Difference between revisions of "Team:Humboldt Berlin/Description"

 
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                             <div class="submenu">
 
                             <div class="submenu">
 
                                 <a href="/Team:Humboldt_Berlin/Team">Team members</a>
 
                                 <a href="/Team:Humboldt_Berlin/Team">Team members</a>
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                                 <a href="/Team:Humboldt_Berlin/Collaborations">Collaboration</a>
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                                 <a href="/Team:Humboldt_Berlin/Collaborations">Collaborations</a>
 
                             </div>
 
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                                 <a class="active" href="/Team:Humboldt_Berlin/Description">Description</a>
 
                                 <a class="active" href="/Team:Humboldt_Berlin/Description">Description</a>
 
                                 <a href="/Team:Humboldt_Berlin/Design">Design</a>
 
                                 <a href="/Team:Humboldt_Berlin/Design">Design</a>
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                                 <a href="/Team:Humboldt_Berlin/Experiments">Experimentals</a>
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                                 <a href="/Team:Humboldt_Berlin/Experiments">Experiments</a>
 
                                 <a href="/Team:Humboldt_Berlin/Notebook">Notebook</a>
 
                                 <a href="/Team:Humboldt_Berlin/Notebook">Notebook</a>
 
                                 <a href="/Team:Humboldt_Berlin/Contribution">Contribution</a>
 
                                 <a href="/Team:Humboldt_Berlin/Contribution">Contribution</a>
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                             <a href="/Team:Humboldt_Berlin/Human_Practices">Human Practices</a>
 
                             <a href="/Team:Humboldt_Berlin/Human_Practices">Human Practices</a>
 
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                                 <a href="/Team:Humboldt_Berlin/Human_Practices">Human Practises</a>
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                                 <a href="/Team:Humboldt_Berlin/Human_Practices">Human Practices</a>
 
                                 <a href="/Team:Humboldt_Berlin/Public_Engagement">Education & Engagement</a>
 
                                 <a href="/Team:Humboldt_Berlin/Public_Engagement">Education & Engagement</a>
 
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                             </div>
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                             <a>Awards</a>
 
                             <a>Awards</a>
 
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                             <div class="submenu">
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                                <a href="/Team:Humboldt_Berlin/Entrepreneurship">Entrepreneurship</a>
 
 
                                 <a href="/Team:Humboldt_Berlin/Hardware">Hardware</a>
 
                                 <a href="/Team:Humboldt_Berlin/Hardware">Hardware</a>
 
                                 <a href="/Team:Humboldt_Berlin/Measurement">Measurement</a>
 
                                 <a href="/Team:Humboldt_Berlin/Measurement">Measurement</a>
 
                                 <a href="/Team:Humboldt_Berlin/Model">Model</a>
 
                                 <a href="/Team:Humboldt_Berlin/Model">Model</a>
 
                                 <a href="/Team:Humboldt_Berlin/Plant">Plant</a>
 
                                 <a href="/Team:Humboldt_Berlin/Plant">Plant</a>
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                                <a href="/Team:Humboldt_Berlin/Software">Software</a>
 
 
                             </div>
 
                             </div>
 
                         </div>
 
                         </div>
 
                         <div class="devider"></div>
 
                         <div class="devider"></div>
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                         <a href="https://igem.org/2019_Judging_Form?team=Humboldt_Berlin">
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                         <a href="https://2019.igem.org/Team:Humboldt_Berlin/Achievements">
 
                             For Judges
 
                             For Judges
 
                         </a>
 
                         </a>
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         </nav>
 
         </nav>
  
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         <section class="image-bg relative-container ar100vh no-filter">
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         <div class="fixed-header-container">
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            <section class="fixed-image-header">
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                <img src="https://static.igem.org/mediawiki/2019/e/e9/T--Humboldt_Berlin--description-header.jpg" alt="notebook" />
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            </section>
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             <h1 class="page-headline description">Description</h1>
 
             <h1 class="page-headline description">Description</h1>
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            <img src="https://static.igem.org/mediawiki/2019/e/e9/T--Humboldt_Berlin--description-header.jpg" alt="notebook" />
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         </div>
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         </section>
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         <section class="page-content">
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         <section class="page-content fixed-header-content">
 
             <h2 class="page-subheadline">How & why</h2>
 
             <h2 class="page-subheadline">How & why</h2>
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             <div class="width-limit">
 
             <div class="width-limit">
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                 <div class="two-columns">
 
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                     <div>
 
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                         <img src="https://static.igem.org/mediawiki/2019/5/54/T--Humboldt_Berlin--ideonella_grafik.png" alt="ideonella grafic" />
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                        <h3 class="headline3">Chlamy who?</h3>
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                        <p class="block-text medium-sized">
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                            Most projects at iGEM and in synthetic biology in general choose to work with bacterial chassis. However, a growing community of plant synthetic biologists have laid the focus increasingly on the utilization of microalgae such as <i>Chlamydomonas reinhardtii</i> as a platform for photosynthetic expression (Jinkerson & Jonikas, 2015; Scaife et al., 2015). Being eukaryotic, this freshwater algae is able to perform post-translational modifications, allowing the expression of more complex proteins, while being easy and cost-efficient to cultivate and to handle (Merchant et al., 2007). A variety of transformation methods, including but not limited to biolistic transformation, glass bead agitation and electroporation are well-established for this model organism (Boynton et al., 1988). Its ability to grow photoautotrophically makes it an ideal chassis to tackle a variety of complex problems in an environmentally-friendly way.
 +
                        </p>
 +
                    </div>
 +
                    <div>
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                         <img class="is-revealing" style="width: 60%; margin-left:auto;margin-right:auto" src="https://static.igem.org/mediawiki/2019/5/5d/T--Humboldt_Berlin--chlamy_wusel.jpeg" alt="chlamy microscope" />
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                    </div>
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                </div>
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<div class="width-limit">
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                <div class="two-columns">
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<div>
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              <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/5/52/T--Humboldt_Berlin--chlamy_hub_vision.png" alt="Vision: ChlamyHUB" />
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                </br>
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            </div>
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<div>
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                        <h3 class="headline3">We present The ChlamyHUB to you</h3>
 +
                        <p class="block-text medium-sized">
 +
                          For the iGEM competition 2019 we developed the project idea of establishing <i>C. reinhardtii</i> in the iGEM community as a viable chassis for protein synthesis through various approaches, summarized under the ChlamyHUB vision. We have have created a toolkit of genetic parts, the ChlamyHUB Collection, which follow the MoClo syntax in the Golden Gate assembly standard. To satisfy the need to reproducibly cultivate photoautotrophic organisms under controlled conditions, we built and optimized a Do-It-Yourself bioreactor, the OpenPBR. Gaining insights into what factors have an influence on algal growth while cultivating under expression of transgenic proteins was achieved through our modeling projects. Lastly, ChlamyHUB aims to demonstrate the advantage of an eco-friendly organism as a platform to degrade PET plastic as a proof-of-concept.
 +
                        </p>
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                    </div>
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                </div>
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 +
 
