 100%;
| + | |
| − | position : absolue;
| + | |
| − | margin-top : 50%;
| + | |
| − | }
| + | |
| − | #sidemenu ul {
| + | |
| − | display : block;
| + | |
| | } | | } |
| − | #sidemenu li { | + | #header4 { |
| − | list-style-type : none;
| + | background-image : url("https://static.igem.org/mediawiki/2017/3/3e/SilicaspongePB.jpeg"); |
| − | }
| + | |
| − | #sidemenu a:hover {
| + | |
| − | border-left : 2px solid #f64553; | + | |
| − | }
| + | |
| − | #sidemenu li a{
| + | |
| − | text-decoration : none;
| + | |
| − | color : black;
| + | |
| − | line-height : 30px;
| + | |
| − | }
| + | |
| − | .liste1 { | + | |
| − | list-style-type: none;
| + | |
| − | line-height : 30px;
| + | |
| − | display : block;
| + | |
| − | }
| + | |
| − | .liste1 a { | + | |
| − | text-decoration : none;
| + | |
| − | color : black;
| + | |
| − | }
| + | |
| − | #element1:focus {
| + | |
| − | color : red;
| + | |
| − | text-decoration : underline;
| + | |
| | } | | } |
| | + | .buttons { |
| | + | width : 300px; |
| | + | height : 100px; |
| | + | position : relative; |
| | + | display : inline-block; |
| | + | margin : 0px auto; |
| | + | text-decoration : none !important; |
| | + | line-height : 100px; |
| | + | font-size : 15px; |
| | + | margin-left : 10px; |
| | + | margin-rigth : 10px; |
| | + | font-size : 25px; |
| | + | border : solid rgb(64,64,64); |
| | | | |
| − | html {
| |
| − | height : 100%;
| |
| | } | | } |
| − | body {
| + | .line { |
| − | margin : 0; | + | display : inline-block; |
| − | pading : 0; | + | width : 100%; |
| − | font-family : lato, sans-serif; | + | height : 100px; |
| − | height : 100%;
| + | margin-top : 50px; |
| − | }
| + | |
| − | #textbody h1 {
| + | |
| | text-align : center; | | text-align : center; |
| − | font-size : 40px;
| |
| − | margin-bottom : 25px;
| |
| − | }
| |
| − | #textbody {
| |
| − | width : 700px;
| |
| − | margin : 0 auto;
| |
| − | text-align : justify;
| |
| − | padding-top : 75px;
| |
| − | }
| |
| − | .block {
| |
| − | width : 100%;
| |
| − | margin-top : 50px;
| |
| − | }
| |
| − | #textbody .maintitle {
| |
| − | width : 100%;
| |
| − | font-size : 50px;
| |
| − | text-align : center !important;
| |
| − | margin-top : 50px;
| |
| − | font-weight : bold;
| |
| | } | | } |
| | </style> | | </style> |
| | | | |
| | <body> | | <body> |
| − | <div id=side>
| + | |
| − | | + | <div id=header1 class="header">BIOMATERIAL</div> |
| − | <li><a href="#PHA">PHA</a>
| + | |
| − | <ul>
| + | < div class= textbody>< div class= line><a href="# header2" class= buttons> PHA</a><a href="# header3" class= buttons>Calcium Carbonate</a><a href="# header4" class= buttons > Polysilicate< /a></ div></ div> |
| − | <li id=element1 class=liste1><a href="#block1">Lorem 1</a></li>
| + | |
| − | <li id=element2 class= liste1><a href="# block2" >Lorem 2</a></li> | + | <div id="header2" class=header>PHA</div> |
| − | <li id=element3 class=liste1><a href="#block3">Lorem 3</a></li>
| + | |
| − | <li id=element4 class=liste1><a href="# block4" >Lorem 4</a></li> | + | <div class=textbody> |
| − | <li id=element5 class=liste1><a href="#block5">Lorem 5</a></li>
| + | <div id=PHA> |
| − | </ul>
| + | |
| − | </li>
| + | <h1>Why P3HB?</h1> |
| − | <li><a href="#Calcium">Calcium</a>
| + | <div class=text1> Poly-3-HydroxyButyrate (P3HB) is the perfect biomaterial to demonstrate our 3D control. It is a bioplastic already used for 3D printing. However, we produced our P3HB with our own <i>E.Coli</i> DH5 alpha strain using the BBa_K1149051 biobrick (Imperial College London 2013) from the iGEM registry. After successfully cloning it into our bacteria and characterising the production with flow cytometry, we modified the biobrick by adding a cell-lysis system.</div> |
| − | <ul>
| + | |
| − | <li id=element1 class=liste1><a href="# block6" >Lorem 1</a></li> | + | |
| − | <li id=element2 class= liste1><a href="#block7"> Lorem 2</ a></ li> | + | |
| − | <li id=element3 class=liste1><a href="#block8">Lorem 3</a></li>
| + | |
| − | <li id=element4 class=liste1><a href="#block9">Lorem 4</a></li>
| + | |
