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| <section class="page-content fixed-header-content"> | | <section class="page-content fixed-header-content"> |
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− | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/2/2f/T--Humboldt_Berlin--chlamy_hub_vision_2columns.png" alt="Vision: ChlamyHUB"/></br> | + | <img src="https://static.igem.org/mediawiki/2019/2/2f/T--Humboldt_Berlin--chlamy_hub_vision_2columns.png" alt="Vision: ChlamyHUB"/></br> |
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| <!-----------------------------------------------------------------------> | | <!-----------------------------------------------------------------------> |
| <!------------------Chlamy to iGEM-----------------------> | | <!------------------Chlamy to iGEM-----------------------> |
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| </p> | | </p> |
| </div> | | </div> |
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| <!--- IMAGE ---> | | <!--- IMAGE ---> |
− | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/3/32/T--Humboldt_Berlin--chlamy2igem.png" alt="Bringing Chlamy to iGEM" /> | + | <img src="https://static.igem.org/mediawiki/2019/3/32/T--Humboldt_Berlin--chlamy2igem.png" alt="Bringing Chlamy to iGEM" /> |
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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" /> | | <img src="https://static.igem.org/mediawiki/2019/1/1b/T--Humboldt_Berlin--designfig1.png" alt="Overview of the hierarchical and modular cloning system" /> |
| <p style="font-size:12px"> | | <p style="font-size:12px"> |
− | <b>Fig. 1. Universal MoClo fusion sites. </b> | + | <figcaption> <b>Fig. 1. Universal MoClo fusion sites. </b> 12 fusion sites (Patron, 2015) for the seamless fusion of different level 0 parts. In general, the fusion sites are grouped into 5’ untranslated regions including promoter sequences (grey), the translated coding sequence CDS (blue) followed by 3’ UTRs ending with a terminator (orange). Within these types, various parts (e.g. three different coding sequences) can be designed and combined into one transcription unit when cloned together into a L1 vector. The bold ATG within the B3 fusion site sets the transcription start.</figcaption> |
− | <figcaption> 12 fusion sites (Patron, 2015) for the seamless fusion of different level 0 parts. In general, the fusion sites are grouped into 5’ untranslated regions including promoter sequences (grey), the translated coding sequence CDS (blue) followed by 3’ UTRs ending with a terminator (orange). Within these types, various parts (e.g. three different coding sequences) can be designed and combined into one transcription unit when cloned together into a L1 vector. The bold ATG within the B3 fusion site sets the transcription start.</figcaption>
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| </div> | | </div> |
| <div> | | <div> |
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| <div> | | <div> |
| <!-- IMAGE WITH CAPTION --> | | <!-- IMAGE WITH CAPTION --> |
− | <figure class="is-revealing"> | + | <figure > |
− | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/d/d9/T--Humboldt_Berlin--designfig2.png" alt="cloning strategy" /> | + | <img src="https://static.igem.org/mediawiki/2019/d/d9/T--Humboldt_Berlin--designfig2.png" alt="cloning strategy" /> |
| </figure> | | </figure> |
| </div> | | </div> |
| <div> | | <div> |
− | <p><b>Fig. 2. Overview of the Golden Gate cloning strategy.</b> | + | <p> |
| <p> | | <p> |
− | <figcaption> Multiple basic genetic elements on a level 0 vector (Phytobricks) can be assembled to a full transcription unit on a level 1 vector. Specific fusion sites (shown in grey) and BpiI recognition sites are added to new genetic elements via PCR. To build a L0 construct the Type IIS restriction enzyme BpiI digests the PCR fragment and the L0 vector. The L0 vector, in turn, contains the recognition site for BsaI. Digestion with BsaI and ligation of several L0 parts with a L1 backbone leads to a L1 transcriptional unit. The different L1 modules of choice can then be assembled via BpiI into the final L2 construct in which no recognition sites for type IIS restriction enzymes are left.</figcaption> | + | <figcaption> <b>Fig. 2. Overview of the Golden Gate cloning strategy.</b> Multiple basic genetic elements on a level 0 vector (Phytobricks) can be assembled to a full transcription unit on a level 1 vector. Specific fusion sites (shown in grey) and BpiI recognition sites are added to new genetic elements via PCR. To build a L0 construct the Type IIS restriction enzyme BpiI digests the PCR fragment and the L0 vector. The L0 vector, in turn, contains the recognition site for BsaI. Digestion with BsaI and ligation of several L0 parts with a L1 backbone leads to a L1 transcriptional unit. The different L1 modules of choice can then be assembled via BpiI into the final L2 construct in which no recognition sites for type IIS restriction enzymes are left.</figcaption> |
