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− | <!--------------------------------------- TWO COLUMN IMG RIGHT END ------------------------------------------------>
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− | <div class="two-columns-headline" id="PtxD">
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− | <!----------------------------------------------------------------------->
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− | <!--------------------PtxD phosphite marker-------------------->
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− | <h3 class="headline3">1.3.3 PtxD - Phosphite Oxidoreductase</h3>
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− | Algae depend on phosphate as their sole phosphorus source. Phosphite, the reduced form of phosphate however, competes with phosphate for transport proteins and therefore is harmful to plant growth.
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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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− | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/d/d5/T--Humboldt_Berlin--screening.jpeg" />
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− | <p><b>Fig. </b>
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− | <figcaption> This figure shows how the phosphite marker works. </figcaption>
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− | The phosphite oxidoreductase from <i>Pseudomonas stutzeri WM88</i> (PtxD) oxidizes phosphite to phosphate using NAD<sup>+</sup> as a co-substrate (Loera-Quezada et al., 2016; White & Metcalf, 2007). We provide our <i>C. reinhardtii</i> strains with the PtxD enzyme to increase its competitiveness against other microorganisms in culture. This way, we enable it to degrade phosphite, presumably as only organism in our culture, avoiding contamination.
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− | </p>
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− | We observe the growth of our engineered <i>C. reinhardtii</i> in media containing phosphite, phosphate or phosphorus to prove the enzyme activity. In the phosphite-supplied media, we expect to observe growth of the strains expressing PtxD and death of wild type algae due to a lack of phosphorus.
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− | <img src="https://static.igem.org/mediawiki/2019/3/3e/T--Humboldt_Berlin--ArrowDown.jpg">
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− | <div class="two-columns-headline" id="Cas9">
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− | <!------------------Cas9 site-directed mutagenesis------------------------------------>
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− | <h3 class="headline3">1.3.4 Cas9/sgRNA-mediated site-directed mutagenesis </h3>
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− | When establishing <i>Chlamydomonas</i> algae as a viable chassis for expression of synthetic constructs, one must consider how to ramp up the expression of such constructs. One solution might be to guide the insertion into a certain genome locus. As has been proven before, gene insertions into the nuclear genome of <i>C. reinhardtii</i> can be achieved by transformation of a recombinant Cas9 protein, combined with a guide RNA (gRNA) (Kelterborn et al., <i>unpublished data</i>).
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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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− | Target recognition inside the nuclear genome of <i>C. reinhardtii</i> relies on the presence of protospacers and protospacer-adjacent motifs (PAM) on the target DNA. A gRNA, which is designed to match specific regions on the genome and bind to them (Mali et al., 2013) is used to direct the Cas9 endonuclease. Upon binding to the target region, a cut of Cas9 creates a double-stranded break (DSB) with blunt ends. Into the DSB artificial DNA could be inserted through DNA repair-mechanisms. We wanted to find out if there were regions in the genome of <i>C. reinhardtii</i> that displayed an increased protein expression. To this end, we designed sgRNA complementary to three different genome regions.
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− | </p>
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− | The first locus is the SNRK locus. Since Kelterborn et al. had implemented a straight-forward screening process, successful cleavages could easily be identified. We want to use these loci mainly as a comparison to different locus insertions, since we do not expect high expression. As we are using the Promoter of the Photosystem Subunit II, we want to check if we are able to increase the expression by inserting our DNA Fragments at the end of the PsaD gene. Strenkert et al. have shown that a light dependent increase of PsaD expression occurs. When grown under synchronized light conditions a higher protein yield may be achieved.
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− | </p>
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− | Ribulose-1,5-bisphosphat-carboxylase (RuBisCo) is often referred to as the most abundant soluble protein. Carrying the function of carbon dioxide fixation it is present in all plants and phototrophic organisms. Due to this frequent appearance, we want to see if insertions inside the RuBisCo locus lead to higher expression. By targeting this locus for the insertion of our constructs, we hope to increase the protein yield, while characterizing the expression pattern of <i>C. reinhardtii</i> to ease the further use for the SynBio community.
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− | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/d/d5/T--Humboldt_Berlin--screening.jpeg" />
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− | <p><b>Fig. </b>
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− | <figcaption> Here should be a figure depicting the Cas9 and/or the RESDA-System. </figcaption>
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− | </figure>
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− | During the process of electroporation transgenic DNA is inserted randomly into the genome of <i>C. reinhardtii</i>. This opens up the possibility that a high expression locus might be found by coincidence during our screening process. This locus could then be considered for further transformations to yield a high protein expression, useful in all genetic engineering projects.