| − | <li id=element5 class=liste1><a href="#block10">Lorem 5</a></li>
| + | |
| − | </ul>
| + | |
| − | </li>
| + | |
| − | <li><a href=#Polysilicate>Polysilicate</a>
| + | |
| − | <ul>
| + | |
| − | <li id=element1 class=liste1><a href="#block11">Lorem 1</a></li>
| + | |
| − | <li id=element2 class=liste1><a href="#block12">Lorem 2</a></li>
| + | |
| − | <li id=element3 class=liste1><a href="#block13">Lorem 3</a></li>
| + | |
| − | <li id=element4 class=liste1><a href="#block14">Lorem 4</a></li>
| + | |
| − | <li id=element5 class=liste1><a href="#block15">Lorem 5</a></li>
| + | |
| − | </ul>
| + | |
| − | </li>
| + | |
| − | </div>
| + | |
| | </div> | | </div> |
| | + | <h1>What is P3HB?</h1> |
| | + | <div class=text2><div class=text2left> P3HB comes from the large family of polymers called polyhydroxyalkanoate (PHA). We were interested in using this biomaterial not only for its mechanical properties, but also for its ecological effects as it is a biodegradable plastic.<br> In nature, microorganisms such as <i>Ralstonia Eutrophus </i> produce P3HB in response to physiological stress. It is used as an energy storage ready to be metabolised when nutrients become scarce.</br></div> |
| | | | |
| − | <div id="header">Find me a header</div> | + | <div class= text2right> The gene comes from <i> Ralstonia Eutrophus</i> H16, a gram-negative bacterium producing P3HB thanks to a 3 enzymes pathway: PhaC, PhaA and PhaB. |
| | + | The first enzyme PhaA codes for 3-ketothiolase. Its role is to combine 2 molecules of Acetyl-Coa into Acetoacetyl-Coa. The newly formed Acetoacetyl-Coa is reduced by Acetylacetyl-Coa reductase, coded by PhaB, into (R) - 3 - Hydroxybutyryl-Coa. At last, P(3HB) synthase, coded by PhaC, polymerises the latter product to form Poly-3-Hydroxybutyrate or P3HB.</div> |
| | + | </div> |
| | + | |
| | + | <h1>Confirmation and characterization</h1> |
| | + | <div class=text2><div class=text2left> We stained our cells using a Nile Red solution (0.3mg/mL in DMSO). Nile red is a lipophilic stain that can be used to detect P3HB presence due to red fluorescence. Thus, to characterize the production of P3HB, we used Fluorescence-activated cell sorting (FACS), specifically the FL2 (575 BP filter) and FL3 (620 BP filter) channels to measure the intensity of the fluorescence of the Nile Red (excitation wavelength between 520 and 550 nm, and emission wavelength between 590 and 630 nm) stained cell containing P3HB.</br> |
| | + | We used Flow Cytometry to characterize the part as we believe it is the best technique compared to Gas Chromatography/ Mass Spectrometry. Using fluorescence-activated cell sorting allowed us to do hundreds of samples a day at minimal price whereas using GC/MS is not only expensive, but you can only run a few samples a day.</div> |
| | | | |
| − | <div id=textbody> | + | |
| − | <div id=PHA><p class=maintitle>PHA</p>
| + | <div class= text2right>< img src= "https://static.igem.org/mediawiki/2017/5/51/P3HBPARISBETTENCOURT.png">< span> Flow cytometer analysis of cell stained with NileRed with BBa_K1149051</ span></div> |
| − | | + | </div> |
| − | <h1>Sub-title1</h1>
| + | |
| − | <p>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Nulla aliquet auctor fringilla. Mauris imperdiet est a risus vulputate, at fringilla nisi vehicula. Aliquam sodales enim vel nibh luctus imperdiet. Phasellus eu felis viverra velit viverra viverra id scelerisque elit. Phasellus sed eros nunc. Duis ipsum metus, tempor vel ipsum vitae, iaculis convallis enim. Praesent rutrum elit sit amet consectetur faucibus. Praesent porttitor non ex et lobortis. Donec malesuada sed odio a scelerisque. Aenean tempor enim sed bibendum malesuada. Nunc pharetra tellus sed massa condimentum, ut placerat lectus vestibulum. Donec ornare pulvinar eros, nec laoreet erat egestas at. Morbi consequat maximus. </p>
| + | <h1>Cell-lysis</h1> |
| − | </div>
| + | <div class=text2><div class=text2left> To link our P3HB production to our project, we needed a way to extract the product without using any chemicals or tampering with the cells. Implementing a cell-lysis system into the bacteria enabled us not only that, but also to fulfill our safety concerns.</div> |
