Mguillaume (Talk | contribs) |
Mguillaume (Talk | contribs) |
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<html> | <html> | ||
<head> | <head> | ||
− | <meta charset="utf-8"> | + | <meta charset="utf-8"/> |
− | + | <meta content="ie=edge" http-equiv="x-ua-compatible"/> | |
− | + | <meta content="width=device-width, initial-scale=1, shrink-to-fit=no" name="viewport"/> | |
− | + | <title> | |
− | + | Lab Journal | |
− | + | </title> | |
− | + | <!-- include normalizer --> | |
− | + | <link href="https://2017.igem.org/Team:Amsterdam/assets/css/normalize?action=raw&ctype=text/css" rel="stylesheet"/> | |
− | + | <!-- include auto generated css --> | |
− | + | <link href="https://2017.igem.org/Team:Amsterdam/assets/css/all?action=raw&ctype=text/css" rel="stylesheet"/> | |
− | + | <!-- include auto generated javascript --> | |
− | + | <script src="https://2017.igem.org/Team:Amsterdam/assets/js/all?action=raw&ctype=text/javascript"> | |
− | + | </script> | |
− | + | <!-- include mathjax --> | |
− | + | <script src="https://cdnjs.cloudflare.com/ajax/libs/mathjax/2.7.2/MathJax.js?config=TeX-MML-AM_CHTML"> | |
− | + | </script> | |
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</head> | </head> | ||
<div class="site-wrapper"> | <div class="site-wrapper"> | ||
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<div class="container-fluid"> | <div class="container-fluid"> | ||
<div class="navbar-header"> | <div class="navbar-header"> | ||
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <img src="https://static.igem.org/mediawiki/2017/8/86/TAmsterdam_pm_logo.png"/> |
− | + | <a class="navbar-brand nav-link" href="https://2017.igem.org/Team:Amsterdam"> | |
− | + | Photosynthetic Magic | |
− | + | </a> | |
− | + | ||
</div> | </div> | ||
<ul class="list-inline nav navbar-right"> | <ul class="list-inline nav navbar-right"> | ||
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</li> | </li> | ||
<li> | <li> | ||
− | <a class="nav-link" href="https://2017.igem.org/Team:Amsterdam/Silver"> | + | <a class="nav-link" href="https://2017.igem.org/Team:Amsterdam/HP/Silver"> |
HP Silver | HP Silver | ||
</a> | </a> | ||
</li> | </li> | ||
<li> | <li> | ||
− | <a class="nav-link" href="https://2017.igem.org/Team:Amsterdam/Gold_Integrated"> | + | <a class="nav-link" href="https://2017.igem.org/Team:Amsterdam/HP/Gold_Integrated"> |
HP Gold | HP Gold | ||
</a> | </a> | ||
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<div class="labj-sum-mod-container"> | <div class="labj-sum-mod-container"> | ||
<div class="labj-sum-icon-container"> | <div class="labj-sum-icon-container"> | ||
− | <img class="labj-sum-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-sum-icon" src="https://static.igem.org/mediawiki/2017/6/62/TAmsterdam_model_icon.png"/> |
− | + | <p class="labj-icon-text"> | |
− | + | Model | |
− | + | </p> | |
− | + | ||
</div> | </div> | ||
<div class="labj-sum-icon-container"> | <div class="labj-sum-icon-container"> | ||
− | <img class="labj-sum-icon" src="https://static.igem.org/mediawiki/2017/6/ | + | <img class="labj-sum-icon" src="https://static.igem.org/mediawiki/2017/6/63/TAmsterdam_shunt_icon.png"/> |
− | + | <p class="labj-icon-text"> | |
− | + | Production | |
− | + | </p> | |
− | + | ||
</div> | </div> | ||
<div class="labj-sum-icon-container"> | <div class="labj-sum-icon-container"> | ||
− | <img class="labj-sum-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-sum-icon" src="https://static.igem.org/mediawiki/2017/b/b3/TAmsterdam_transporter_icon.png"/> |
− | + | <p class="labj-icon-text"> | |
− | + | Exportation | |
− | + | </p> | |
− | + | ||
</div> | </div> | ||
<div class="labj-sum-icon-container"> | <div class="labj-sum-icon-container"> | ||
− | <img class="labj-sum-icon" src="https://static.igem.org/mediawiki/2017/3/ | + | <img class="labj-sum-icon" src="https://static.igem.org/mediawiki/2017/3/3a/TAmsterdam_sensor_icon.png"/> |
− | + | <p class="labj-icon-text"> | |
− | + | Detection | |
− | + | </p> | |
− | + | ||