 +
                <div class="two-columns">
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                    <div>
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                        <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/0/0e/T--Humboldt_Berlin--chlamydomonas_schaubild_eng.png" alt="chlamydomonas schaubild" />
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                    </div>
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                    <div>
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                        <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/9/9e/T--Humboldt_Berlin--800px-Chlamydomonas6-1.png" alt="Chlamydomonas" />
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                    </div>
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                </div>
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 +
                <div class="two-columns big-border-left">
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                    <div class="image-column">
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                        <img src="https://static.igem.org/mediawiki/2019/4/49/T--Humboldt_Berlin--e_coli.png" alt="e-coli illustration" />
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                    </div>
 +
                    <div class="big-text">
 +
<b>Bacteria</b></br>
 +
                        <b class="plus">Fast growth</b><br />
 +
                        <b class="minus">Inclusion bodies</b><br />
 +
                        <b class="minus">lack of eucaryotic posttranslational modification</b><br />
 +
                    </div>
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                </div>
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                <div class="two-columns big-border-left">
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                    <div class="image-column">
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                        <img src="https://static.igem.org/mediawiki/2019/b/bd/T--Humboldt_Berlin--eucaryotic_cells.png" alt="eucaryotic cells illustration" />
 +
                    </div>
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                    <div class="big-text">
 +
<b>Yeast & Tissue Cells</b></br>
 +
                        <b class="plus">Post-translational modification</b><br />
 +
                        <b class="minus">Expensive cultivation</b><br />
 +
                        <b class="minus">No motility</b><br />
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                    </div>
 +
                </div>
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 +
                <div class="two-columns big-border-left">
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                    <div class="image-column">
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                        <img src="https://static.igem.org/mediawiki/2019/2/23/T--Humboldt_Berlin--chlamy_nur_so.png" alt="chlamy illustration" />
 +
                    </div>
 +
                    <div class="big-text">
 +
<b>Microalgae</b></br>
 +
                        <b class="plus">Inexpensive & easy cultivation</b><br />                     
 +
                        <b class="plus">Post-translational modification</b><br />
 +
                        <b class="plus">Photosynthesis</b><br />
 +
                        <b class="plus">Enviromental-safe</b><br />
 +
                        <b class="plus">Two expression compartments (nucleus & chloroplast)</b><br />
 +
                    </div>
 +
                </div>
 +
 
 +
                <div class="two-columns">
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                    <div>
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                        <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/b/b4/T--Humboldt_Berlin--ablauf_sakaiensis.png" alt="process sakaiensis" />
 
                     </div>
 
                     </div>
 
                     <div>
 
                     <div>
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                         <!----------------------------------------------------------------------->
 
                         <!----------------------------------------------------------------------->
 
                         <h3 class="headline3">Our Inspiration</h3>
 
                         <h3 class="headline3">Our Inspiration</h3>
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                         <p class="block-text medium-sized">
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                         <p class="medium-sized block-text">
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                             In 2016 a bacterium, <i>Ideonella sakaiensis</i>, was found that is able to
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                             The unicellular green alga <i>C. reinhardtii</i> has an impressive history. It is used since the 1950s as a model organism to not only elucidate the structure of flagellar and basic plant cellular processes, such as photosynthesis and light perception, but also in research regarding phototaxis, circadian rhythmicity, cell cycle and mating mechanisms (Harris et al., 1989; Harris, 2009). However, <i>C. reinhardtii</i> has not only been used for fundamental research but is also a model for microalgal biotechnology and has become a photosynthetic expression platform to synthesize recombinant proteins. <i>C. reinhardtii</i> has been engineered to produce biomass for biofuels such as the biodiesel-precursor bisabolene (Wichmann et al., 2018). <i>Chlamydomonas</i> has also been used in therapeutic applications, having been modified to express an HIV antigen (Barahimipour et al., 2016).<br><br> In 2016, the bacterium <i>Ideonella sakaiensis</i> which is able to use polyethylene terephthalate (PET) as a primary carbon and energy source was discovered (Yoshida et al., 2016). This bacterium secretes two different hydrolases that perform the first two steps in PET degradation (Yoshida et al., 2016). The first hydrolase, PETase, breaks down PET to mono(2-hydroxyethyl) terephtalic acid (MHET). The second hydrolase, the MHETase, then digests MHET to terephthalic acid (TPA) and ethylene glycole (EG). In 2018, the PETase was characterized and engineered to improve its performance (Austin et al., 2018). Astonished by the possibility to degrade one of the most commonly used plastics, we were inspired to try to integrate the PETase and MHETase enzymes into <i>C. reinhardtii</i> in order to illustrate the capabilities of our favorite algae using a toolkit of various genetic parts.<br><br> One of the issues of the gravest impact on our generation is the environmental pollution. Especially the production of synthetic polymers, like plastic, has increased considerably since the 20th century (Andrady, 2011). Plastic is the most frequent material collected in studies on the surface of the Oceans (Law et al., 2010) and is also observed on the seafloor (Galgani et al., 2000). Even though the exact implication of microplastic to organisms is still unknown, the appearance of microplastic inside a wide variety of marine organisms is significant (Murray & Cowie, 2011).
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                            use polyethylene terephthalate (PET) as a primary carbon and energy
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                            source (Yoshida et al., 2016). This bacterium secrets two different
+
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                            hydrolases into the exterior to execute the first two PET degradation
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                            steps (Yoshida et al., 2016). The first hydrolase, PETase, mainly
+
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                            breaks down PET to mono(2-hydroxyethyl) terephtalic acid (MHET).
+
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                            The second hydrolase, the MHETase, then digests MHET to terephthalic
+
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                            acid (TPA) and ethylene glycole (EG). The bacterium itself grows
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                            optimally within a pH range of 7-7,5 and a temperature of 30-37°C
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                            (Tanasupawat, Takehana, Yoshida, Hiraga, & Oda, 2016). It was also
+
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                            demonstrated that it cannot grow anaerobically and has a GC-rich
+
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                            genome (70,4%) (Tanasupawat et al., 2016; Yoshida et al., 2016).
+
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                            Later in 2018, the PETase was characterized and engineered to improve
+
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                            its performance (Austin et al., 2018).  
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                         </p>
 