| − | | + | <div class= text2right> By shining lights on our cells producing P3HB, the cell- lysis system is activated, meaning it breaks down the bacteria, therefore releasing the product out of the cell. The P3HB will then form an aggregate with the other P3HB granules around it. By orientating the lasers to specific positions, the P3HB keeps on aggregating until we have the final product.</ div> |
| − | <h1>Sub-title2</h1>
| + | </div> |
| − | <p>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Nulla aliquet auctor fringilla. Mauris imperdiet est a risus vulputate, at fringilla nisi vehicula. Aliquam sodales enim vel nibh luctus imperdiet. Phasellus eu felis viverra velit viverra viverra id scelerisque elit. Phasellus sed eros nunc. Duis ipsum metus, tempor vel ipsum vitae, iaculis convallis enim. Praesent rutrum elit sit amet consectetur faucibus. Praesent porttitor non ex et lobortis. Donec malesuada sed odio a scelerisque. Aenean tempor enim sed bibendum malesuada. Nunc pharetra tellus sed massa condimentum, ut placerat lectus vestibulum. Donec ornare pulvinar eros, nec laoreet erat egestas at. Morbi consequat maximus. </p>
| + | <h1>Application</h1> |
| − | </div>
| + | <div class=text2> P3HB has a range of application from <a href="https://www.hindawi.com/journals/ijps/2014/789681/#B17">medical </a>to<a href="lhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4307263/"> bio packaging </a>. As it is biodegradable and renewable when composted, P3HB gets a lot of attention, and for the right reasons. Many new companies are now producing the thermoplastic, so much that it reaches a production capacity of over 10,000 tons per year.</br> |
| − | <div id=block3>
| + | Therefore, we believe P3HB and PHAs in general will be a material of the future. This is one of the reasons why we chose to use this biomaterial for our proof-of-concept, on top of its physical properties that would allow the consumer to use our P3HB as a regular material for 3D printing.</div></div></div> |
| − | <h1>Sub-title3</h1>
| + | |
| − | <h4> something</h4>
| + | |
| − | <p>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Donec ultrices nulla tincidunt, hendrerit diam at, condimentum sem. Quisque ac condimentum tellus, at viverra risus. Aenean venenatis at est sed rhoncus. Suspendisse libero sapien, dictum quis convallis tempus, dapibus et tortor. Etiam tincidunt ut turpis quis tincidunt. Donec vitae suscipit arcu. Nullam finibus purus leo, sed vestibulum mi sodales a. Ut vestibulum quis metus eu ullamcorper. Morbi elementum tellus eu nibh bibendum, non laoreet lorem molestie. Nam interdum dictum leo, at malesuada nisi fermentum quis. Aliquam cursus non ex vitae euismod. In eget mi et ligula viverra cursus venenatis et justo. Proin sed lacus aliquam, interdum metus a, tempor sapien. Maecenas non cursus sem. Nulla auctor sollicitudin ullamcorper. Pellentesque faucibus sed est in varius. Vestibulum ullamcorper elementum molestie. Maecenas posuere lectus ut placerat volutpat. Vivamus vulputate, nisi in efficitur sodales, felis ex rhoncus neque, sed sollicitudin nibh purus nec tellus. Donec. </p>
| + | |
| − | </div>
| + | |
| − | <div id=block4>
| + | |
| − | <h1>Sub-title4</h1>
| + | </div> |
| − | <p>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Pellentesque vel vehicula felis. Aenean non elit magna. Integer tincidunt cursus fringilla. Vestibulum ante ipsum primis in faucibus orci luctus et ultrices posuere cubilia Curae; Quisque tempor vitae sem at varius. Donec sed rutrum justo. Cras ut elementum sem. Cras tempus, nunc eget tempor feugiat, quam diam aliquet nisi, id pharetra mauris tortor vitae nibh. Curabitur id turpis eget nibh vehicula cursus nec et sapien. Aenean laoreet aliquam gravida. Donec a ex tempus, varius augue quis, pharetra massa. Proin in gravida urna. Pellentesque dapibus elit quis enim mattis, a porttitor purus rutrum. Donec eget consectetur nisl, sed commodo dui. Suspendisse lobortis justo ac ornare lacinia. Pellentesque quis arcu justo. Praesent pulvinar, lorem ut