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− | </p>
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− | To identify such a locus we try to amplify this genomic region with a <b>restriction enzyme site-directed amplification PCR (RESDA-PCR)</b>. This polymerase chain reaction uses primers that are designed in a way that they anneal at restriction enzyme recognition sites of the enzymes <i>Alu</i>I, <i>Sac</i>II, PstI or <i>Taq</i>I. These sites are widely spread inside the genome. This increases the probability that the transgene DNA is inserted next to one of these sites, so that a PCR-product containing the genome sequences might be amplified and sequenced.
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− | <img src="https://static.igem.org/mediawiki/2019/3/3e/T--Humboldt_Berlin--ArrowDown.jpg">
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− | <div class="two-columns-headline" id="GrowthModeling">
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− | <!--------------------Modeling photoautotrophic growth-------------------->
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− | <h3 class="headline3">1.3.5. Modeling photoautotrophic growth of Chlamy</h3>
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− | In a first step to facilitate the ‚Design - Build - Test - Learn‘ cycle, we wanted to create a model that has the aim to give an overview of metabolic processes, genes and other parameters necessary for photosynthetic growth.
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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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− | <!-- IMAGE WITH CAPTION -->
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− | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/1/16/T--Humboldt_Berlin--GrowthModeling.jpeg" />
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− | <p><b>Fig. Overview of the components of a growth model.</b>
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− | <p>
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− | <figcaption> Each component of our analysis is derived from the function it has in Chlamy's growth. Adapting the parameters can give insight into the best way of cultivation for the optimal growth conditions, for example, in a bioreactor. </figcaption>
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− | </figure>
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− | We use the syntax defined in the Constraint Based Reconstruction Analysis (COBRA) Toolbox for Python (Ebrahim et al. 2013), already existing metabolic reconstructions of Chlamy (Imam et al 2015, Kliphuis et al. 2011), the fully sequenced genome (Merchant et. al 2007 ) and a recent -omics dataset (Strenkert et al. 2019) to define the components of the model. In a second step we combine these components in such a way that we are able to assess how they work together to give rise to growth of a Chlamy culture. The model should also provide the synthetic biologist with information useful for performing specific tasks in <i>C. reinhardtii</i>.
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− | </p>
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− | <p>
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− | Unfortunately, we could not finish the model because there was not enough time. We still were able to use knowledge gathered along the way, though.
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− | </p>
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− | <h3 class="headline3">2. Working on PET-Degradation as a proof-of-concept</h3> | + | <h3 class="headline3">2 Compnents overview</h3> |
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| <h3><a href="#PETModelling">2.1 Modeling PET degradation by <i>C. reinhardtii</i> using an optimized PETase</a></h3> | | <h3><a href="#PETModelling">2.1 Modeling PET degradation by <i>C. reinhardtii</i> using an optimized PETase</a></h3> |
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| <h3><a href="#ToxTest">2.3 Testing the toxicity of TPA and EG for <i>C. reinhardtii</i></a></h3> | | <h3><a href="#ToxTest">2.3 Testing the toxicity of TPA and EG for <i>C. reinhardtii</i></a></h3> |
− | </LI>
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− | <LI>
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− | <h3><a href="#selectionCassette">2.4 Selection cassette construction</a></h3>
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− | </LI>
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− | <h3><a href="#Concentration">2.5 Measuring concentration of TPA and EG in medium (quantitative activity test)</a></h3>
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− | </LI>
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− | <h3><a href="#Assay">2.6 PnpB assay to test enzyme activity (quantitative test)</a></h3>
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− | </LI>
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− | <h3><a href="#ChlamyiGEM">2.7 Purification of PETase and MHETase from <i>E. coli</i> in order to characterize and compare enzyme activity</a></h3>
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| </LI> | | </LI> |
| </UL> | | </UL> |
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| <!------------------Bioreactor-----------------------> | | <!------------------Bioreactor-----------------------> |
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− | <h3 class="headline3" :>3. Designing and building a bioreactor</h3> | + | <h3 class="headline3" :>3 Assembly instructions</h3> |
| </div> | | </div> |
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