                         </p>
 
                     </div>
 
                     </div>
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                 <blockquote class="quote image-left">
 
                 <blockquote class="quote image-left">
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                     <img src="https://static.igem.org/mediawiki/2019/2/2d/T--Humboldt_Berlin--flasche_igem.png" alt="plastic bottle illustration" />
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                     <div class="quote-image-container">
 +
                        <img src="https://static.igem.org/mediawiki/2019/2/2d/T--Humboldt_Berlin--flasche_igem.png" alt="plastic bottle illustration" />
 +
                    </div>
 
                     <div class="block-text big-underline big-text">
 
                     <div class="block-text big-underline big-text">
 
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                         <!----------------------------------------------------------------------->
 
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                         <p><b>
 
                         <p><b>
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                            We know that we are not after something
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                          We know that we are not after something
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                             completely new. But we want to do this
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                             completely new, but we want to do this
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                             right. so we chose a different organism
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                             right. So we chose a chassis, which is commonly found in freshwater and environmentally-safe and tried to tackle obstacles other teams
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                            and tried to tackle obstacles other teams
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                             failed to solve.
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                             failed to solve.</b>
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                        </b></p>
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                        </p>
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                     </div>
 
                     </div>
 
                 </blockquote>
 
                 </blockquote>
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                         <!----------------------------------------------------------------------->
 
                         <!----------------------------------------------------------------------->
 
                         <h3 class="headline3">The iGEM projects that inspired us</h3>
 
                         <h3 class="headline3">The iGEM projects that inspired us</h3>
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                         <p class="block-text medium-sized">
+
                         <p class="medium-sized block-text">
−
                             Degrading microplastic is not a new idea when it comes to iGEM projects.
+
                             Degrading microplastic is not a new idea when it comes to iGEM projects. Similarly inspired by the works of Yoshida and his colleagues (Yoshida et al., 2016) a multitude of different teams have worked on comparable topics. We know that we are not after something completely new. But we wanted to do this right. So we chose a different organism and tried to tackle obstacles other teams failed to solve. <br><br> Our work was inspired by the team <a href="https://2016.igem.org/Team:TJUSLS_China/Description" target="blank" rel="noopener">TJUSLS project</a> on PETase (1), who improved the activity levels of the PETase through direct mutagenesis. It was through their project that we started looking into improved versions of the PETase first discovered by Yoshida et al. We were intrigued by the effort Harvard BioDesign 2016 put into their project <a href="https://2016.igem.org/Team:Harvard_BioDesign" target="blank" rel="noopener">“Plastiback” </a>(2), pushing us towards the direction of degrading microplastic in an aquatic environment secreting PETase into the enclosed system of a bioreactor. We also integrated the separation of both PET degradation products TPA and EG, useful precursors in the synthesis of new PET, in our bioreactor. The project of <a href="https://2016.igem.org/Team:ASIJ_Tokyo/Results" target="blank" rel="noopener">ASIJ Tokyo</a> in 2016 (3) inspired our module design greatly. Their characterization of the expression strength of PETase using different promoters and the secretion tests conducted under different secretion signals struck us as good practices to optimize the expression of complex constructs. Therefore, while designing our transcriptional units and choosing possible parts we aimed for a high variety of interchangeable modules to compare their functions. The team from <a href="https://2016.igem.org/Team:Tianjin/Description" target="blank" rel="noopener">Tianjin </a> in the year 2016 (4) combined photosynthetically active organisms and the degradation of PET, lending their reactor the capacity of fixating carbon dioxide using the energy from sunlight. The approaches by the team of <a href="https://2017.igem.org/Team:ITB_Indonesia/Description" target="blank" rel="noopener">ITB</a> 2017 (5) aided us in our project design as well, inspiring us with their use of biofilms on plastics. Looking for microorganisms with the abillity of binding to the surface of PET, we came across flagellar adhesion of <i>C. reinhardtii</i> to surfaces, which is even light-switchable (Kreis et al., 2018). <a href="https://2018.igem.org/Team:Yale" target="blank" rel="noopener">iGEM Team Yale´s</a> (6) focus on improving functionality of the enzymes also greatly impacted our work.
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                            Similarly inspired by the works of Yoshida and his colleagues (Yoshida
+
−
                            et al., 2016) a multitude of different teams have worked on comparable
+
−
                            topics. We know that we are not after something completely new.
+
−
                            But we wanted to do this right. So we chose a different organism
+
−
                            and tried to tackle obstacles other teams failed to solve. Our work
+
−
                            was inspired by TJUSLS project on PETase 2016 (1), we are intrigued
+
−
                            by the effort Harvard BioDesign 2016 put into their project “Plastikback”
+
−
                            (2) and the project of ASIJ Tokyo in 2016 struck the same nerve (3).
+
−
                            The approaches by the Teams of Tianjin 2016 (4) and ITB 2017 (5) have
+
−
                            gravely encouraged our project as well.
+
 
                         </p>
 
                         </p>
 
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                     <div>
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                         <img src="https://static.igem.org/mediawiki/2019/b/bc/T--Humboldt_Berlin--microplastic_icon.png" alt="microplastic icon" />
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                         <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/b/bc/T--Humboldt_Berlin--microplastic_icon.png" alt="microplastic icon" />
 