consequat tincidunt, augue nunc laoreet orci, quis pharetra arcu augue eu ligula. Vestibulum ante ipsum primis in faucibus orci luctus et ultrices posuere cubilia Curae; Pellentesque fermentum mattis ipsum, et tincidunt dolor tempus nec. Maecenas ac arcu eu lacus fringilla ornare non ac erat. Orci varius natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Sed vehicula euismod placerat. Ut volutpat felis vitae lorem pulvinar, eget finibus dui molestie. Vestibulum ante ipsum primis in faucibus orci luctus et ultrices posuere cubilia Curae; Nulla egestas est vitae odio sagittis condimentum euismod et magna. Vivamus porttitor diam tortor, quis tincidunt dolor pulvinar quis. Suspendisse tincidunt ornare eros. Nullam at lobortis lectus. Integer sit amet bibendum turpis. Suspendisse enim elit, placerat ac iaculis sed, maximus vel lectus. Praesent fermentum facilisis eros. Pellentesque tincidunt, enim vel porta commodo, justo massa laoreet lorem, ac interdum massa augue in turpis. Fusce elit est, rutrum sit amet velit ac, ultrices pellentesque metus. Cras viverra rutrum eros id vestibulum. Donec at luctus neque, a pellentesque diam. Donec vel congue nibh, in auctor tellus. Duis nisi lectus, placerat vel odio et, dapibus pharetra dui. Mauris sagittis purus non ipsum eleifend, ut interdum neque tincidunt.</p>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <h1>Sub-title5</h1>
| + | |
| − | <p>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Nulla aliquet auctor fringilla. Mauris imperdiet est a risus vulputate, at fringilla nisi vehicula. Aliquam sodales enim vel nibh luctus imperdiet. Phasellus eu felis viverra velit viverra viverra id scelerisque elit. Phasellus sed eros nunc. Duis ipsum metus, tempor vel ipsum vitae, iaculis convallis enim. Praesent rutrum elit sit amet consectetur faucibus. Praesent porttitor non ex et lobortis. Donec malesuada sed odio a scelerisque. Aenean tempor enim sed bibendum malesuada. Nunc pharetra tellus sed massa condimentum, ut placerat lectus vestibulum. Donec ornare pulvinar eros, nec laoreet erat egestas at. Morbi consequat maximus. </p>
| + | <div id="header3" class=header>CALCIUM CARBONATE</div> |
| − | </div>
| + | <article class="textbody"> |
| − | </ div> <div id=Calcium><p class= maintitle>Calcium Carbonate</p> | + | <section> |
| − | | + | <h1>Introduction</h1> |
| − | <p> In recent years, the interest in obtaining microbial cement gained its popularity along with such problems as fractures and fissures in concrete structures which is created by weathering, land subsidence, faults, earthquakes and human activities. Synthetic biology proposed a novel way to repair or remediate caused problems - is biomineralization of calcium carbonate using microbes such as Bacillus species. The application of microbial concrete to construction may simplify some of the existing construction processes and revolutionize the ways of new construction process. </p> | + | |
| − | </div>
| + | |
| − | | + | |
| − | <p> Though multiple mechanisms are proposed to participate calcium carbonate, most efforts focused on the ureolysis driven precipitation from different specie origins ( P Anbu et al. 2016). The limit to apply this method in diverse scenario could be the additional precursor urea itself. Also, because the precipitation is physico- chemically dominant, when the environment is too acidic, ureolysis driven mechanism is likely to fail. </p> | + | In recent years, the interest in obtaining microbial cement has gained popularity. This is in part because of the potential of microbial cement to overcome problems such as fractures and fissures in concrete structures which are created by weathering, land subsidence, faults, earthquakes and human activities. Synthetic biology has proposed a novel way to repair and remediate these problems. One of the possible solutions is biomineralization of calcium carbonate using microbes such as <i>Bacillus species</i>.The application of microbial concrete in construction may simplify some of the existing construction processes and revolutionise them. |
| − | </div>
| + | </p> |
