|
|
| (216 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"); |
| − | margin-top : 65px;
| + | |
| − | text-align : center;
| + | |
| − | font-size : 20px;
| + | |
| | } | | } |
| − | #headerimg { | + | |
| − | width : 100%; | + | |
| | + | #header2 { |
| | + | background-image : url("https://static.igem.org/mediawiki/2017/b/b7/PHA2.png"); |
| | } | | } |
| − | body {
| + | #header3 { |
| − | margin : 0; | + | background-image : url("https://static.igem.org/mediawiki/2017/0/0c/CaCO3_Alizarin.png"); |
| − | pading : 0;
| + | |
| | } | | } |
| − | h1 {
| + | #header4 { |
| − | text-align : center; | + | background-image : url("https://static.igem.org/mediawiki/2017/3/3e/SilicaspongePB.jpeg"); |
| | } | | } |
| − | .leftparagraphe { | + | .buttons { |
| − | float : left;
| + | width : 300px; |
| − | margin-left : 5%;
| + | height : 100px; |
| − | width : 40%; | + | |
| − | text-align : justify;
| + | |
| − | }
| + | |
| − | .rightimg {
| + | |
| − | float : right;
| + | |
| − | margin-right : 5%;
| + | |
| − | width : 40%;
| + | |
| − | height : 100%; | + | |
| − | background : red;
| + | |
| − | }
| + | |
| − | .rightparagraphe {
| + | |
| − | float : right;
| + | |
| − | margin-right : 5%;
| + | |
| − | width : 40%;
| + | |
| − | text-align : justify;
| + | |
| | position : relative; | | 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); |
| | + | |
| | } | | } |
| − | .leftimg { | + | .line { |
| − | float : left; | + | display : inline-block; |
| − | margin-left : 5%;
| + | |
| − | width : 40%;
| + | |
| − | height : 100%;
| + | |
| − | background : red;
| + | |
| − | position : relative;
| + | |
| − | }
| + | |
| − | .block {
| + | |
| | width : 100%; | | width : 100%; |
| − | height : 400px; | + | height : 100px; |
| | margin-top : 50px; | | margin-top : 50px; |
| − | overflow : hidden; | + | text-align : center; |
| | } | | } |
| | </style> | | </style> |
| | | | |
| − | <head> | + | <body> |
| | | | |
| − | <div id="header"><img id="headerimg" src="">Find me a header</div> | + | <div id=header1 class="header">BIOMATERIAL</div> |
| | | | |
| − | </head> | + | <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> |
| | | | |
| − | <body> | + | <div id="header2" class=header>PHA</div> |
| | | | |
| − | <h1>Project X</h1> | + | <div class=textbody> |
| | + | <div id=PHA> |
| | + | |
| | + | <h1>Why P3HB?</h1> |
| | + | <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> |
| | + | </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 class=block><div class=leftparagraphe>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Nam sit amet sodales ex. Sed tempus nisl at diam tempus facilisis. In pellentesque vulputate erat a vulputate. In maximus risus et accumsan scelerisque. Nullam vel porttitor metus. Donec varius risus eu urna sodales, ac convallis metus faucibus. Sed quis sapien tellus. Cras efficitur arcu et tellus vehicula maximus. Ut tincidunt dui ut sollicitudin malesuada. Quisque maximus, ex in ultrices tristique, purus felis ultrices mauris, ut sagittis lorem turpis et lorem. Etiam quis arcu aliquam, molestie risus vel, aliquam sapien. Ut metus arcu, sodales eget ex ac, lobortis venenatis lorem. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos. Vivamus tempus mollis lorem, eget accumsan orci venenatis in. Fusce id sapien mi. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Nam et magna orci. Sed sodales ante elit, id porttitor arcu rutrum in. Nunc porta non tortor eu rutrum. Praesent mollis tincidunt tortor id sodales. Mauris nibh arcu, tincidunt sollicitudin mi nec, volutpat blandit sem. Aenean condimentum ante non interdum tincidunt. In vel nisl nulla. Morbi sodales massa eget justo tempus, sit amet tristique libero. </div><img class=rightimg src=""></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 class=block><img class=leftimg src=""><div class=rightparagraphe>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Donec consectetur vitae enim suscipit rutrum. Phasellus aliquam, mi molestie pretium porta, orci turpis condimentum leo, sit amet consequat dui est vitae lorem. Morbi enim