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                         <img src="https://static.igem.org/mediawiki/2019/c/c5/T--Humboldt_Berlin--chlamy_organism.png" alt="chlamy organism" />
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                         <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/c/c5/T--Humboldt_Berlin--chlamy_organism.png" alt="chlamy organism" />
 
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                         <!----------------------------------------------------------------------->
 
                         <!----------------------------------------------------------------------->
 
                         <h3 class="headline3">Chlamydomonas as a model organism</h3>
 
                         <h3 class="headline3">Chlamydomonas as a model organism</h3>
−
                         <p class="block-text medium-sized">
+
                         <p class="medium-sized block-text">
−
                             We propose that combining a photosynthesis active organism with at least
+
                             We propose that by combining a photosynthetically active organism with at least the optimized PETase and the MHETase we can create a new way of recycling PET or even degrading PET completely to CO₂ and H₂O. The organism could then able to use the CO₂ obtained from the plastic as its carbon source. We immediately thought of <i>Chlamydomonas reinhardtii</i> as a chassis, as it proliferates quickly under energy-efficient conditions. Being not only able to conduct photosynthesis, fixing carbon dioxide and using it as its carbon source with the power of light, <i>C. reinhardtii</i> can also live off acetate as its carbon source.
−
                            the optimized PETase and the MHETase we can create a new way of recycling
+
−
                            PET or even degrade PET completely to CO2 and H2O. The organism is then
+
−
                            able to use the CO2 coming from the plastic as carbon source.
+
−
                            We immediately thought of <i>Chlamydomonas reinhardtii</i> as an organism
+
−
                            as it grows fast under energy-efficient conditions.
+
 
                         </p>
 
                         </p>
 
                     </div>
 
                     </div>
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                         <!----------------------------------------------------------------------->
 
                         <!----------------------------------------------------------------------->
 
                         <p>
 
                         <p>
−
                             <img class="check-icon" src="https://static.igem.org/mediawiki/2019/b/b8/T--Humboldt_Berlin--chlamy_check_bullet.png"/>
+
                             <img class="check-icon" src="https://static.igem.org/mediawiki/2019/b/b8/T--Humboldt_Berlin--chlamy_check_bullet.png" /> easy to cultivate & phototrophic
−
                            easy to cultivate & phototrophic
+
 
                         </p>
 
                         </p>
 
                         <p>
 
                         <p>
−
                             <img class="check-icon" src="https://static.igem.org/mediawiki/2019/b/b8/T--Humboldt_Berlin--chlamy_check_bullet.png"/>
+
                             <img class="check-icon" src="https://static.igem.org/mediawiki/2019/b/b8/T--Humboldt_Berlin--chlamy_check_bullet.png" /> one organism = single cell
−
                            one organism = single cell
+
 
                         </p>
 
                         </p>
 
                         <p>
 
                         <p>
−
                             <img class="check-icon" src="https://static.igem.org/mediawiki/2019/b/b8/T--Humboldt_Berlin--chlamy_check_bullet.png"/>
+
                             <img class="check-icon" src="https://static.igem.org/mediawiki/2019/b/b8/T--Humboldt_Berlin--chlamy_check_bullet.png" /> well established as model organism
−
                            well established as model organism
+
 
                         </p>
 
                         </p>
 
                     </div>
 
                     </div>
 
                     <img class="small-image" src="https://static.igem.org/mediawiki/2019/2/23/T--Humboldt_Berlin--chlamy_nur_so.png" alt="chlamy" />
 
                     <img class="small-image" src="https://static.igem.org/mediawiki/2019/2/23/T--Humboldt_Berlin--chlamy_nur_so.png" alt="chlamy" />
 
                 </blockquote>
 
                 </blockquote>
 +
            </div>
 +
        </section>
  
−
                <div class="two-columns block-text medium-sized not-centered">
+
        <!----------------------------------------------------------------------->
−
                    <!----------------------------------------------------------------------->
+
        <!------------------------------- LEARN MORE ---------------------------->
−
                    <!--------------------- GOLDEN GATE MODULAR CLONING --------------------->
+
        <!----------------------------------------------------------------------->
−
                    <!----------------------------------------------------------------------->
+
−
                    <div>
+
−
                        <h3 class="headline3">Golden Gate Modular Cloning for Chlamydomonas reinhardtii</h3>
+
−
                        <p>
+
−
                            To synthesize and assemble all the needed gene constructs to let <i>Chlamydomonas</i>
+
−
                            express the PETase and MHETase together with different promotors, terminators,
+
−
                            secretion signals, tags and selection markers cloning was done using the Golden
+
−
                            Gate Modular Cloning (referred to hereafter as “MoClo”) toolkit for C. reinhardtii
+
−
                            (Crozet et al., 2018). The <i>Chlamydomonas</i> MoClo kit is standardized to fit the
+
−
                            syntax of the plant synthetic biology community (Patron et al., 2015).
+
−
                        </p>
+
−
                        <p>
+
−
                            MoClo is an assembly method using type IIS restriction sites first introduced
+
−
                            by (Weber, Engler, Gruetzner, Werner, & Marillonnet, 2011). Type IIS
+
−
                            restriction enzymes (BpiI; BsaI) cleave outside of their recognition site
+
−
                            leaving a four base pair overhang also called a fusion site (Engler, Kandzia,
+
−
                            & Marillonnet, 2008). Placing those restriction sites before the 5’ beginning
+
−
                            and the 3’ end of a desired DNA fragment in inverse orientation will allow
+
−
                            ligation of DNA fragments with compatible fusion sites to be correctly
+
−
                            assembled (Weber et al., 2011). Type IIS restriction sites can be constructed
+
−
                            to build different overhangs making an assembly of multiple fragments possible
+
−
                            (Weber et al., 2011).
+
−
                        </p>
+
  