| − | | + | </div> |
| − | <p> Instead of calcite precipitation from natural microbial, many other organisms also have the power to produce calcium carbonate, such as corals. In stony coral, Stylophora pistillata, 4 acid-rich proteins (CARPs 1–4; GenBank accession numbers KC148537–KC148539 and KC493647) were identified to be responsible for calcium carbonate precipitation ( Tail Mass et al. 2013). </p> | + | |
| − | </div>
| + | <div> |
| − | | + | <img src="https://static.igem.org/mediawiki/2017/b/be/CaCO3.png" /> |
| − | <p>The putative mechanism of calcium carbonate nucleation is that the highly acidic pocket of CARPs localize the substrate, buffer and catalyze the reaction between calcium ion and carboxylate (Figure 3). In a high-resolution magnetic resonance spectroscopy analyses, evidences have been shown that the calcification in stony coral is mainly biologically controlled and relatively robust, due to the template-induced nucleation mediated by the skeleton organic matrix, in particular, acid-rich proteins like CARPs (Stanislas Von Euw et al. 2017) .</p>
| + | </div> |
| − | <img src="https:// 2017.igem.org/ File:CARP_work.png " alt="альтернативный текст" /> | + | |
| − | </div>
| + | <span><b>Figure 1:</b> Chemical structure of calcium carbonate.</span> |
| − | <div id=block10>
| + | </div> |
| − | <p>As such, bioreaction of aragonite formation is far from the thermodynamic equilibrium. It may even compromise with acidification and very low mineral saturation state (E. Tambutté & A. A. Venn et al. 2015). All CARP 1-4 can be cloned and expressed in E.coli BL21 strain and remain the activity. Each of them is able to independently catalyze the calcification in artificial seawater. This method we decided to use and improve in our project/ due to the our project. </p>
| + | |
| − | </div>
| + | </div> |
| − | </div>
| + | </section> |
| − | <div id= Polysilicate><p class=maintitle> Polysilicate</p> | + | <section> |
| − | | + | <h1>Back to basics</h1> |
| − | <h1>Sub-title1</h1>
| + | <div class="text2"> |
| − | <p>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Nulla aliquet auctor fringilla. Mauris imperdiet est a risus vulputate, at fringilla nisi vehicula. Aliquam sodales enim vel nibh luctus imperdiet. Phasellus eu felis viverra velit viverra viverra id scelerisque elit. Phasellus sed eros nunc. Duis ipsum metus, tempor vel ipsum vitae, iaculis convallis enim. Praesent rutrum elit sit amet consectetur faucibus. Praesent porttitor non ex et lobortis. Donec malesuada sed odio a scelerisque. Aenean tempor enim sed bibendum malesuada. Nunc pharetra tellus sed massa condimentum, ut placerat lectus vestibulum. Donec ornare pulvinar eros, nec laoreet erat egestas at. Morbi consequat maximus. </p>
| + | <div class="text2left"> |
| − | </div>
| + | |
| − | <div id=block12>
| + | Biomineralization is process by which living organisms are naturally able to produce minerals. |
| − | <h1>Sub-title2</h1>
| + | Production of microbial calcium carbonate (CaCO3) is a widely studied and a promising technology with various engineering applications. The use of CaCo3 include: treatment of concrete, manufacturing of construction materials (such as building bricks and fillers for rubber), synthesis of plastics and inks. <br> |
| − | <p>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Nulla aliquet auctor fringilla. Mauris imperdiet est a risus vulputate, at fringilla nisi vehicula. Aliquam sodales enim vel nibh luctus imperdiet. Phasellus eu felis viverra velit viverra viverra id scelerisque elit. Phasellus sed eros nunc. Duis ipsum metus, tempor vel ipsum vitae, iaculis convallis enim. Praesent rutrum elit sit amet consectetur faucibus. Praesent porttitor non ex et lobortis. Donec malesuada sed odio a scelerisque. Aenean tempor enim sed bibendum malesuada. Nunc pharetra tellus sed massa condimentum, ut placerat lectus vestibulum. Donec ornare pulvinar eros, nec laoreet erat egestas at. Morbi consequat maximus. </p>
| + | There are three distinct pathways of bacterial calcium carbonate precipitation: |
| − | </div>
| + | </p> |