| </div> | | </div> |
| </div> | | </div> |
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| </p> | | </p> |
| <img src="https://static.igem.org/mediawiki/2019/5/5e/T--Humboldt_Berlin--SelKas_overview.png" alt="SelKas" /> | | <img src="https://static.igem.org/mediawiki/2019/5/5e/T--Humboldt_Berlin--SelKas_overview.png" alt="SelKas" /> |
| + | <figcaption> <b>Overview of the construction of selection cassettes. </b> |
| </div> | | </div> |
| <div class="expandable-more"> | | <div class="expandable-more"> |
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| <!--- IMAGE ---> | | <!--- IMAGE ---> |
− | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/b/bc/T--Humboldt_Berlin--Selection_cassette.jpeg" alt="Selection cassette" /> | + | <img src="https://static.igem.org/mediawiki/2019/b/bc/T--Humboldt_Berlin--Selection_cassette.jpeg" alt="Selection cassette" /> |
− | <p><b>Fig. 3. Cloning process to insert a selection cassette into the L1c-RFP_ampR/Ori plasmid.</b> | + | <p> |
− | <p>
| + | <figcaption> <b>Fig. 3 - Cloning process to insert a selection cassette into the L1c-RFP_ampR/Ori plasmid.</b> The selection transcription unit (PsaD-B2Linker-hygR-Rbcs2) inside the pICH47732 backbone can be amplified with primers containing restriction sites for <i>BamH</i>I and <i>Xho</i>I to receive a PCR product linkable with the new constructed L1 plasmid L1c-RFP_ampR/Ori when digested with <i>BamH</i>I and <i>Xho</i>I. The resulting plasmid L1c-RFP-HygR represents a level 1 Golden Gate cloning vector specified for <i>C. reinhardtii</i>. </figcaption> |
− | <figcaption> The selection transcription unit (PsaD-B2Linker-hygR-Rbcs2) inside the pICH47732 backbone can be amplified with primers containing restriction sites for <i>BamH</i>I and <i>Xho</i>I to receive a PCR product linkable with the new constructed L1 plasmid L1c-RFP_ampR/Ori when digested with <i>BamH</i>I and <i>Xho</i>I. The resulting plasmid L1c-RFP-HygR represents a level 1 Golden Gate cloning vector specified for <i>C. reinhardtii</i>. </figcaption>
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| </div> | | </div> |
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| <div> | | <div> |
− | <figure class="is-revealing"> | + | <figure > |
− | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/d/d5/T--Humboldt_Berlin--screening.jpeg" /> | + | <img src="https://static.igem.org/mediawiki/2019/d/d5/T--Humboldt_Berlin--screening.jpeg" /> |
| + | <figcaption><b>Fig 4 - Visualization of the YFP selection process of transformation clones. </b> After growing on a selective media, the clones are screened for expression of the transformed transgenes. The screening methods are dependent on the intended function of each construct. |
| </figure> | | </figure> |
| </div> | | </div> |
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| <!--- IMAGE ---> | | <!--- IMAGE ---> |
| <img src="https://static.igem.org/mediawiki/2019/6/6c/T--Humboldt_Berlin--secretionChlamy.png"/> | | <img src="https://static.igem.org/mediawiki/2019/6/6c/T--Humboldt_Berlin--secretionChlamy.png"/> |
| + | <figcaption><b>Fig. 5 - Secretion of transgene proteins into the media by <i>C. reinhardtii</i>. </b> Secretion tags in form of signalling peptides added to expressed proteins in the ribosomes. These lead to secretion of said protein through exporters, a process during which the secretion tag is lost.</figcaption> |
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| </div> | | </div> |
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| <div> | | <div> |
− | <figure class="is-revealing"> | + | <figure > |
− | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/d/dd/T--Humboldt_Berlin--phosphite_to_phosphate_comparison.png" /> | + | <img src="https://static.igem.org/mediawiki/2019/d/dd/T--Humboldt_Berlin--phosphite_to_phosphate_comparison.png" /> |
− | <p><b>Fig. </b> | + | <figcaption><b>Fig. 6 - Growth comparison of wildtype <i>C. reinhardtii</i> and PtxD-expressing mutant in phosphite media.</b> In the case of the wildtype algae, growth is inhibited because the metabolization of phosphite is not possible and these strains therefore lack phosphorous. The PtxD-containing strains however are able to import and successfully integrate phosphite into their phosphorous metabolism.</figcaption> |