−
                        <img src="https://static.igem.org/mediawiki/2019/4/42/T--Humboldt_Berlin--chlamy_overview-cloning-system.png" alt="Pverview of the hierarchical and modular cloning system"/>
+
        <section class="learn-more">
−
                        <p>
+
            <h3>Learn more...</h3>
−
                            <b>Fig. 1</b><i> Overview of the hierarchical and modular cloning system (Weber et
+
            <div class="learn-more-links">
−
                            al., 2011). (A) Level 0 plasmid modules containing cloned and sequenced
+
                <a href="/Team:Humboldt_Berlin/Design">
−
                            promoters (P), 5′ untranslated regions (U), coding sequences (CDS) and
+
                    <img src="https://static.igem.org/mediawiki/2019/6/6d/T--Humboldt_Berlin--Header_design.jpeg" alt="design preview" />
−
                            terminators (T). Because of the standardization of the toolkit the desired
+
                    <h4>Design</h4>
−
                            transcription units can be assembled from selected level 0 plasmids in a
+
                    <p class="block-text">
−
                            one-step digestion and ligation reaction into a level 1 vector. (B) Scheme
+
                        The <b>MoClo</b> syntax is based on the Golden Gate cloning standard, that uses Type IIS restriction sites. The <i>Chlamydomonas</i> MoClo kit is standardized to fit the syntax of the plant synthetic biology community.
−
                            of a Level 0 (L0) and L1 module. The gene part of a L0 module is flanked
+
                    </p>
−
                            by compatible fusion sites providing the correct assembly of these modules
+
                </a>
−
                            when cloned into a L1 destination vector. The fusion sites are four
+
                <a href="/Team:Humboldt_Berlin/Experiments" class="white-text">
−
                            nucleotides long (1-4 and 5-8) and flanked by a BsaI recognition sites.</i>
+
                    <img src="https://static.igem.org/mediawiki/2019/4/43/T--Humboldt_Berlin--design.jpg" alt="experiments preview" />
−
                        </p>
+
                     <h4>Experiments</h4>
−
                    </div>
+
                    <p class="block-text">
−
                    <div>
+
                        If you are interested in our day-to-day lab protocols or standard workflows - our Experiments page lists all resources you might need.
−
                        <p>
+
                    </p>
−
                            The MoClo toolkit for Chlamydomonas consists of three different cloning
+
                 </a>
−
                            vectors called level 0, 1 and 2. They are used in consecutive assembly
+
−
                            steps. Level 0 (referred to hereafter as “L0”) destination vectors contain
+
−
                            a selection marker gene such as lacZ or RFP surrounded by two cloning sites
+
−
                            (BpiI; BsaI). It is possible to insert gene parts into L0 plasmids such as
+
−
                            promotors, coding sequences or UTRs with specific fusion sites and surrounded
+
−
                            by BsaI restriction sites (Fig. 1 B). The specific fusion sites and BsaI
+
−
                            restriction sites make it possible to correctly assemble those L0 modules
+
−
                            onto a next plasmid level 1 (referred to hereafter as “L1”) in one step
+
−
                            generating a transcriptional unit (Fig. 1, Tab. 1).
+
−
                        </p>
+
−
                        <p>
+
−
                            Each assembly is performed in a single reaction mix with the desired insert,
+
−
                            the destination vector, DNA ligase and one type IIS restriction enzyme.
+
−
                            Correctly assembled L1 modules can then be transformed into C. reinhardtii.
+
−
                        </p>
+
−
                        <p>
+
−
                            Each L0 module has designated nucleotides as their fusion site determining their
+
−
                            cloning position in a L1. The kit offers ten different options for positioning in
+
−
                            a L1 plasmid. Those cloning positions for each gene part are defined by its
+
−
                            function and altogether its position in a L1 plasmid (Tab. 1).
+
−
                        </p>
+
−
 
+
−
                        <span>
+
−
                            <b>Tab. 1</b><i> MoClo L0 to L1 cloning; L1 fusion sites
+
−
                            establishing a complete transcriptional unit (TU).</i>
+
−
                        </span>
+
−
                        <img src="https://static.igem.org/mediawiki/2019/2/21/T--Humboldt_Berlin--chlamy_table_moclo.png" alt="MoClo L0 to L1 cloning table" />
+
−
                     </div>
+
−
                 </div>
+
 
             </div>
 
             </div>
 
         </section>
 
         </section>
  
−
        <div class="greyblue-devider"></div>
+
<div class="greyblue-devider">
 +
 
 +
<!--------------------------------------- TO TOP LINK ----------------------------------------------->
 +
                <a href="#" class="to-top-link">
 +
                    <img src="https://static.igem.org/mediawiki/2019/3/3e/T--Humboldt_Berlin--ArrowDown.jpg" /> Go to top
 +
                </a>
 +
                <!--------------------------------------- TO TOP LINK END ------------------------------------------->
 +
 
