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| Right sided introduction here | | Right sided introduction here |
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| <a href="#design"> | | <a href="#design"> |
| <h3 style="color:black"></h3> | | <h3 style="color:black"></h3> |
− | </a>
| + | Design introduction here |
− | <LI>
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− | <h3><a href=""> </a> </h3>
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− | <LI>
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− | <h3><a href="#compo">1.2. Selection cassette construction</a></h3>
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− | </LI>
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− | <h3><a href="">1.3.1 Promoter-comparison-tests</a></h3>
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− | </LI>
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− | <h3><a href="">1.3.2 Secretion signal</a></h3>
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− | </LI>
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− | <h3><a href="assembly">1.3 Protein expression analysis - testing our transgenic proteins</a></h3>
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− | <UL class="no-bulletpoint">
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− | <h3><a href="#ChlamyiGEM">1.3.1 Promoter-comparison-tests</a></h3>
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− | </LI>
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− | <LI>
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− | <h3><a href="#Secretion">1.3.2 Secretion signal</a></h3>
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| </UL> | | </UL> |
| </p> | | </p> |
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| <!-----------------------------------------------------------------------> | | <!-----------------------------------------------------------------------> |
| <div class="two-columns-headline" id="GoldenGate"> | | <div class="two-columns-headline" id="GoldenGate"> |
− | <h3 class="headline3">1.1 Golden Gate Modular Cloning for <i>Chlamydomonas reinhardtii</i></h3> | + | <h3 class="headline3"> Design of the open PBR</h3> |
| </div> | | </div> |
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| <p class="medium-sized"></h3> | | <p class="medium-sized"></h3> |
− | To synthesize and assemble the genetic elements for the Chlamy-HUB Collection, we applied the Type IIS “Golden Gate” cloning strategy (Engler et al., 2008). We used the Modular Cloning (MoClo) toolkit optimized for <i>C. reinhardtii</i> (Crozet et al., 2018), which follows the MoClo syntax of the plant synthetic biology community (Patron et al., 2015). | + | Design intro here |
| </p> | | </p> |
| <img src="https://static.igem.org/mediawiki/2019/1/1b/T--Humboldt_Berlin--designfig1.png" alt="Overview of the hierarchical and modular cloning system" /> | | <img src="https://static.igem.org/mediawiki/2019/1/1b/T--Humboldt_Berlin--designfig1.png" alt="Overview of the hierarchical and modular cloning system" /> |
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| <!------------------Selection Cassette construction-----------------------> | | <!------------------Selection Cassette construction-----------------------> |
| <!-----------------------------------------------------------------------> | | <!-----------------------------------------------------------------------> |
− | <h3 class="headline3">1.2 Construction of a selection cassette</h3> | + | <h3 class="headline3">2 Overview of single components</h3> |
| </div> | | </div> |
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| <!------------------Expression Analysis------------------------------------> | | <!------------------Expression Analysis------------------------------------> |
| <!-----------------------------------------------------------------------> | | <!-----------------------------------------------------------------------> |
− | <h3 class="headline3">1.3 Expression analysis - testing our transgenic proteins</h3> | + | <h3 class="headline3">3 Assembly instructions</h3> |
| </div> | | </div> |
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| <div class="expandable-preview"> | | <div class="expandable-preview"> |
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− | The composite parts we created contained different markers, antibiotic resistances or the phosphite marker PtxD, that allow for selection of transformed algae clones. Depending on their intended utility for <i>C. reinhardtii</i>, each of our constructs is screened in a different way to prove their intended function. | + | Assembly preview here |
| </p> | | </p> |
| <img src="https://static.igem.org/mediawiki/2019/1/1b/T--Humboldt_Berlin--designfig1.png" alt="Overview of the hierarchical and modular cloning system" /> | | <img src="https://static.igem.org/mediawiki/2019/1/1b/T--Humboldt_Berlin--designfig1.png" alt="Overview of the hierarchical and modular cloning system" /> |
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− | <h3 class="headline3" :>2.3 Testing the toxicity of TPA and EG for <i>C. reinhardtii</i></h3>
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− | <!-------------------Toxicity Test---------------------->
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− | A <i>C. reinhardtii</i> which expresses and secretes the enzymes PETase and MHETase could pose as a solution for the problem of micro-plastic polluted water. Nevertheless, the viability of PET-degradation by <i>C. reinhardtii</i> at a larger scale is yet unknown.
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− | </p>
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− | <img src="https://static.igem.org/mediawiki/2019/1/1b/T--Humboldt_Berlin--designfig1.png" alt="Overview of the hierarchical and modular cloning system" />
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− | As it is our goal to grow <i>C. reinhardtii</i> in a bioreactor in which it secretes PETase and MHETase we need to understand how it can deal with the produced degradation products terephthalic acid (TPA) and ethylene glycol (EG). Within this framework we measured the growth rates of several <i>C. reinhardtii</i> strains in a series of experiments. <br> </br>
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− | With the help of the Multi Cultivator MC 1000 we can test four different <i>C. reinhardtii</i> strains on these reagents to find out which one is the most suitable for further experiments and for transformation.
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− | <!--- IMAGE --->
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− | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/3/32/T--Humboldt_Berlin--modell_uebersicht.png" alt="PET degradation model" />
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− | <h3 class="headline3" :>2.4 Selection cassete construction</h3>
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− | <!----------------------------------------------------------------------->
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− | <!-------------------Selection Cassete---------------------->
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− | <img src="https://static.igem.org/mediawiki/2019/5/5d/T--Humboldt_Berlin--chlamy_wusel.jpeg" alt="preview" />
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− | <h3 class="headline3" :>2.5 Measuring concentration of TPA and EG in medium (quantitative activity test)</h3>
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− | <!-------------------Concentration Measurement---------------------->
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− | To test the quantitative power of PETase and MHETase we designed several experiments. Overall we aimed to measure the concentration of the degraded products TPA and EG. This is how we can calculate the degradation rate/enzymes activity.
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− | </p>
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− | <img src="https://static.igem.org/mediawiki/2019/5/5d/T--Humboldt_Berlin--chlamy_wusel.jpeg" alt="preview" />
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− | <h3 class="headline3" :>2.6 PnpB assay to test enzyme activity (quantitative test)</h3>
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− | <!----------------------------------------------------------------------->
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− | <!-------------------Assay---------------------->
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− | <!----------------------------------------------------------------------->
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− | The pNP (p-nitrophenol) assay can be used to quantify lipase activity.
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− | <img src="https://static.igem.org/mediawiki/2019/5/5d/T--Humboldt_Berlin--chlamy_wusel.jpeg" alt="preview" />
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− | The pNP (p-nitrophenol) assay can be used to quantify lipase activity. Because PETase is a kind of lipase, it is possible to determine the ability of PETase to cleave proteins. The substrate pNPB (p-nitrophenol butyrate) can be hydrolyzed by PETase to pNP which has a maximum absorption at 405 nm which can be measured. The absorption is closely monitored starting at 30 minutes after the addition of enzyme by an ELISA reader and was established by Wei in 2014 (Wei et al., 2014).
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− | <img src="https://static.igem.org/mediawiki/2019/3/3e/T--Humboldt_Berlin--ArrowDown.jpg">
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