velit, euismod at leo malesuada, malesuada tempor justo. Cras vel augue nec leo hendrerit scelerisque. Praesent venenatis nunc sed tellus lacinia sodales. Pellentesque non sagittis magna, ac aliquet lacus. Suspendisse eu eros nisl. Nunc tincidunt tempor mauris. Vestibulum condimentum ac arcu ac pretium. Cras fermentum efficitur tristique. Fusce ac nibh a ex hendrerit vulputate. Aliquam aliquet a felis vitae ullamcorper. Suspendisse pellentesque libero id mi commodo ultrices. Nullam vel nisi dapibus, convallis ex sit amet, rhoncus mi. Etiam nec diam fermentum nulla vulputate volutpat vitae eu nulla. Pellentesque non dignissim arcu, accumsan dapibus ex. Donec rhoncus orci et quam maximus tempor. Vestibulum malesuada dui at dapibus dictum. Duis convallis faucibus sem eu iaculis. Vestibulum vel arcu ex. Sed porta lacus ut libero imperdiet, pellentesque tempor ante luctus. Mauris porta augue dui, sed commodo magna cursus vitae. Phasellus sollicitudin, ex in imperdiet dictum, mauris diam tempus libero, vel tincidunt nulla magna in enim. Donec et leo malesuada, sodales mi ac, vestibulum massa. Suspendisse potenti. Curabitur dignissim lorem id. </div></div>
| |
| − | <div class=block><div class=leftparagraphe>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Praesent gravida dui et nisi egestas, sit amet volutpat orci posuere. Integer vel pulvinar sem, mattis vulputate dolor. Interdum et malesuada fames ac ante ipsum primis in faucibus. Vestibulum vestibulum leo eu ante ullamcorper, quis posuere velit faucibus. Fusce quis metus diam. In et est quam. Suspendisse molestie vel nunc nec dignissim. Maecenas ex felis, porta et ipsum ut, interdum tempor elit. Maecenas sagittis suscipit diam in fringilla. Phasellus consectetur rutrum sodales. Cras interdum elit sed euismod elementum. Sed bibendum risus mi, eu laoreet eros lacinia sed. Phasellus a laoreet turpis. Donec ullamcorper est vel risus sagittis, sit amet bibendum eros condimentum. Aliquam vitae pellentesque tortor, non condimentum ligula. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Sed eget tempus magna. Vivamus diam massa, iaculis sit amet lacus ut, tempus blandit ex. Mauris posuere convallis ligula a egestas. Proin tempus elementum tempor. Praesent eu feugiat ante. Aenean in porttitor ante. Aliquam auctor ornare massa vel iaculis. Aliquam ut nulla vel est vulputate tempus. Curabitur vitae magna id augue vestibulum consectetur euismod et odio. Mauris ultrices gravida odio, ac porttitor purus pharetra nec. In hac habitasse platea dictumst. </div><img class=rightimg src=""></div>
| |
| − | <div class=block><div class=rightparagraphe>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Praesent gravida dui et nisi egestas, sit amet volutpat orci posuere. Integer vel pulvinar sem, mattis vulputate dolor. Interdum et malesuada fames ac ante ipsum primis in faucibus. Vestibulum vestibulum leo eu ante ullamcorper, quis posuere velit faucibus. Fusce quis metus diam. In et est quam. Suspendisse molestie vel nunc nec dignissim. Maecenas ex felis, porta et ipsum ut, interdum tempor elit. Maecenas sagittis suscipit diam in fringilla. Phasellus consectetur rutrum sodales. Cras interdum elit sed euismod elementum. Sed bibendum risus mi, eu laoreet eros lacinia sed. Phasellus a laoreet turpis. Donec ullamcorper est vel risus sagittis, sit amet bibendum eros condimentum. Aliquam vitae pellentesque tortor, non condimentum ligula. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Sed eget tempus magna. Vivamus diam massa, iaculis sit amet lacus ut, tempus blandit ex. Mauris posuere convallis ligula a egestas. Proin tempus elementum tempor. Praesent eu feugiat ante. Aenean in porttitor ante. Aliquam auctor ornare massa vel iaculis. Aliquam ut nulla vel est vulputate tempus. Curabitur vitae magna id augue vestibulum consectetur euismod et odio. Mauris ultrices gravida odio, ac porttitor purus pharetra nec. In hac habitasse platea dictumst. </div><img class=leftimg src=""></div>
| |