 +
</div>
  
 
         <section class="width-limit">
 
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−
                     <p>Austin, H. P., Allen, M. D., Donohoe, B. S., Rorrer, N. A., Kearns, F. L., Silveira, R. L., . . . Beckham, G. T. (2018). Characterization and engineering of a plastic-degrading aromatic polyesterase. Proceedings of the National Academy of Sciences, 115(19), E4350. Retrieved from http://www.pnas.org/content/115/19/E4350.abstract. doi:10.1073/pnas.1718804115</p>
+
                     <p> Austin, H. P., Allen, M. D., Donohoe, B. S., Rorrer, N. A., Kearns, F. L., Silveira, R. L., . . . Beckham, G. T. (2018). Characterization and engineering of a plastic-degrading aromatic polyesterase. Proceedings of the National Academy of Sciences, 115(19), E4350. Retrieved from http://www.pnas.org/content/115/19/E4350.abstract. doi:10.1073/pnas.1718804115</p>
−
                     <p>Crozet, P., Navarro, F. J., Willmund, F., Mehrshahi, P., Bakowski, K., Lauersen, K. J., . . . Lemaire, S. D. (2018). Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii. ACS Synthetic Biology, 7(9), 2074-2086. Retrieved from https://doi.org/10.1021/acssynbio.8b00251. doi:10.1021/acssynbio.8b00251</p>
+
                     <p> Andrady, A. L. (2011). Microplastics in the marine environment. Marine Pollution Bulletin, 62(8), 1596-1605. Retrieved from http://www.sciencedirect.com/science/article/pii/S0025326X11003055. doi:https://doi.org/10.1016/j.marpolbul.2011.05.030</p>
−
                     <p>Engler, C., Kandzia, R., & Marillonnet, S. (2008). A one pot, one step, precision cloning method with high throughput capability. PLOS ONE, 3(11), e3647. doi:10.1371/journal.pone.0003647</p>
+
                    <p> Barahimipour, R., Neupert, J., & Bock, R. (2016). Efficient expression of nuclear transgenes in the green alga Chlamydomonas: synthesis of an HIV antigen and development of a new selectable marker. Plant Molecular Biology, 90(4), 403-418. Retrieved from https://doi.org/10.1007/s11103-015-0425-8. doi:10.1007/s11103-015-0425-8</p>
−
                     <p>Patron, N. J., Orzaez, D., Marillonnet, S., Warzecha, H., Matthewman, C., Youles, M., . . . Haseloff, J. (2015). Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist, 208(1), 13-19. Retrieved from https://nph.onlinelibrary.wiley.com/doi/abs/10.1111/nph.13532. doi:10.1111/nph.13532</p>
+
                     <p> Boynton, J. E., Gillham, N. W., Harris, E. H., Hosler, J. P., Johnson, A. M., Jones, A. R., . . . et al. (1988). Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science, 240(4858), 1534-1538.</p>
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                     <p>Purton, S., Szaub, J., Wannathong, T., Young, R., & Economou, C. (2013). Genetic engineering of algal chloroplasts: progress and prospects. Russian Journal of Plant Physiology, 60(4), 491-499.</p>
+
                     <p> Galgani, F., Leaute, J. P., Moguedet, P., Souplet, A., Verin, Y., Carpentier, A., . . . Nerisson, P. (2000). Litter on the Sea Floor Along European Coasts. Marine Pollution Bulletin, 40(6), 516-527. Retrieved from http://www.sciencedirect.com/science/article/pii/S0025326X99002349. doi:https://doi.org/10.1016/S0025-326X(99)00234-9</p>
 +
                    <p> Harris, E. H. (2009). The Chlamydomonas Sourcebook: Introduction to Chlamydomonas and Its Laboratory Use: Elsevier Science.</p>
 +
                    <p> Harris, E. H., Stern, D. B., & Witman, G. B. (1989). The chlamydomonas sourcebook (Vol. 2): Academic Press San Diego.</p>
 +
                    <p> Jinkerson, R. E., & Jonikas, M. C. (2015). Molecular techniques to interrogate and edit the Chlamydomonas nuclear genome. Plant J, 82(3), 393-412. doi:10.1111/tpj.12801</p>
 +
                     <p> Kreis, C. T., Le Blay, M., Linne, C., Makowski, M. M., & Bäumchen, O. (2018). Adhesion of Chlamydomonas microalgae to surfaces is switchable by light. Nature Physics, 14(1), 45–49. https://doi.org/10.1038/nphys4258</p>
 
                 </div>
 
                 </div>
 
                 <div>
 
                 <div>
−
                     <p>Tanasupawat, S., Takehana, T., Yoshida, S., Hiraga, K., & Oda, K. (2016). Ideonella sakaiensis sp. nov., isolated from a microbial consortium that degrades poly(ethylene terephthalate). Int J Syst Evol Microbiol, 66(8), 2813-2818. doi:10.1099/ijsem.0.001058</p>
+
                    <p> Law, K. L., Morét-Ferguson, S., Maximenko, N. A., Proskurowski, G., Peacock, E. E., Hafner, J., & Reddy, C. M. (2010). Plastic Accumulation in the North Atlantic Subtropical Gyre. Science, 329(5996), 1185. Retrieved from http://science.sciencemag.org/content/329/5996/1185.abstract. doi:10.1126/science.1192321</p>
−
                     <p>Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLOS ONE, 6(2), e16765. Retrieved from https://doi.org/10.1371/journal.pone.0016765. doi:10.1371/journal.pone.0016765</p>
+
                    <p> Merchant, S. S., Prochnik, S. E., Vallon, O., Harris, E. H., Karpowicz, S. J., Witman, G. B., . . . Grossman, A. R. (2007). The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science, 318(5848), 245-250. doi:10.1126/science.1143609</p>
−
                     <p>Yoshida, S., Hiraga, K., Takehana, T., Taniguchi, I., Yamaji, H., Maeda, Y., . . . Oda, K. (2016). A bacterium that degrades and assimilates poly(ethylene terephthalate). Science, 351(6278), 1196-1199. doi:10.1126/science.aad6359</p>
+
                    <p> Murray, F., & Cowie, P. R. (2011). Plastic contamination in the decapod crustacean Nephrops norvegicus (Linnaeus, 1758). Marine Pollution Bulletin, 62(6), 1207-1217. Retrieved from http://www.sciencedirect.com/science/article/pii/S0025326X11001755. doi:https://doi.org/10.1016/j.marpolbul.2011.03.032</p>
 +
                    <p> Scaife, M. A., Nguyen, G. T., Rico, J., Lambert, D., Helliwell, K. E., & Smith, A. G. (2015). Establishing Chlamydomonas reinhardtii as an industrial biotechnology host. Plant J, 82(3), 532-546. doi:10.1111/tpj.12781</p>
 +
                     <p> Tanasupawat, S., Takehana, T., Yoshida, S., Hiraga, K., & Oda, K. (2016). Ideonella sakaiensis sp. nov., isolated from a microbial consortium that degrades poly(ethylene terephthalate). Int J Syst Evol Microbiol, 66(8), 2813-2818. doi:10.1099/ijsem.0.001058</p>
 +
                     <p> Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLOS ONE, 6(2), e16765. Retrieved from https://doi.org/10.1371/journal.pone.0016765. doi:10.1371/journal.pone.0016765</p>
 +
                    <p> Wichmann, J., Baier, T., Wentnagel, E., Lauersen, K. J., & Kruse, O. (2018). Tailored carbon partitioning for phototrophic production of (E)-alpha-bisabolene from the green microalga Chlamydomonas reinhardtii. Metab Eng, 45, 211-222. doi:10.1016/j.ymben.2017.12.010</p>
 +
                     <p> Yoshida, S., Hiraga, K., Takehana, T., Taniguchi, I., Yamaji, H., Maeda, Y., . . . Oda, K. (2016). A bacterium that degrades and assimilates poly(ethylene terephthalate). Science, 351(6278), 1196-1199. doi:10.1126/science.aad6359</p>
 
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Latest revision as of 11:21, 12 December 2019

notebook

Description

How & why

Chlamy who?

