|
|
| Line 9: |
Line 9: |
| | background-color: #292929; | | background-color: #292929; |
| | } | | } |
| − | #banner { | + | #bannerchanged{ |
| − | background-image: ;
| + | width: 100%; |
| | + | overflow: hidden; |
| | + | margin: 0 auto; |
| | } | | } |
| | @media screen and (max-width: 760px) { | | @media screen and (max-width: 760px) { |
| − | #banner { | + | /*#wrapper { |
| − | background-image: ;
| + | width: 100%; |
| | + | overflow: hidden; |
| | + | } |
| | + | #container { |
| | + | width: 100%; |
| | + | margin: 0 auto; |
| | + | }*/ |
| | + | .banner-img { |
| | + | width: 100%; |
| | } | | } |
| | } | | } |
| | + | |
| | ul li{ | | ul li{ |
| | list-style: disc; | | list-style: disc; |
| Line 22: |
Line 33: |
| | </style> | | </style> |
| | | | |
| − | <div id="banner"> | + | <div id="bannerchanged"> |
| − | <h1>GROWING BACK NERVES</h1> | + | <img class="banner-img" src="https://static.igem.org/mediawiki/2018/b/be/T--Pasteur_Paris--Banner_Overview.jpg"> |
| | + | </div> |
| | + | <h1></h1> |
| | </div> | | </div> |
| | | | |
| | | | |
| | <div id="GeneralContent"> | | <div id="GeneralContent"> |
| − | <div id="index" class="block">
| |
| − | <div id="indexContent">
| |
| − | <p><a href="#RIP" class="link">Neurotrophins</a></p>
| |
| − | <p><a href="#NGF" class="link">NGF / Pro-NGF</a></p>
| |
| − | <p><a href="#Kill" class="link">Production & Secretion</a></p>
| |
| − | <p><a href="#References" class="link">References</a></p>
| |
| − | </div>
| |
| − | <div id="indexRight">
| |
| − | <div id="littleButton"></div>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div id="MainContent">
| |
| − | <div class="block title">
| |
| − | <h1>CONTEXT</h1>
| |
| − | <p>Protheses are aimed to enhance the quality of life, independence, mobility, and safety for amputees. The worldwide frequency of amputations has created an increased demand for improved prostheses technologies. Within developing countries, the part of the population with physical disabilities in demand of a prosthesis is estimated at about 0.5%. In 2050, approximatively 3 million people, in the United States, will live with a limb loss <sup>[1]</sup>.</p>
| |
| − |
| |
| − | <p>Over the past decade, many types of prostheses have been created. One of them is the myoelectric prosthesis, which captures the electromyography (EMG) signal of residual limb muscle through surface electrodes on the skin. These kinds of robotic prostheses allow amputees to recover some autonomy and to accomplish simple everyday gestures. Yet, they are still limited by the number of controllable moves and by the lack of sensory feedback <sup>[1]</sup>.</p>
| |
| − |
| |
| − | <p>Today, the direct attachment of the prosthesis to the skeleton introduces the concept of osseointegration. This kind of prosthesis provides the patient with precise and reliable control of the prosthesis, regardless of the environmental conditions or the limb position. The opportunity to record and stimulate the neuromuscular system allows the intuitive control and a better understanding of sensory perception <sup>[2]</sup>.</p>
| |
| − |
| |
| − | <p>The central nervous system (CNS), made up of the brain and the spinal cord aids to integrate, influence and coordinate activities across the whole organism. The neural tissues bring information from one region of the body to another by sending electrical signals along the axon <sup>[3]</sup>. The motor or efferent neurons of the peripherical nervous system (PNS) communicate with muscle and are under voluntary control. They go from the spinal cord to the arms, hands, legs, feet and allow to transmit nerve impulses away from the CNS, leading to an action <sup>[3]</sup>. To increase the electrical signal speed, the axon is surrounded by myelin, produced by another type of cells called Schwann cells <sup>[4]</sup>. These cells twist around the axon and prevent the loss of electrical signal. After limb amputation, the main issue of the human-machine communication is due to nerves damage. Indeed, the electrical signal cannot be transmitted because the peripheral nerve cells are unable to activate target muscle from the limbs with signal from the brain <sup>[4]</sup>.</p>
| |
| − |
| |
| | <div id="MainContent"> | | <div id="MainContent"> |
| − | <div class="block title">
| |
| − | <h1>OUR SOLUTION</h1>
| |