</div> | </div> | ||
<div class="labj-sum-icon-container"> | <div class="labj-sum-icon-container"> | ||
− | <img class="labj-sum-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-sum-icon" src="https://static.igem.org/mediawiki/2017/7/78/TAmsterdam_cellfactory_wiki.png"/> |
− | + | <p class="labj-icon-text"> | |
− | + | General Topics | |
− | + | </p> | |
− | + | ||
</div> | </div> | ||
</div> | </div> | ||
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<div class="labj-mod-holder" id="stability"> | <div class="labj-mod-holder" id="stability"> | ||
<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/62/TAmsterdam_model_icon.png"/> |
− | + | ||
</div> | </div> | ||
<div class="labj-mod-cont"> | <div class="labj-mod-cont"> | ||
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/63/TAmsterdam_shunt_icon.png"/> |
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</div> | </div> | ||
<div class="labj-mod-cont"> | <div class="labj-mod-cont"> | ||
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/b/b3/TAmsterdam_transporter_icon.png"/> |
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</div> | </div> | ||
<div class="labj-mod-cont"> | <div class="labj-mod-cont"> | ||
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<div class="labj-mod-holder" id="sensor"> | <div class="labj-mod-holder" id="sensor"> | ||
<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/3a/TAmsterdam_sensor_icon.png"/> |
− | + | ||
</div> | </div> | ||
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/7/78/TAmsterdam_cellfactory_wiki.png"/> |
− | + | ||
</div> | </div> | ||
<div class="labj-mod-cont"> | <div class="labj-mod-cont"> | ||
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<div class="labj-mod-holder" id="stability"> | <div class="labj-mod-holder" id="stability"> | ||
<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/62/TAmsterdam_model_icon.png"/> |
− | + | ||
</div> | </div> | ||
<div class="labj-mod-cont"> | <div class="labj-mod-cont"> | ||
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/63/TAmsterdam_shunt_icon.png"/> |
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</div> | </div> | ||
<div class="labj-mod-cont"> | <div class="labj-mod-cont"> | ||
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/b/b3/TAmsterdam_transporter_icon.png"/> |
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</div> | </div> | ||
<div class="labj-mod-cont"> | <div class="labj-mod-cont"> | ||
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</li> | </li> | ||
</ul> | </ul> | ||
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <img src="https://static.igem.org/mediawiki/2017/2/2a/TAmsterdam_week15t.gif"/> |
− | + | ||
</div> | </div> | ||
</div> | </div> | ||
<div class="labj-mod-holder" id="sensor"> | <div class="labj-mod-holder" id="sensor"> | ||
<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/3a/TAmsterdam_sensor_icon.png"/> |
− | + | ||
</div> | </div> | ||
<div class="labj-mod-cont"> | <div class="labj-mod-cont"> | ||
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<div class="labj-mod-holder" id="stability"> | <div class="labj-mod-holder" id="stability"> | ||
<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/62/TAmsterdam_model_icon.png"/> |
− | + | ||
</div> | </div> | ||
<div class="labj-mod-cont"> | <div class="labj-mod-cont"> | ||
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/63/TAmsterdam_shunt_icon.png"/> |
− | + | ||
</div> | </div> | ||
<div class="labj-mod-cont"> | <div class="labj-mod-cont"> | ||
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<div class="labj-mod-holder" id="transporter"> | <div class="labj-mod-holder" id="transporter"> | ||
<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/b/b3/TAmsterdam_transporter_icon.png"/> |
− | + | ||
</div> | </div> | ||
<div class="labj-mod-cont"> | <div class="labj-mod-cont"> | ||
New pipeline: | New pipeline: | ||
− | <br> | + | <br/> |
− | + | <img src="https://static.igem.org/mediawiki/2017/b/bd/TAmsterdam_week16t.png"/> | |
− | + | <br/> | |