| − | | + | <p>1) biologically controlled - cellular specific control of formation of the mineral (exoskeleton, bone or teeth) ,</p> |
| − | <h1>Sub-title3</h1>
| + | <p>2) biologically - influenced - passive mineral precipitation caused through the presence on the surface of the cell of organic matter and </p> |
| − | <p>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Donec ultrices nulla tincidunt, hendrerit diam at, condimentum sem. Quisque ac condimentum tellus, at viverra risus. Aenean venenatis at est sed rhoncus. Suspendisse libero sapien, dictum quis convallis tempus, dapibus et tortor. Etiam tincidunt ut turpis quis tincidunt. Donec vitae suscipit arcu. Nullam finibus purus leo, sed vestibulum mi sodales a. Ut vestibulum quis metus eu ullamcorper. Morbi elementum tellus eu nibh bibendum, non laoreet lorem molestie. Nam interdum dictum leo, at malesuada nisi fermentum quis. Aliquam cursus non ex vitae euismod. In eget mi et ligula viverra cursus venenatis et justo. Proin sed lacus aliquam, interdum metus a, tempor sapien. Maecenas non cursus sem. Nulla auctor sollicitudin ullamcorper. Pellentesque faucibus sed est in varius. Vestibulum ullamcorper elementum molestie. Maecenas posuere lectus ut placerat volutpat. Vivamus vulputate, nisi in efficitur sodales, felis ex rhoncus neque, sed sollicitudin nibh purus nec tellus. Donec. </p>
| + | <p>3) biologically- induced - which is the chemical alteration of an environment by biological activity. </p> |
| − | </div>
| + | |
| − | | + | </div> |
| − | <h1>Sub-title4</h1>
| + | <div class="text2right"> |
| − | <p>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Pellentesque vel vehicula felis. Aenean non elit magna. Integer tincidunt cursus fringilla. Vestibulum ante ipsum primis in faucibus orci luctus et ultrices posuere cubilia Curae; Quisque tempor vitae sem at varius. Donec sed rutrum justo. Cras ut elementum sem. Cras tempus, nunc eget tempor feugiat, quam diam aliquet nisi, id pharetra mauris tortor vitae nibh. Curabitur id turpis eget nibh vehicula cursus nec et sapien. Aenean laoreet aliquam gravida. Donec a ex tempus, varius augue quis, pharetra massa. Proin in gravida urna. Pellentesque dapibus elit quis enim mattis, a porttitor purus rutrum. Donec eget consectetur nisl, sed commodo dui. Suspendisse lobortis justo ac ornare lacinia. Pellentesque quis arcu justo. Praesent pulvinar, lorem ut consequat tincidunt, augue nunc laoreet orci, quis pharetra arcu augue eu ligula. Vestibulum ante ipsum primis in faucibus orci luctus et ultrices posuere cubilia Curae; Pellentesque fermentum mattis ipsum, et tincidunt dolor tempus nec. Maecenas ac arcu eu lacus fringilla ornare non ac erat. Orci varius natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Sed vehicula euismod placerat. Ut volutpat felis vitae lorem pulvinar, eget finibus dui molestie. Vestibulum ante ipsum primis in faucibus orci luctus et ultrices posuere cubilia Curae; Nulla egestas est vitae odio sagittis condimentum euismod et magna. Vivamus porttitor diam tortor, quis tincidunt dolor pulvinar quis. Suspendisse tincidunt ornare eros. Nullam at lobortis lectus. Integer sit amet bibendum turpis. Suspendisse enim elit, placerat ac iaculis sed, maximus vel lectus. Praesent fermentum facilisis eros. Pellentesque tincidunt, enim vel porta commodo, justo massa laoreet lorem, ac interdum massa augue in turpis. Fusce elit est, rutrum sit amet velit ac, ultrices pellentesque metus. Cras viverra rutrum eros id vestibulum. Donec at luctus neque, a pellentesque diam. Donec vel congue nibh, in auctor tellus. Duis nisi lectus, placerat vel odio et, dapibus pharetra dui. Mauris sagittis purus non ipsum eleifend, ut interdum neque tincidunt.</p>
| + | <div><img src="https://static.igem.org/mediawiki/2017/0/0b/Microscope_CaCO3.png" /></div> |
| − | </div>
| + | |
| − | <div id=block15>
| + | <span><b>Figure 2:</b> Alizarin Red staining for detection of calcium carbonate composites in the precipitated proteins: A) stained calcium carbonate powder - positive control, B) stained sample of BL21 extracted protein precipitation in CaCl 1M solution - negative control , C) stained sample of CARPs extracted protein precipitation in CaCl 1M solution.</span> |