| − | <div class=block><div class=leftparagraphe>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Praesent gravida dui et nisi egestas, sit amet volutpat orci posuere. Integer vel pulvinar sem, mattis vulputate dolor. Interdum et malesuada fames ac ante ipsum primis in faucibus. Vestibulum vestibulum leo eu ante ullamcorper, quis posuere velit faucibus. Fusce quis metus diam. In et est quam. Suspendisse molestie vel nunc nec dignissim. Maecenas ex felis, porta et ipsum ut, interdum tempor elit. Maecenas sagittis suscipit diam in fringilla. Phasellus consectetur rutrum sodales. Cras interdum elit sed euismod elementum. Sed bibendum risus mi, eu laoreet eros lacinia sed. Phasellus a laoreet turpis. Donec ullamcorper est vel risus sagittis, sit amet bibendum eros condimentum. Aliquam vitae pellentesque tortor, non condimentum ligula. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Sed eget tempus magna. Vivamus diam massa, iaculis sit amet lacus ut, tempus blandit ex. Mauris posuere convallis ligula a egestas. Proin tempus elementum tempor. Praesent eu feugiat ante. Aenean in porttitor ante. Aliquam auctor ornare massa vel iaculis. Aliquam ut nulla vel est vulputate tempus. Curabitur vitae magna id augue vestibulum consectetur euismod et odio. Mauris ultrices gravida odio, ac porttitor purus pharetra nec. In hac habitasse platea dictumst. </div><img class=rightimg src=""></div>
| |
| − | <div class=block><div class=rightparagraphe>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Praesent gravida dui et nisi egestas, sit amet volutpat orci posuere. Integer vel pulvinar sem, mattis vulputate dolor. Interdum et malesuada fames ac ante ipsum primis in faucibus. Vestibulum vestibulum leo eu ante ullamcorper, quis posuere velit faucibus. Fusce quis metus diam. In et est quam. Suspendisse molestie vel nunc nec dignissim. Maecenas ex felis, porta et ipsum ut, interdum tempor elit. Maecenas sagittis suscipit diam in fringilla. Phasellus consectetur rutrum sodales. Cras interdum elit sed euismod elementum. Sed bibendum risus mi, eu laoreet eros lacinia sed. Phasellus a laoreet turpis. Donec ullamcorper est vel risus sagittis, sit amet bibendum eros condimentum. Aliquam vitae pellentesque tortor, non condimentum ligula. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Sed eget tempus magna. Vivamus diam massa, iaculis sit amet lacus ut, tempus blandit ex. Mauris posuere convallis ligula a egestas. Proin tempus elementum tempor. Praesent eu feugiat ante. Aenean in porttitor ante. Aliquam auctor ornare massa vel iaculis. Aliquam ut nulla vel est vulputate tempus. Curabitur vitae magna id augue vestibulum consectetur euismod et odio. Mauris ultrices gravida odio, ac porttitor purus pharetra nec. In hac habitasse platea dictumst. </div><img class=leftimg src=""></div>
| |
| − | <div class=block><div class=leftparagraphe>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Praesent gravida dui et nisi egestas, sit amet volutpat orci posuere. Integer vel pulvinar sem, mattis vulputate dolor. Interdum et malesuada fames ac ante ipsum primis in faucibus. Vestibulum vestibulum leo eu ante ullamcorper, quis posuere velit faucibus. Fusce quis metus diam. In et est quam. Suspendisse molestie vel nunc nec dignissim. Maecenas ex felis, porta et ipsum ut, interdum tempor elit. Maecenas sagittis suscipit diam in fringilla. Phasellus consectetur rutrum sodales. Cras interdum elit sed euismod elementum. Sed bibendum risus mi, eu laoreet eros lacinia sed. Phasellus a laoreet turpis. Donec ullamcorper est vel risus sagittis, sit amet bibendum eros condimentum. Aliquam vitae pellentesque tortor, non condimentum ligula. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Sed eget tempus magna. Vivamus diam massa, iaculis sit amet lacus ut, tempus blandit ex. Mauris posuere convallis ligula a egestas. Proin tempus elementum tempor. Praesent eu feugiat ante. Aenean in porttitor ante. Aliquam auctor ornare massa vel iaculis. Aliquam ut nulla vel est vulputate tempus. Curabitur vitae magna id augue vestibulum consectetur euismod et odio. Mauris ultrices gravida odio, ac porttitor purus pharetra nec. In hac habitasse platea dictumst. </div><img class=rightimg src=""></div>