− | + | New appointment with Anton | |
− | + | ||
− | + | ||
− | + | ||
</div> | </div> | ||
</div> | </div> | ||
<div class="labj-mod-holder" id="sensor"> | <div class="labj-mod-holder" id="sensor"> | ||
<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/3a/TAmsterdam_sensor_icon.png"/> |
− | + | ||
</div> | </div> | ||
<div class="labj-mod-cont"> | <div class="labj-mod-cont"> | ||
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/62/TAmsterdam_model_icon.png"/> |
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</div> | </div> | ||
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/63/TAmsterdam_shunt_icon.png"/> |
− | + | ||
</div> | </div> | ||
<div class="labj-mod-cont"> | <div class="labj-mod-cont"> | ||
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/b/b3/TAmsterdam_transporter_icon.png"/> |
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</div> | </div> | ||
<div class="labj-mod-cont"> | <div class="labj-mod-cont"> | ||
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/3a/TAmsterdam_sensor_icon.png"/> |
− | + | ||
</div> | </div> | ||
<div class="labj-mod-cont"> | <div class="labj-mod-cont"> | ||
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/62/TAmsterdam_model_icon.png"/> |
− | + | ||
</div> | </div> | ||
<div class="labj-mod-cont"> | <div class="labj-mod-cont"> | ||
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</li> | </li> | ||
</ul> | </ul> | ||
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <img src="https://static.igem.org/mediawiki/2017/2/21/TAmsterdam_week18st.png"/> |
− | + | ||
</div> | </div> | ||
</div> | </div> | ||
<div class="labj-mod-holder" id="shunt"> | <div class="labj-mod-holder" id="shunt"> | ||
<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/63/TAmsterdam_shunt_icon.png"/> |
− | + | ||
</div> | </div> | ||
<div class="labj-mod-cont"> | <div class="labj-mod-cont"> | ||
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</li> | </li> | ||
</ul> | </ul> | ||
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <img src="https://static.igem.org/mediawiki/2017/b/b0/TAmsterdam_week18gl.png"/> |
− | + | ||
</div> | </div> | ||
</div> | </div> | ||
<div class="labj-mod-holder" id="transporter"> | <div class="labj-mod-holder" id="transporter"> | ||
<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/b/b3/TAmsterdam_transporter_icon.png"/> |
− | + | ||
</div> | </div> | ||
<div class="labj-mod-cont"> | <div class="labj-mod-cont"> | ||
Selected genes and project overview on drive. | Selected genes and project overview on drive. | ||
− | <br> | + | <br/> |
− | + | <img src="https://static.igem.org/mediawiki/2017/1/16/TAmsterdam_week18tr.png"/> | |
− | + | <br/> | |
− | + | Josine needs conclusions on which ones to use and which ones not to use. | |
Make a meeting where you present what you found so far and which transporters specifically you want to express in which strains. Critically go through what you found. Josine has to convince us. | Make a meeting where you present what you found so far and which transporters specifically you want to express in which strains. Critically go through what you found. Josine has to convince us. | ||
− | + | <br/> | |
− | + | You are planning to overexpress this is in Synechocystis as a final product. First, knock this promoter out to see if it has an effect on fumarate transportation. See what the impact is. | |
− | + | <br/> | |
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</div> | </div> | ||
</div> | </div> | ||
<div class="labj-mod-holder" id="sensor"> | <div class="labj-mod-holder" id="sensor"> | ||
<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/3a/TAmsterdam_sensor_icon.png"/> |
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</div> | </div> | ||
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/62/TAmsterdam_model_icon.png"/> |
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</div> | </div> | ||
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− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/63/TAmsterdam_shunt_icon.png"/> |
