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− | As many enzymes function outside of its host organism, we want to recreate the secretion of heterologous expressed proteins in <i>C. reinhardtii</i>. Secretion is possible due to a signal sequence attached to the protein at its 3’UTR that leads it to the secretory pathway. We take native signals from arylsulphatase 1 (ARS1) and a putative signal sequence of gametolysin (GLE) (Ramos-Martinez, Fimognari, & Sakuragi, 2017; Rasala et al., 2012) and build L0 modules with them. As the yield of heterologous expressed proteins fused to these secretion signals was previously described as relatively low, we additionally designed a 5’UTR glycomodule (SP<sub>20</sub>) as a L0 part which also enhances the stability of the attached protein when secreted (Ramos-Martinez, Fimognari, & Sakuragi, 2017; Rasala et al., 2012).
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− | </p>
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− | <p>
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− | In order to test these secretion signals, we tagged them with a YFP protein, which can be observed under a fluorescence microscope (excitation peak at 480 nm and emission peak at 528 nm). If secreted, the medium outside the cells is expected to emit fluorescence as opposed to the medium surrounding wild type algae.
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| <h3 class="headline3">1.3.3 PtxD - Phosphite Oxidoreductase</h3> | | <h3 class="headline3">1.3.3 PtxD - Phosphite Oxidoreductase</h3> |
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| + | <p class="medium-sized"> |
| + | 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. |
| + | </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" /> |
| + | </div> |
| + | <div class="expandable-more"> |
| + | <div class="two-columns block-text medium-sized not-centered no-margin-top" style="margin-bottom:0px"> |
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| <figure class="is-revealing"> | | <figure class="is-revealing"> |
| <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/d/d5/T--Humboldt_Berlin--screening.jpeg" /> | | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/d/d5/T--Humboldt_Berlin--screening.jpeg" /> |
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− | <p>
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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. 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. | + | 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. |
| </p> | | </p> |
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| + | </div> |
| + | <div class="expandable-readall"> |
| + | <span class="readmore">Read more</span> |
| + | <span class="readless">Read less</span> |
| + | <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"> | | <div class="two-columns-headline" id="Cas9"> |
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| <h3 class="headline3">1.3.4 Cas9/sgRNA-mediated site-directed mutagenesis </h3> | | <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>).
| + | <div class="expandable-preview"> |
− | </p>
| + | <p class="medium-sized"> |
− | <p> | + | 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>). |
| + | </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" /> |
| + | </div> |
| + | <div class="expandable-more"> |
| + | <div class="two-columns block-text medium-sized not-centered no-margin-top" style="margin-bottom:0px"> |
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| + | <div> |
| + | <p> |
| 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. | | 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. |
| </p> | | </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. | | 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. |
− | </p>
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− | <p>
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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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− | <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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| </p> | | </p> |
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| <figure class="is-revealing"> | | <figure class="is-revealing"> |
| <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/d/d5/T--Humboldt_Berlin--screening.jpeg" /> | | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/d/d5/T--Humboldt_Berlin--screening.jpeg" /> |
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| <figcaption> Here should be a figure depicting the Cas9 and/or the RESDA-System. </figcaption> | | <figcaption> Here should be a figure depicting the Cas9 and/or the RESDA-System. </figcaption> |
| </figure> | | </figure> |
| + | |
| + | <p> |
| + | 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. |
| + | </p> |
| + | <p> |
| + | 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. |
| + | </p> |
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| + | <div class="expandable-readall"> |
| + | <span class="readmore">Read more</span> |
| + | <span class="readless">Read less</span> |
| + | <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"> | | <div class="two-columns-headline" id="GrowthModeling"> |
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