| |
| | | | |
| − | <img > | + | <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>Cell-lysis</h1> |
| | + | <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> |
| | + | </div> |
| | + | <h1>Application</h1> |
| | + | <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> |
| | + | 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> |
| | + | |
| | + | |
| | + | |
| | + | |
| | + | |
| | + | </div> |
| | + | |
| | + | |
| | + | |
| | + | |
| | + | <div id="header3" class=header>CALCIUM CARBONATE</div> |
| | + | <article class="textbody"> |
| | + | <section> |
| | + | <h1>Introduction</h1> |
| | + | <div class="text2"> |
| | + | <div class="text2left"> |
| | + | <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. |
| | + | </p> |
| | + | </div> |
| | + | <div class="text2right"> |
| | + | <div> |
| | + | <img src="https://static.igem.org/mediawiki/2017/b/be/CaCO3.png" /> |
| | + | </div> |
| | + | |
| | + | <span><b>Figure 1:</b> Chemical structure of calcium carbonate.</span> |
| | + | </div> |
| | + | |
| | + | </div> |
| | + | </section> |
| | + | <section> |
| | + | <h1>Back to basics</h1> |
| | + | <div class="text2"> |
| | + | <div class="text2left"> |
| | + | <p> |
| | + | Biomineralization is process by which living organisms are naturally able to produce minerals. |
| | + | 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> |
| | + | There are three distinct pathways of bacterial calcium carbonate precipitation: |
| | + | </p> |
| | + | <p>1) biologically controlled - cellular specific control of formation of the mineral (exoskeleton, bone or teeth) ,</p> |
| | + | <p>2) biologically - influenced - passive mineral precipitation caused through the presence on the surface of the cell of organic matter and </p> |
| | + | <p>3) biologically- induced - which is the chemical alteration of an environment by biological activity. </p> |
| | + | |
| | + | </div> |
| | + | <div class="text2right"> |
| | + | <div><img src="https://static.igem.org/mediawiki/2017/0/0b/Microscope_CaCO3.png" /></div> |
| | + | |
| | + | <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> |
| | + | </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="text2"> |
| | + | <div class="text2left"> |
| | + | <p> |
| | + | 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 class="text2right"> |
| | + | <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> |
| | + | <div class="text1"> |
| | + | <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> |
| | + | <div> |
| | + | <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 id=Polysilicate> |
| | + | <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 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> |
| | + | <!-- If you need more block/subdivision of a block, ask me--> |
| | + | |
| | + | <script src="http://ajax.googleapis.com/ajax/libs/jquery/1.8.3/jquery.min.js"></script> |
| | + | |
| | | | |
| | </body> | | </body> |
| | | | |
| | </html> | | </html> |
| | + | |
| | + | |
| | | | |
| | {{Paris_Bettencourt/footer}} | | {{Paris_Bettencourt/footer}} |
Why P3HB?
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 E.Coli 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.
What is P3HB?
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.
In nature, microorganisms such as Ralstonia Eutrophus produce P3HB in response to physiological stress. It is used as an energy storage ready to be metabolised when nutrients become scarce.
The gene comes from Ralstonia Eutrophus 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.
Confirmation and characterization
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.
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.
Flow cytometer analysis of cell stained with NileRed with BBa_K1149051Cell-lysis
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.
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.
Application
P3HB has a range of application from
medical to
bio packaging . 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.
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.