| − | <p><i>The central idea of our project is to find a way to have motor nerves of amputees grow back and connect to our interface. Literature studies led us to think of neurotrophins as the perfect molecules to start our research on. Indeed, this family of peptides are growth factors specialized in the regulation of neuronal development, survival, plasticity and nervous system function<sup>[5]</sup>.</i></p>
| |
| − | </div>
| |
| − | <div class="block title"><h3 style="text-align: left;">What are neurotrophins? </h3></div>
| |
| − | <div class="block full">
| |
| − | <p>The first neurotrophin to be discovered was the Nerve Growth Factor (NGF). It was described in 1952 by Rita Levy Montalcini and Viktor Hamburger<sup>[6]</sup>. Since then, many other neurotrophins were discovered (BDNF, NT-3, NT-4, NT-6)<sup>[7]</sup> and much progress has been achieved towards understanding how they work. Yet, NGF is still considered a “prototype neurotrophin”[5] and is generally used as an example to describe their function. For this reason, we chose to clone this polypeptide into our bacteria, creating the first component of the NeuronArch project.</p>
| |
| − | </div>
| |
| − | <div class="block title"><h3 style="text-align: left;">The ambiguity between NGF and pro-NGF.</h3></div>
| |
| − | <div class="block half">
| |
| − | <p>The mature-NGF protein (or <FONT face="Raleway">β</FONT>-NGF) results from the cleavage of <FONT face="Raleway">β</FONT>-NGF from a bigger protein called pro-NGF, which contains both a pro-sequence and <FONT face="Raleway">β</FONT>-NGF (see figure 1). Both pro-NGF and <FONT face="Raleway">β</FONT>-NGF proteins are biologically active, and there is, to this date, some sort of uncertainty about the exact effect of pro-NGF. </p>
| |
| − | </div>
| |
| − | <div class="block half">
| |
| − | <img src="https://static.igem.org/mediawiki/2018/2/29/T--Pasteur_Paris--proNGFstructure.png">
| |
| − | <div class="legend"><b>Figure 1: </b>Structure of the Pro-NGF gene. Adapted from Ivanisevic, 2013.</div>
| |
| − | </div>
| |
| − | <div class="block full">
| |
| − | <p>Indeed, if there is no doubt that <FONT face="Raleway">β</FONT>-NGF is a neurotrophic factor, it has still not been determined exactly whether pro-NGF is a neurotrophic or an apoptotic factor<sup>[8]</sup>. Many articles are still in contradiction on this matter and part of the reason comes from the biological pathways by which both proteins act on neurons. Two receptors are involved in their signaling: tropomyosin-related kinase A (TrkA) and the p75 neurotrophin receptor (p75<sup>NTR</sup>). The two proteins (proNGF and <FONT face="Raleway">β</FONT>-NGF) are able to bind both receptors, but it is globally accepted that <FONT face="Raleway">β</FONT>-NGF has a higher affinity for TrkA and pro-NGF has a higher affinity for p75<sup>NTR</sup>. Figure 2 shows a big picture of how the two signaling pathways are thought to work (adapted from[9]).</p>
| |
| − | </div>
| |
| − | <div class="block two-third center">
| |
| − | <img src="https://static.igem.org/mediawiki/2018/d/d2/T--Pasteur_Paris--NGFvsProNGF.png">
| |
| − | <div class="legend"><b>Figure 2: </b>NGF versus pro-NGF signaling pathways. Adapted from Wang, 2014.</div>
| |
| − | </div>
| |
| − | <div class="block full">
| |
| − | <p>
| |
| − | At this point, it might seem that choosing to clone <FONT face="Raleway">β</FONT>-NGF in the bacteria of our interface would be a better idea than pro-NGF. Yet, a few other information led us to think the other way around. <br>
| |
| − | First, even despite of the existing controversy, pro-NGF has been proven to exhibit a neurotrophic activity on some neural cells, even though it is not as strong as mature <FONT face="Raleway">β</FONT>-NGF (there is a fivefold activity difference)<sup>[10]</sup>. This neurotrophic activity is likely generated by a p75<sup>NTR</sup>-dependant mechanism, and depends on the proportion of TrkA and p75<sup>NTR</sup> in the neural cells<sup>[11]</sup>. Moreover, TrkA is mainly expressed in three types of neurons: peripheral sensory neurons, sympathetic neurons and basal forebrain cholinergic neurons, whereas p75<sup>NTR</sup> is more evenly dispersed in different types of neurons<sup>[9]</sup>. Thus, expressing pro-NGF in our case seems like a good choice. <br>
| |
| − | Furthermore, several articles support the idea that the pro-sequence of NGF facilitates, and is even absolutely necessary, for the proper folding of the protein when cloned into bacteria, which is particularly relevant as this protein contains three disulfide bonds and is particularly difficult to express using synthetic biology while keeping its function<sup>[12]</sup>,<sup>[13]</sup>.<br><br></p>