| − | <h1>Sub-title5</h1>
| + | </div> |
| − | <p>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Nulla aliquet auctor fringilla. Mauris imperdiet est a risus vulputate, at fringilla nisi vehicula. Aliquam sodales enim vel nibh luctus imperdiet. Phasellus eu felis viverra velit viverra viverra id scelerisque elit. Phasellus sed eros nunc. Duis ipsum metus, tempor vel ipsum vitae, iaculis convallis enim. Praesent rutrum elit sit amet consectetur faucibus. Praesent porttitor non ex et lobortis. Donec malesuada sed odio a scelerisque. Aenean tempor enim sed bibendum malesuada. Nunc pharetra tellus sed massa condimentum, ut placerat lectus vestibulum. Donec ornare pulvinar eros, nec laoreet erat egestas at. Morbi consequat maximus. </p>
| + | |
| − | </div>
| + | </div> |
| | + | <div class="text1"> |
| | + | <div><img style="width:596px" src="https://static.igem.org/mediawiki/2017/8/81/CARPs_precipitation.png" /></div> |
| | + | |
| | + | <span><b>Figure 3:</b> Stained calcium carbonate deposits formed in the present of CARPs in artificial seawater(ASW).</span> |
| | + | </div> |
| | + | <div class="text1"> |
| | + | <p>The most commonly found mechanism in bacteria for CaCO3 precipitation has been to generate an alkaline environment through different physiological actions. Precipitation of CaCO3 by ureolytic bacteria is the most straightforward and most easily controlled mechanism of microbially induced calcium carbonate precipitation. It also has the potential to produce high amounts of carbonates in short period of time.</p> |
| | + | </div> |
| | + | </section> |
| | + | <section> |
| | + | <h1>Alternative </h1> |
| | + | |
| | + | <div class="text2left"> |
| | + | |
| | + | Besides the CaCO3 precipitation induced naturally by microbes, many other organisms also have the power to produce calcium carbonate, such as corals. In the stony coral, <i>Stylophora pistillata</i>, 4 acid-rich proteins (CARPs 1–4; GenBank accession numbers KC148537–KC148539 and KC493647) were identified to be responsible for calcium carbonate precipitation. These proteins were found in the study of changes in the growth of corals with increasing of acidity in the ocean. |
| | + | |
| | + | </p> |
| | + | <p>As such, bioreaction of calcite formation is far from the thermodynamic equilibrium. It may even compromise with acidification and very low mineral saturation state (E. Tambutté & A. A. Venn et al. 2015). </p> |
| | + | </div> |
| | + | |
| | + | <div><img src="https://static.igem.org/mediawiki/2017/5/5c/CaCO3-2.png" /></div> |
| | + | |
| | + | <span><b>Figure 4:</b>The pathway of calcium carbonate precipitation through production of coral acid-rich proteins in <i>E.Coli</i>. </span> |
| | + | </div> |
| | + | |
| | + | </div> |
| | + | <div class="text1"> |
| | + | <p>In our project, coral acid-rich proteins (CARPs) was cloned and expressed in <i>E.coli</i> BL21 strain. They were characterized for their ability to induce calcium carbonate precipitation. |
| | + | </p> |
| | + | </div> |
| | + | <div class="text2"> |
| | + | <div class="text2left"> |
| | + | <p> |
| | + | According to the putative mechanism of calcium carbonate nucleation by CARP, a highly acidic pocket brings together a calcium ion and a carboxylate molecule thus favouring their reaction (Figure 5). Evidence based on high-resolution magnetic resonance spectroscopy has shown that the calcification in stony coral is mainly controlled by CARPS embedded in skeleton organic matrix. |
| | + | |
| | + | </p> |
| | + | </div> |
| | + | <div class="text2right"> |
| | + | <div><img src="https:// static.igem.org/ mediawiki/2017/2/25/CARP_work.png" /></div> |
| | + | |
| | + | <span><b>Figure 5:</b>The highly acidic regions of the proteins interact with calcium ions (grey spheres) via coordination chemistry allowing the carboxylate groups to attract and localize calcium ions in a microenvironment, enhancing the local ionic strength. This local interaction results in a shift in pKa, favouring the formation of carbonate. Being a stronger Lewis base, with greater negative charge, carbonates displace carboxyl groups from the proteins to form stable coordination bonds with the calcium on the protein scaffold. |