Most projects at iGEM and in synthetic biology in general choose to work with bacterial chassis. However, a growing community of plant synthetic biologists have laid the focus increasingly on the utilization of microalgae such as Chlamydomonas reinhardtii as a platform for photosynthetic expression (Jinkerson & Jonikas, 2015; Scaife et al., 2015). Being eukaryotic, this freshwater algae is able to perform post-translational modifications, allowing the expression of more complex proteins, while being easy and cost-efficient to cultivate and to handle (Merchant et al., 2007). A variety of transformation methods, including but not limited to biolistic transformation, glass bead agitation and electroporation are well-established for this model organism (Boynton et al., 1988). Its ability to grow photoautotrophically makes it an ideal chassis to tackle a variety of complex problems in an environmentally-friendly way.

chlamy microscope
Vision: ChlamyHUB

We present The ChlamyHUB to you

For the iGEM competition 2019 we developed the project idea of establishing C. reinhardtii in the iGEM community as a viable chassis for protein synthesis through various approaches, summarized under the ChlamyHUB vision. We have have created a toolkit of genetic parts, the ChlamyHUB Collection, which follow the MoClo syntax in the Golden Gate assembly standard. To satisfy the need to reproducibly cultivate photoautotrophic organisms under controlled conditions, we built and optimized a Do-It-Yourself bioreactor, the OpenPBR. Gaining insights into what factors have an influence on algal growth while cultivating under expression of transgenic proteins was achieved through our modeling projects. Lastly, ChlamyHUB aims to demonstrate the advantage of an eco-friendly organism as a platform to degrade PET plastic as a proof-of-concept.

chlamydomonas schaubild
Chlamydomonas
e-coli illustration
Bacteria
Fast growth
Inclusion bodies
lack of eucaryotic posttranslational modification
eucaryotic cells illustration
Yeast & Tissue Cells
Post-translational modification
Expensive cultivation
No motility
chlamy illustration
Microalgae
Inexpensive & easy cultivation
Post-translational modification
Photosynthesis
Enviromental-safe
Two expression compartments (nucleus & chloroplast)
process sakaiensis

Our Inspiration

The unicellular green alga C. reinhardtii has an impressive history. It is used since the 1950s as a model organism to not only elucidate the structure of flagellar and basic plant cellular processes, such as photosynthesis and light perception, but also in research regarding phototaxis, circadian rhythmicity, cell cycle and mating mechanisms (Harris et al., 1989; Harris, 2009). However, C. reinhardtii has not only been used for fundamental research but is also a model for microalgal biotechnology and has become a photosynthetic expression platform to synthesize recombinant proteins. C. reinhardtii has been engineered to produce biomass for biofuels such as the biodiesel-precursor bisabolene (Wichmann et al., 2018). Chlamydomonas has also been used in therapeutic applications, having been modified to express an HIV antigen (Barahimipour et al., 2016).

In 2016, the bacterium Ideonella sakaiensis which is able to use polyethylene terephthalate (PET) as a primary carbon and energy source was discovered (Yoshida et al., 2016). This bacterium secretes two different hydrolases that perform the first two steps in PET degradation (Yoshida et al., 2016). The first hydrolase, PETase, breaks down PET to mono(2-hydroxyethyl) terephtalic acid (MHET). The second hydrolase, the MHETase, then digests MHET to terephthalic acid (TPA) and ethylene glycole (EG). In 2018, the PETase was characterized and engineered to improve its performance (Austin et al., 2018). Astonished by the possibility to degrade one of the most commonly used plastics, we were inspired to try to integrate the PETase and MHETase enzymes into C. reinhardtii in order to illustrate the capabilities of our favorite algae using a toolkit of various genetic parts.

One of the issues of the gravest impact on our generation is the environmental pollution. Especially the production of synthetic polymers, like plastic, has increased considerably since the 20th century (Andrady, 2011). Plastic is the most frequent material collected in studies on the surface of the Oceans (Law et al., 2010) and is also observed on the seafloor (Galgani et al., 2000). Even though the exact implication of microplastic to organisms is still unknown, the appearance of microplastic inside a wide variety of marine organisms is significant (Murray & Cowie, 2011).

plastic bottle illustration

We know that we are not after something completely new, but we want to do this right. So we chose a chassis, which is commonly found in freshwater and environmentally-safe and tried to tackle obstacles other teams failed to solve.

The iGEM projects that inspired us

Degrading microplastic is not a new idea when it comes to iGEM projects. Similarly inspired by the works of Yoshida and his colleagues (Yoshida et al., 2016) a multitude of different teams have worked on comparable topics. We know that we are not after something completely new. But we wanted to do this right. So we chose a different organism and tried to tackle obstacles other teams failed to solve.

Our work was inspired by the team TJUSLS project on PETase (1), who improved the activity levels of the PETase through direct mutagenesis. It was through their project that we started looking into improved versions of the PETase first discovered by Yoshida et al. We were intrigued by the effort Harvard BioDesign 2016 put into their project “Plastiback” (2), pushing us towards the direction of degrading microplastic in an aquatic environment secreting PETase into the enclosed system of a bioreactor. We also integrated the separation of both PET degradation products TPA and EG, useful precursors in the synthesis of new PET, in our bioreactor. The project of ASIJ Tokyo in 2016 (3) inspired our module design greatly. Their characterization of the expression strength of PETase using different promoters and the secretion tests conducted under different secretion signals struck us as good practices to optimize the expression of complex constructs. Therefore, while designing our transcriptional units and choosing possible parts we aimed for a high variety of interchangeable modules to compare their functions. The team from Tianjin in the year 2016 (4) combined photosynthetically active organisms and the degradation of PET, lending their reactor the capacity of fixating carbon dioxide using the energy from sunlight. The approaches by the team of ITB 2017 (5) aided us in our project design as well, inspiring us with their use of biofilms on plastics. Looking for microorganisms with the abillity of binding to the surface of PET, we came across flagellar adhesion of C. reinhardtii to surfaces, which is even light-switchable (Kreis et al., 2018). iGEM Team Yale´s (6) focus on improving functionality of the enzymes also greatly impacted our work.