| |
| − | <p>Considering all this information, we finally chose to clone pro-NGF in our interface.</p>
| |
| − | </div>
| |
| − | <div class="block title"><h3 style="text-align: left;">How to produce and secrete pro-NGF from an <i>E. coli</i> biofilm?</h3></div>
| |
| − | <div class="block full">
| |
| − | <img src="https://static.igem.org/mediawiki/2018/1/16/T--Pasteur_Paris--proNGFprodandsec.png">
| |
| − | <div class="legend"><b>Figure 3: </b>pro-NGF production and secretion from <i>E. coli</i></div>
| |
| − | <p>The composition of our final composite biobrick <b><a href="">BBa_K2616000 </a></b> is detailed in the <a href="https://2018.igem.org/Team:Pasteur_Paris/Composite_Part">PARTS</a> submenu of this wiki. </p>
| |
| − | <p>Concretely, it expresses two main proteins:
| |
| − | <ul style="text-align: left;">
| |
| − | <li>pro-NGF, linked to HlyA, a type I secretion system export signal in <i>E. coli.</i> Between the two, we added a TEV protease cleavage site. </li>
| |
| − | <li>TEV protease, a protein from Tobacco Etch Virus that recognizes a specific sequence and cleaves it. We also linked the TEV protease to the same export signal.</li>
| |
| − | </ul>
| |
| − | </p>
| |
| − | <p>Once exported from the cell, the TEV protease can cleave the pro-NGF from HlyA and free the pro-neurotrophin in the external medium.</p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="block title" id="Results">
| |
| − | <h1>RESULTS</h1>
| |
| − | <i style="text-align: left;"><p>Achievements:<br>
| |
| − | <ul>
| |
| − | <li>Successfully cloned a part coding for secretion of NGF in pET43.1a and iGEM plasmid backbone, creating a <b>new composite part</b></li>
| |
| − | <li>Successfully co-transform E. coli with plasmid secreting NGF and plasmid expressing the secretion system, creating bacteria <b>capable of secreting NGF</b> in the medium</li>
| |
| − | <li>Successfully characterized production of NGF thanks to mass spectrometry</li>
| |
| − | <li>Successfully <b>observe axon growth</b> in microfluidic chip in presence of commercial NGF</li>
| |
| − | </ul><br></p>
| |
| − | <p>Next steps:<br>
| |
| − | <ul>
| |
| − | <li><b>Purify</b> secreted NGF, and characterize its effects on neuron growth thanks to our microfluidic device </li>
| |
| − | <li><b>Global proof of concept</b> in a microfluidic device containing neurons in one of the chamber, and our engineered bacteria in the other</li>
| |
| − | </ul>
| |
| − | </p></i>
| |
| − | </div>
| |
| − | <div class="block separator-mark"></div>
| |
| − | <div class="block title"><h1>REFERENCES</h1></div>
| |
| | <div class="block full"> | | <div class="block full"> |
| − | <ul style="text-align: left;"> | + | <p>In science fiction movies, some amputees are equiped with incredibly efficient bionic prostheses that enable them to accomplish everyday gestures like any valid person would. At the beginning of our project, we wanted to understand why this kind of technologies were not yet available. There are millions of amputees around the world, and presently, the very best equipment that can be offered to them is still far from equaling expectations or those seen in the movies. </p> |
| − | <li style="list-style-type: decimal;">P. F. Pasquina, B. N. Perry, M. E. Miller, G. S. F. Ling, and J. W. Tsao, “Recent advances in bioelectric prostheses,” Neurol. Clin. Pract., vol. 5, no. 2, pp. 164–170, Apr. 2015.<br><br></li>
| + | <p>To solve this problem, our team of biologists, physicists, mathematicians, designers and lawyers decided to tackle the problem from several angles. We had less than a year to develop our project, and we were resolute to come up with an innovation worthy of this major stake! </p> |
| − | <li style="list-style-type: decimal;">Y. Li and R. Brånemark, “Osseointegrated prostheses for rehabilitation following amputation,” Unfallchirurg, vol. 120, no. 4, pp. 285–292, Apr. 2017.<br><br></li> | + | <p>We decided to create a universal biological interface that would be able to connect the residual nerves from the amputees’ limbs and the prosthesis. The idea was to express neurotrophins (NGF) from the inside to help the nerves grow back to the prosthesis. However, putting bacteria at a prosthetic interface was both a technical and ethical challenge. Moreover, with this innovation came the necessity to have the device surgically osseointegrated to the patient. This opened our minds to a huge challenge of orthopedic implants: infectious biofilms. These frequently develop around implants and cause heavy infections, very resistant to antibiotics. We decided to tackle both problems at the same time, using synthetic biology to add a barrier of protection against pathogenic bacteria directly into our device. </p> |