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</div> | </div> | ||
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</li> | </li> | ||
</ul> | </ul> | ||
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</div> | </div> | ||
</div> | </div> | ||
<div class="labj-mod-holder" id="transporter"> | <div class="labj-mod-holder" id="transporter"> | ||
<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/b/b3/TAmsterdam_transporter_icon.png"/> |
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</div> | </div> | ||
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
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</div> | </div> | ||
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
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− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/3a/TAmsterdam_sensor_icon.png"/> |
− | + | ||
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− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/3a/TAmsterdam_sensor_icon.png"/> |
− | + | ||
</div> | </div> | ||
<div class="labj-mod-cont"> | <div class="labj-mod-cont"> | ||
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/62/TAmsterdam_model_icon.png"/> |
− | + | ||
</div> | </div> | ||
<div class="labj-mod-cont"> | <div class="labj-mod-cont"> | ||
All single knockouts | All single knockouts | ||
− | <br> | + | <br/> |
− | + | <img src="https://static.igem.org/mediawiki/2017/c/c6/TAmsterdam_week22st1.png"/> | |
− | + | <br/> | |
− | + | R_RBCh: 1 O2 1 D-Ribulose 1,5-bisphosphate ==> 2 H+ 1 3-Phospho-D-glycerate 1 2-Phosphoglycolate | |
− | + | <br/> | |
− | + | R_PGLYCP: 1 H2O 1 2-Phosphoglycolate ==> 1 Phosphate 1 Glycolate | |
− | + | <br/> | |
− | + | <img src="https://static.igem.org/mediawiki/2017/1/14/TAmsterdam_week22st2.png"/> | |
− | + | <br/> | |
− | + | R_DDPGA - 1 4-Hydroxy-2-oxoglutarate <=> 1 Pyruvate 1 Glyoxylate | |
− | + | <br/> | |
− | + | R_EHGLAT - 1 2-Oxoglutarate 1 L-erythro-4-Hydroxyglutamate ==> 1 L-Glutamate 1 4-Hydroxy-2-oxoglutarate | |
− | + | <br/> | |
− | + | R_PROAKGOX1r - 1 O2 1 L-Proline 1 2-Oxoglutarate ==> 1 Succinate 1 CO2 1 trans-4-Hydroxy-L-proline | |
− | + | <br/> | |
− | + | R_PHCD - 1 Nicotinamide adenine dinucleotide 2 H2O 1 L-1-Pyrroline-3-hydroxy-5-carboxylate ==> 1 H+ 1 Nicotinamide adenine dinucleotide - reduced 1 L-erythro-4-Hydroxyglutamate | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
</div> | </div> | ||
</div> | </div> | ||
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/63/TAmsterdam_shunt_icon.png"/> |
− | + | ||
</div> | </div> | ||
<div class="labj-mod-cont"> | <div class="labj-mod-cont"> | ||
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</li> | </li> | ||
</ul> | </ul> | ||
− | <img src="https:// | + | <img src="https://2017.igem.org/Team:Amsterdam/images/labjournal/week22sh.png"/> |
− | + | <ul> | |
− | + | <b> | |
− | + | Untargeted | |
− | + | </b> | |
− | + | <li> | |
− | + | Making a detailed schedule with Patricia, including non-lab work to speed things up and get a clear overview | |
− | + | </li> | |
− | + | <li> | |
− | + | Again transformation of MazF cassette into ∆fumC | |
− | + | </li> | |
− | + | </ul> | |
− | + | ||
</div> | </div> | ||
</div> | </div> | ||
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</ul> | </ul> | ||
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</div> | </div> | ||
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
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</div> | </div> | ||
<div class="labj-mod-cont"> | <div class="labj-mod-cont"> | ||
Back to where I was, now with full back-up stocks of DNA, constructs, everything PCRred to the max. | Back to where I was, now with full back-up stocks of DNA, constructs, everything PCRred to the max. | ||
− | <br> | + | <br/> |
− | + | Will do ligation today. | |
− | + | ||
</div> | </div> | ||
</div> | </div> | ||
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</div> | </div> | ||
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