microplastic icon
chlamy organism

Chlamydomonas as a model organism

We propose that by combining a photosynthetically active organism with at least the optimized PETase and the MHETase we can create a new way of recycling PET or even degrading PET completely to CO₂ and H₂O. The organism could then able to use the CO₂ obtained from the plastic as its carbon source. We immediately thought of Chlamydomonas reinhardtii as a chassis, as it proliferates quickly under energy-efficient conditions. Being not only able to conduct photosynthesis, fixing carbon dioxide and using it as its carbon source with the power of light, C. reinhardtii can also live off acetate as its carbon source.

easy to cultivate & phototrophic

one organism = single cell

well established as model organism

chlamy

Learn more...

design preview

Design

The MoClo syntax is based on the Golden Gate cloning standard, that uses Type IIS restriction sites. The Chlamydomonas MoClo kit is standardized to fit the syntax of the plant synthetic biology community.

experiments preview

Experiments

If you are interested in our day-to-day lab protocols or standard workflows - our Experiments page lists all resources you might need.

Go to top

Austin, H. P., Allen, M. D., Donohoe, B. S., Rorrer, N. A., Kearns, F. L., Silveira, R. L., . . . Beckham, G. T. (2018). Characterization and engineering of a plastic-degrading aromatic polyesterase. Proceedings of the National Academy of Sciences, 115(19), E4350. Retrieved from http://www.pnas.org/content/115/19/E4350.abstract. doi:10.1073/pnas.1718804115

Andrady, A. L. (2011). Microplastics in the marine environment. Marine Pollution Bulletin, 62(8), 1596-1605. Retrieved from http://www.sciencedirect.com/science/article/pii/S0025326X11003055. doi:https://doi.org/10.1016/j.marpolbul.2011.05.030

Barahimipour, R., Neupert, J., & Bock, R. (2016). Efficient expression of nuclear transgenes in the green alga Chlamydomonas: synthesis of an HIV antigen and development of a new selectable marker. Plant Molecular Biology, 90(4), 403-418. Retrieved from https://doi.org/10.1007/s11103-015-0425-8. doi:10.1007/s11103-015-0425-8

Boynton, J. E., Gillham, N. W., Harris, E. H., Hosler, J. P., Johnson, A. M., Jones, A. R., . . . et al. (1988). Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science, 240(4858), 1534-1538.

Galgani, F., Leaute, J. P., Moguedet, P., Souplet, A., Verin, Y., Carpentier, A., . . . Nerisson, P. (2000). Litter on the Sea Floor Along European Coasts. Marine Pollution Bulletin, 40(6), 516-527. Retrieved from http://www.sciencedirect.com/science/article/pii/S0025326X99002349. doi:https://doi.org/10.1016/S0025-326X(99)00234-9

Harris, E. H. (2009). The Chlamydomonas Sourcebook: Introduction to Chlamydomonas and Its Laboratory Use: Elsevier Science.

Harris, E. H., Stern, D. B., & Witman, G. B. (1989). The chlamydomonas sourcebook (Vol. 2): Academic Press San Diego.

Jinkerson, R. E., & Jonikas, M. C. (2015). Molecular techniques to interrogate and edit the Chlamydomonas nuclear genome. Plant J, 82(3), 393-412. doi:10.1111/tpj.12801

Kreis, C. T., Le Blay, M., Linne, C., Makowski, M. M., & Bäumchen, O. (2018). Adhesion of Chlamydomonas microalgae to surfaces is switchable by light. Nature Physics, 14(1), 45–49. https://doi.org/10.1038/nphys4258

Law, K. L., Morét-Ferguson, S., Maximenko, N. A., Proskurowski, G., Peacock, E. E., Hafner, J., & Reddy, C. M. (2010). Plastic Accumulation in the North Atlantic Subtropical Gyre. Science, 329(5996), 1185. Retrieved from http://science.sciencemag.org/content/329/5996/1185.abstract. doi:10.1126/science.1192321

Merchant, S. S., Prochnik, S. E., Vallon, O., Harris, E. H., Karpowicz, S. J., Witman, G. B., . . . Grossman, A. R. (2007). The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science, 318(5848), 245-250. doi:10.1126/science.1143609

Murray, F., & Cowie, P. R. (2011). Plastic contamination in the decapod crustacean Nephrops norvegicus (Linnaeus, 1758). Marine Pollution Bulletin, 62(6), 1207-1217. Retrieved from http://www.sciencedirect.com/science/article/pii/S0025326X11001755. doi:https://doi.org/10.1016/j.marpolbul.2011.03.032

Scaife, M. A., Nguyen, G. T., Rico, J., Lambert, D., Helliwell, K. E., & Smith, A. G. (2015). Establishing Chlamydomonas reinhardtii as an industrial biotechnology host. Plant J, 82(3), 532-546. doi:10.1111/tpj.12781

Tanasupawat, S., Takehana, T., Yoshida, S., Hiraga, K., & Oda, K. (2016). Ideonella sakaiensis sp. nov., isolated from a microbial consortium that degrades poly(ethylene terephthalate). Int J Syst Evol Microbiol, 66(8), 2813-2818. doi:10.1099/ijsem.0.001058

Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLOS ONE, 6(2), e16765. Retrieved from https://doi.org/10.1371/journal.pone.0016765. doi:10.1371/journal.pone.0016765

Wichmann, J., Baier, T., Wentnagel, E., Lauersen, K. J., & Kruse, O. (2018). Tailored carbon partitioning for phototrophic production of (E)-alpha-bisabolene from the green microalga Chlamydomonas reinhardtii. Metab Eng, 45, 211-222. doi:10.1016/j.ymben.2017.12.010

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Weblinks:
1 https://2016.igem.org/Team:TJUSLS_China/Description
2 https://2016.igem.org/Team:Harvard_BioDesign
3 https://2016.igem.org/Team:ASIJ_Tokyo/Results
4 https://2016.igem.org/Team:Tianjin/Description
5 https://2017.igem.org/Team:ITB_Indonesia/Description
6 https://2018.igem.org/Team:Yale