| − | <li style="list-style-type: decimal;">A. Farley, C. Johnstone, C. Hendry, and E. McLafferty, “Nervous system: part 1,” Nurs. Stand., vol. 28, no. 31, pp. 46–51, Apr. 2014.<br><br></li> | + | <p>We designed this interface as something that could become the new standard, something that would then be connected to any bionic prosthesis, and that would allow a much greater control on the movement. We mixed synthetic biology with disciplines like physics and industrial design to come up with a prototype (fig. 1). </p> |
| − | <li style="list-style-type: decimal;">“Schwann Cells,” PubMed Health. [Online]. Available: https://www.ncbi.nlm.nih.gov/pubmedhealth/PMHT0025728/. [Accessed: 27-Sep-2018].<br><br></li> | + | <img src="https://static.igem.org/mediawiki/2018/e/e0/T--Pasteur_Paris--Device.svg"> |
| − | <li style="list-style-type: decimal;">L. Ivanisevic and H. U. Saragovi, “Neurotrophins,” in <i>Handbook of Biologically Active Peptides</i>, Elsevier, 2013, pp. 1639–1646.<br><br></li>
| + | <div class="legend"><b>Figure 1: </b>Exploded drawing of NeuronArch's interface device</div> |
| − | <li style="list-style-type: decimal;">S. Cohen, R. Levi-Montalcini, and V. Hamburger, “A nerve growth-stimulating factor isolated from sarcom AS 37 and 180,” <i>Proc. Natl. Acad. Sci. U. S. A.</i>, vol. 40, no. 10, pp. 1014–8, Oct. 1954.<br><br></li>
| + | <p>To do all of that, we decided to cover the inner part of the device with an engineered biofilm of E. coli bacteria. We gave them two main functions: the secretion of NGF and the inhibition of S. aureus quorum sensing (fig. 2). </p> |
| − | <li style="list-style-type: decimal;">S. Razavi, G. Nazem, M. Mardani, E. Esfandiari, S. Esfahani, and H. Salehi, “Neurotrophic factors and their effects in the treatment of multiple sclerosis,” <i>Adv. Biomed. Res.</i>, vol. 4, no. 1, p. 53, 2015.v<br><br></li>
| + | <img src="https://static.igem.org/mediawiki/2018/1/13/T--Pasteur_Paris--ProjectOverview.svg"> |
| − | <li style="list-style-type: decimal;">M. Fahnestock, G. Yu, and M. D. Coughlin, “ProNGF: a neurotrophic or an apoptotic molecule?,” 2004, pp. 101–110.<br><br></li>
| + | <div class="legend"><b>Figure 2: </b>Overview of the biological functions of our genetically modified bacteria.</div> |
| − | <li style="list-style-type: decimal;">H. Wang et al., “The Nerve Growth Factor Signaling and Its Potential as Therapeutic Target for Glaucoma,” <i>Biomed Res. Int.</i>, vol. 2014, pp. 1–10, 2014.<br><br></li>
| + | <p>Since we began working on NeuronArch, we have all endeavored as much as possible to make it become something real. We hope you’ll have as much fun discovering our project through our wiki as we had making it! </p> |
| − | <li style="list-style-type: decimal;">M. Fahnestock et al., “The nerve growth factor precursor proNGF exhibits neurotrophic activity but is less active than mature nerve growth factor,” <i>J. Neurochem.</i>, vol. 89, no. 3, pp. 581–592, 2004.<br><br></li>
| + | <p><u>Team iGEM Pasteur Paris 2018</u></p> |
| − | <li style="list-style-type: decimal;">L. Howard, S. Wyatt, G. Nagappan, and A. M. Davies, “ProNGF promotes neurite growth from a subset of NGF-dependent neurons by a p75<sup>NTR</sup>-dependent mechanism,” <i>Development</i>, vol. 140, no. 10, pp. 2108–2117, 2013.<br><br></li>
| + | |
| − | <li style="list-style-type: decimal;">A. Rattenholl, H. Lilie, A. Grossmann, A. Stern, E. Schwarz, and R. Rudolph, “The pro-sequence facilitates folding of human nerve growth factor from <i>Escherichia coli</i> inclusion bodies,” <i>Eur. J. Biochem.</i>, vol. 268, no. 11, pp. 3296–3303, 2001.<br><br></li>
| + | |
| − | <li style="list-style-type: decimal;">M. Kliemannel et al., “The mature part of proNGF induces the structure of its pro-peptide,” <i>FEBS Lett.</i>, vol. 566, no. 1–3, pp. 207–212, May 2004.<br><br></li>
| + | |
| − | </ul>
| + | |
| | </div> | | </div> |
| | </div> | | </div> |
| | </div> | | </div> |
| | </html> | | </html> |