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</li> | </li> | ||
</ul> | </ul> | ||
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <img src="https://static.igem.org/mediawiki/2017/7/7a/TAmsterdam_week26tr1.png"/> |
− | + | <ul> | |
− | + | <li> | |
− | + | sll1314 | |
− | + | </li> | |
− | + | </ul> | |
− | + | <img src="https://static.igem.org/mediawiki/2017/f/fc/TAmsterdam_week26tr2.png"/> | |
− | + | <ul> | |
− | + | <li> | |
− | + | MazF cassette ligated → transformed → colony PCR: confirmed right construct. | |
− | + | </li> | |
− | + | <li> | |
− | + | Monday start with transformation to Synechocystis (now preparing strains and media). | |
− | + | </li> | |
− | + | <b> | |
− | + | Start new project | |
− | + | </b> | |
− | + | <li> | |
− | + | Primers designed for the overexpression of DcuB and DcuC from E.coli | |
− | + | </li> | |
− | + | <li> | |
− | + | Designed gene for synthesis (DcuC from N. Spumigena, 1.300 bp) | |
− | + | </li> | |
− | + | </ul> | |
− | + | ||
− | + | ||
</div> | </div> | ||
</div> | </div> | ||
<div class="labj-mod-holder" id="sensor"> | <div class="labj-mod-holder" id="sensor"> | ||
<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
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− | + | ||
</div> | </div> | ||
<div class="labj-mod-cont"> | <div class="labj-mod-cont"> | ||
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/62/TAmsterdam_model_icon.png"/> |
− | + | ||
</div> | </div> | ||
<div class="labj-mod-cont"> | <div class="labj-mod-cont"> | ||
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<div class="labj-mod-holder" id="shunt"> | <div class="labj-mod-holder" id="shunt"> | ||
<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/63/TAmsterdam_shunt_icon.png"/> |
− | + | ||
</div> | </div> | ||
<div class="labj-mod-cont"> | <div class="labj-mod-cont"> | ||
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</li> | </li> | ||
</ul> | </ul> | ||
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <img src="https://static.igem.org/mediawiki/2017/4/48/TAmsterdam_week27sh1.png"/> |
− | + | <ul> | |
− | + | <li> | |
− | + | Re-inoculate deltaFumC-zwf and WT-Omega | |
− | + | </li> | |
− | + | <li> | |
− | + | I was not able to digest the vector pFLXN with 4 enzymes. After 3 tries I found that the NedI enzyme is not cutting the plasmid. Since it is FLXN it should contain it. So now checking if a new enzyme stock works. | |
− | + | </li> | |
− | + | <li> | |
− | + | I go on holiday this saturday. I arranged a planning with Wen Yang to continue the steps in Synechocystis. | |
− | + | </li> | |
− | + | </ul> | |
− | + | <img src="https://static.igem.org/mediawiki/2017/0/00/TAmsterdam_week27sh2.png"/> | |
− | + | <ul> | |
− | + | <b> | |
− | + | Untargeted | |
− | + | </b> | |
− | + | <li> | |
− | + | ∆fumC mazFΩ is ready: glycerol stocks in -80C | |
− | + | </li> | |
− | + | <li> | |
− | + | Get also evidence without marker | |
− | + | </li> | |
− | + | <li> | |
− | + | Planned to digest gDNA with Sau3AI and MS-ICL with BglII this week and ligate over the weekend -but Sau3AI is not in yet.. | |
− | + | </li> | |
− | + | <li> | |
− | + | Missed part for sequencing, send again - no results yet | |
− | + | </li> | |
− | + | <li> | |
− | + | Making competent cells from different protocols to test efficiency: iGEM Kit Competent Cells test (-20C). Made an excel file that calculates the efficiency from the amount of colonies using this kit. | |
− | + | </li> | |
− | + | <b> | |
− | + | Tip to check concentration: | |
− | + | </b> | |
− | + | <li> | |
− | + | Load 5 uL ladder, 1 uL sample. You would at least have the same concentration of your band and your sample, because then it is five times higher than your ladder (compare bands). Filipe says your concentration must be 100. | |
− | + | </li> | |
− | + | </ul> | |
− | + | ||
− | + | ||
</div> | </div> | ||
</div> | </div> | ||
<div class="labj-mod-holder" id="transporter"> | <div class="labj-mod-holder" id="transporter"> | ||