| | + | </span> |
| | + | </div> |
| | + | |
| | + | </div> |
| | + | |
| | + | <p> The key advantage of CARPs is their power to bypass the acidification of the growth medium and the urea synthesis associated with the classical urease pathway. Furthermore, CaCO3 precipitation with CARPs occurs in one enzymatic step, greatly reducing the metabolic cost for the cell. |
| | + | </p> |
| | + | |
| | + | <img src="https://static.igem.org/mediawiki/2017/e/e6/PAGE-GEL.png" /> |
| | + | </div> |
| | + | <span> |
| | + | <b>Figure 6:</b> |
| | + | SDS-PAGE separated CARP1-CARP4 proteins according to their molecular weight, based on their differential rates of migration through a sieving matrix (a gel) under the influence of an applied electrical field. |
| | + | </span> |
| | + | </div> |
| | + | </section> |
| | + | |
| | + | </article> |
| | + | |
| | + | |
| | + | |
| | + | |
| | + | |
| | + | |
| | + | |
| | + | |
| | + | |
| | + | |
| | + | |
| | + | |
| | + | |
| | + | |
| | + | |
| | + | </div> |
| | + | </div> |
| | + | <div id="header4" class=header>POLYSILICATE</div> |
| | + | <div class=textbody> |
| | + | |
| | + | <div class=text1>Silicate (Si<sub>m</sub>O<sub>n</sub>), the main component of the planet’s crust is also known to be naturally precipitated biologically. It is extensively used for electronic and biologic microimplants. The physical properties of this mineral depend entirely on the microstructure of the silica crystals forming quartz, glass or others. Mineral polysilicate is formed with a great pressure and temperature. It is the variation of those two factors that induce the formation of different kinds of rocks. Some living organisms take advantage of the abundance of silicate in their environment and use it to create their skeleton and shell. Some sponges which can grow up to 3m have a skeleton made of polysilicate. Diatoms, unicellular microalgea, can also cover their cell wall in silica. The formation processes in sponge and diatom are fairly well known. The pathways require multiple proteins, but the key factors have been successfully expressed in <i> E.coli</i> (W MULLER & al). |
| | + | </div> |
| | + | <div class= text2>< div class=text2left> We decided to use Silicatein α from the sponge < i> Suberites domuncula < /i> because it has already been used in iGEM before. First, we used the biobrick Bba_K1890000 from the 2016 TU Delft team, that they kindly sent to us. We created a construct in <a href="http://parts. igem. org/ Part:pSB4K5"> PsB4K5</ a> using < a href= "http://parts.igem.org/Part:BBa_K206000"> Pbad< /a> as a promoter and < a href="http:/ /parts.igem.org/Part:Bba_B0015"> p0015< /a>. After the production culture, we stained the cells with rhodamine 123 (C. -W. Li &all) to test the presence of poysilicate. As shown on the figure, the three populations supposed to produce silicateinα don't show any fluorescence that would indicate the presence of polysilicate. |
| | + | </div> |
| | + | <div class=text2right><img src="https://static.igem.org/mediawiki/2017/7/79/SilicateprodDelftPB.png"</div> |
| | </div> | | </div> |
| | + | <div class=text2><div class=text2left><img src="https://static.igem.org/mediawiki/2017/e/ec/SilicateprodpasteurPB.png"></div> |
| | + | <div class=text2right> Subsequently, we used a biobrick from iGEM Pasteur, that was designed in previous years but never used nor submitted. This biobrick was designed by replacing the protein region responsible for cellulose synthesis by the protein region responsible for the synthesis of Silicatein α. The FACS results we obtain from those cells clearly show a different population between the stained control and the three cell lines with </div> |
| | + | </div></div> |
| | + | </div> |
| | </div> | | </div> |
| | <!-- If you need more block/subdivision of a block, ask me--> | | <!-- If you need more block/subdivision of a block, ask me--> |