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− | + | ||
</div> | </div> | ||
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</b> | </b> | ||
− | <br> | + | <br/> |
− | + | <b> | |
− | + | Ecoli. | |
− | + | </b> | |
− | + | <li> | |
− | + | pFL-XN-H1H2-0168 without restriction sites is constructed and is send for sequencing to check the direction of the ligation. | |
− | + | </li> | |
− | + | <li> | |
− | + | IDT sequences arrived, picked them up this morning. I ordered my sequence in three parts. I will dilute them and try to clone the three parts together. (With Wei if possible) | |
− | + | </li> | |
− | + | <b> | |
− | + | Synechocystis. | |
− | + | </b> | |
− | + | <li> | |
− | + | Waiting for full segregation of WT-zwf | |
− | + | </li> | |
− | + | <li> | |
− | + | Waiting for full segregation of deltaFUMC-zwf-Mazf | |
− | + | </li> | |
− | + | <li> | |
− | + | WT-MazF-zwf and Fumc-zwf are not growing, so maybe try once more at perfect OD for the third time. (With Wei if possible) | |
− | </li> | + | </li> |
− | </ | + | </ul> |
+ | <img src="https://static.igem.org/mediawiki/2017/f/fc/TAmsterdam_week29sh.png"/> | ||
+ | <ul> | ||
+ | <b> | ||
+ | Untargeted | ||
+ | </b> | ||
+ | <li> | ||
+ | Started segregation experiment for Synechocystis with Patricia - plate everyday (Wednesday, Thursday, Friday, Saturday) and then every other day (Monday, Wednesday etc). | ||
+ | </li> | ||
+ | <li> | ||
+ | I will make a plan with Patricia for when she is on a holiday to continue some running experiments. | ||
+ | </li> | ||
+ | <li> | ||
+ | Digested the gDNA and my plasmid with the glyoxylate shunt genes and ligated together. Going to transform today. | ||
+ | </li> | ||
+ | <li> | ||
+ | Made competent cells from Mara's protocol (XL1Blue). Tested efficiency - looked online for heat shock protocol. | ||
+ | </li> | ||
+ | <li> | ||
+ | 90 sec is standard in the lab | ||
+ | </li> | ||
+ | <li> | ||
+ | 45 sec was recommended | ||
+ | </li> | ||
+ | <li> | ||
+ | >> better efficiency with 45 sec heat shock | ||
+ | </li> | ||
+ | <li> | ||
+ | gDNA extraction: 10 min vortex vs 10 min beadbeater | ||
+ | </li> | ||
+ | <li> | ||
+ | Testing now | ||
+ | </li> | ||
+ | <li> | ||
+ | reinoculated ∆fumC mazFΩ into BG-11 without antibiotics to check segregation - how long should this grow? | ||
+ | </li> | ||
</ul> | </ul> | ||
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</li> | </li> | ||
</ul> | </ul> | ||
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− | + | <ul> | |
− | + | <b> | |
− | + | Untargeted | |
− | + | </b> | |
− | + | <li> | |
− | + | Segregation ∆fumC mazFΩ | |
− | + | </li> | |
− | + | <li> | |
− | + | Gel picture shows full segregation. It is a population, so there might be a 0.1% change there is a synechocystis that is not fully segregated. Discussed with Wei: the steps after (transformation of glyoxylate shunt) will even increase segregation. | |
− | + | </li> | |
− | + | <li> | |
− | + | In parallel I will still inoculate from a single fully segregated colony. Plated liquid culture - picked colonies today and plated on a grid. After 3-4 days I will do a colony PCR and inoculate the fully segregated one. | |
− | + | </li> | |
− | + | <b> | |
− | + | Library construction | |
− | + | </b> | |
− | + | <li> | |
− | + | I digested my gDNA with Sau3AI and my plasmid with the glyoxylate shunt enzymes (pFLXN h1h2 MS-ICL) with BglII. I ligated them and transformed them: I have a lot of colonies (>1000) :D :D | |
− | + | </li> | |
− | + | <li> | |
− | + | Did ligation in different ratio's and did a colony PCR. | |
− | + | </li> | |
− | + | <li> | |
− | + | Washed colonies of plate (from protocols on internet) : this is my library in E. coili. | |
− | + | </li> | |
− | + | <li> | |
− | + | made a glycerol stock of this library | |
− | + | </li> | |
− | + | <li> | |
− | + | inoculated and grown overnight | |
− | + | </li> | |
− | + | <li> | |
− | + | Today: miniprep - this will be my library | |
− | + | </li> | |
− | + | <li> | |
− | + | Friday: transform library into Synechocystis | |
− | + | </li> | |
− | + | <b> | |
− | + | Competent cells | |
− | + | </b> | |
− | + | <li> | |
− | + | I made competent cells with XL1Blue. Tested efficiency with iGEM kit: transformation efficiency: 1.12 E+8. Protocol that works best: 45 seconds heat shock for 50 µl vials. | |
− | + | </li> | |
− | + | <b> | |
− | + | Segregation experiment: plating | |
− | + | </b> | |
− | + | <li> | |
− | + | Plated 8 days, then stopped because culture looks dead. Tycho saw results within 7 days, so it should be sufficient. | |
− | + | </li> | |
− | + | </ul> | |
− | + | ||
</div> | </div> | ||
</div> | </div> | ||
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Project overexpression | Project overexpression | ||
</b> | </b> | ||
− | <br> | + | <br/> |
− | + | <b> | |
− | + | E. coli transporters | |
− | + | </b> | |
− | + | <li> | |
− | + | DcuB-ec & DcuC-ec colony PCR with fwd and rev primers on my gene looked correct, but I was not able to cut with NdeI and sequencing results showed undigested T-vector. | |
− | + | </li> | |
− | + | <li> | |
− | + | Explanation: Colony PCR is on E.coli, so I amplified from genomic DNA. | |
− | + | </li> | |
− | + | <li> | |
− | + | Replated all colonies on cam-plates → 2 colonies did not grow so they probably contain my insert. | |
− | + | </li> | |
− | + | <li> | |
− | + | New colony-PCR to check, plasmid isolation, will send it for sequencing today. | |
− | + | </li> | |
− | + | <b> | |
− | + | NS transporter | |
− | + | </b> | |
− | + | <li> | |
− | + | (NS = Nodularia Spumigena) | |
− | + | </li> | |
− | + | <li> | |
− | + | DcuC-NS transformation to E.coli succeeded → colony PCR confirmation with primers on T-vector show the right construct. Will send it for sequencing today. | |
− | + | </li> | |
− | + | ||
</ul> | </ul> | ||
</div> | </div> | ||
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I am working on Age's project while he is on holiday. Last week he managed to get a right colony for the pCI-dcuSZ7 (we had cake!!) using my transformation protocol and competent cells. So yesterday I did transformation of all other 9 in exactly the same way. I have some colonies. | I am working on Age's project while he is on holiday. Last week he managed to get a right colony for the pCI-dcuSZ7 (we had cake!!) using my transformation protocol and competent cells. So yesterday I did transformation of all other 9 in exactly the same way. I have some colonies. | ||
− | <br> | + | <br/> |
− | + | Next: screening if the colonies contain the right insert. | |
− | + | ||
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− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/3a/TAmsterdam_sensor_icon.png"/> |
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− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/62/TAmsterdam_model_icon.png"/> |
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− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/63/TAmsterdam_shunt_icon.png"/> |
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− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/b/b3/TAmsterdam_transporter_icon.png"/> |
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/3a/TAmsterdam_sensor_icon.png"/> |
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− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/62/TAmsterdam_model_icon.png"/> |
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− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/63/TAmsterdam_shunt_icon.png"/> |
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− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/b/b3/TAmsterdam_transporter_icon.png"/> |
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/3a/TAmsterdam_sensor_icon.png"/> |
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− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/62/TAmsterdam_model_icon.png"/> |
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− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/63/TAmsterdam_shunt_icon.png"/> |
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/b/b3/TAmsterdam_transporter_icon.png"/> |
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/3a/TAmsterdam_sensor_icon.png"/> |
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/62/TAmsterdam_model_icon.png"/> |
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− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/63/TAmsterdam_shunt_icon.png"/> |
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/b/b3/TAmsterdam_transporter_icon.png"/> |
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/3a/TAmsterdam_sensor_icon.png"/> |
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/62/TAmsterdam_model_icon.png"/> |
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/63/TAmsterdam_shunt_icon.png"/> |
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/b/b3/TAmsterdam_transporter_icon.png"/> |
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/3a/TAmsterdam_sensor_icon.png"/> |
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/62/TAmsterdam_model_icon.png"/> |
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− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/63/TAmsterdam_shunt_icon.png"/> |
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/3a/TAmsterdam_sensor_icon.png"/> |
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/7/78/TAmsterdam_cellfactory_wiki.png"/> |
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/62/TAmsterdam_model_icon.png"/> |
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/b/b3/TAmsterdam_transporter_icon.png"/> |
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/3a/TAmsterdam_sensor_icon.png"/> |
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/62/TAmsterdam_model_icon.png"/> |
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/6/63/TAmsterdam_shunt_icon.png"/> |
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<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/b/b3/TAmsterdam_transporter_icon.png"/> |
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<div class="labj-mod-holder" id="sensor"> | <div class="labj-mod-holder" id="sensor"> | ||
<div class="labj-mod-icon"> | <div class="labj-mod-icon"> | ||
− | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/ | + | <img class="labj-icon" src="https://static.igem.org/mediawiki/2017/3/3a/TAmsterdam_sensor_icon.png"/> |
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<div class="footer-col-left"> | <div class="footer-col-left"> | ||
<a href="https://www.aimms.vu.nl/en/index.aspx" style="order:9" target="_blank"> | <a href="https://www.aimms.vu.nl/en/index.aspx" style="order:9" target="_blank"> | ||
− | <img alt="Aimms1" class="footer-sponsor-img" id="aimms" src="https://static.igem.org/mediawiki/2017/ | + | <img alt="Aimms1" class="footer-sponsor-img" id="aimms" src="https://static.igem.org/mediawiki/2017/8/8d/TAmsterdam_aimms1.png"> |
+ | </img> | ||
</a> | </a> | ||
<a href="https://www.amsterdamsciencepark.nl/" style="order:11" target="_blank"> | <a href="https://www.amsterdamsciencepark.nl/" style="order:11" target="_blank"> | ||
− | <img alt="Sciencepark" class="footer-sponsor-img" id="sciencepark" src="https://static.igem.org/mediawiki/2017/ | + | <img alt="Sciencepark" class="footer-sponsor-img" id="sciencepark" src="https://static.igem.org/mediawiki/2017/b/b2/TAmsterdam_sciencepark.png"> |
+ | </img> | ||
</a> | </a> | ||
<a href="http://www.auf.nl/" style="order:1" target="_blank"> | <a href="http://www.auf.nl/" style="order:1" target="_blank"> | ||
− | <img alt="Auf" class="footer-sponsor-img" id="auf" src="https://static.igem.org/mediawiki/2017/ | + | <img alt="Auf" class="footer-sponsor-img" id="auf" src="https://static.igem.org/mediawiki/2017/5/5c/TAmsterdam_auf.png"> |
+ | </img> | ||
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<a href="https://www.dsm.com/" style="order:5" target="_blank"> | <a href="https://www.dsm.com/" style="order:5" target="_blank"> | ||
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