− | <p>
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− | <figcaption> This figure shows how the phosphite marker works. </figcaption>
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| </figure> | | </figure> |
| </div> | | </div> |
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| To test if they behave as expected, we designed constructs making it possible to directly compare them by expressing YFP as fluorescent marker protein under the regulation of both promoters. Using a plate reader and fluorescence microscope made comparing numerous AR- and PsaD-constructs very easily. | | To test if they behave as expected, we designed constructs making it possible to directly compare them by expressing YFP as fluorescent marker protein under the regulation of both promoters. Using a plate reader and fluorescence microscope made comparing numerous AR- and PsaD-constructs very easily. |
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− | We also planned to compare expression rates of constructs containing the PsaD promoter with and without introns. PsaD with introns was designed taking the AR promoter http://parts.igem.org/Part:BBa_K2984046 as a model. The AR promoter is composed of the HSP70 promoter fused to the RbcS2 intron. We followed a similar approach by fusing the RbcS2 intron to the end of the PsaD promoter http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984008, hoping to achieve a higher expression yield. | + | We also planned to compare expression rates of constructs containing the PsaD promoter with and without introns. PsaD with introns was designed taking the <a href="http://parts.igem.org/Part:BBa_K2984046">AR promoter</a> as a model. The AR promoter is composed of the HSP70 promoter fused to the RbcS2 intron. We followed a similar approach by fusing the RbcS2 intron to the end of the <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984008">PsaD promoter</a>, hoping to achieve a higher expression yield. |
| Unfortunately, we were not able to compare expression driven by these three variants of promoters in the wet lab. | | Unfortunately, we were not able to compare expression driven by these three variants of promoters in the wet lab. |
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| <!--- IMAGE ---> | | <!--- IMAGE ---> |
| <img src="https://static.igem.org/mediawiki/2019/d/df/T--Humboldt_Berlin--promotor_vs-plate_reader-microscope.png"/> | | <img src="https://static.igem.org/mediawiki/2019/d/df/T--Humboldt_Berlin--promotor_vs-plate_reader-microscope.png"/> |
− | | + | <figcaption><b>Fig. 7 - Comparing the efficiency of PsaD promoter with and without intron.</b> The presence of YFP protein expressed under each promoter variant is assessed by fluorescence microscopy first. Then, the amount of YFP is determined by measuring with a plate reader using a calibration curve of known protein amount-fluorescence measurements.</figcaption> |
| </div> | | </div> |
| </div> | | </div> |
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| </p> | | </p> |
| <p> | | <p> |
− | 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. | + | 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. (2019) have shown that a light dependent increase of PsaD expression occurs. When grown under synchronized light conditions a higher protein yield may be achieved. |
| </p> | | </p> |
| <p> | | <p> |
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| <div> | | <div> |
− | <figure class="is-revealing"> | + | <figure > |
− | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/6/6c/T--Humboldt_Berlin--cas9_resda.png" /> | + | <img src="https://static.igem.org/mediawiki/2019/6/6c/T--Humboldt_Berlin--cas9_resda.png" /> |
− | <p><b>Fig. </b>
| + | <figcaption> <b>Fig. 8 Crisp Cas9 and RESDA-PCR </b> Inserting genes through Cas9 and verifying their position through RESDA-PCR </figcaption> |
− | <p>
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− | <figcaption> Inserting genes through Cas9 and verifying their position through RESDA-PCR </figcaption>
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| </figure> | | </figure> |
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| <!-- IMAGE WITH CAPTION --> | | <!-- IMAGE WITH CAPTION --> |
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− | <figure class="is-revealing"> | + | <figure > |
− | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/1/16/T--Humboldt_Berlin--GrowthModeling.jpeg" /> | + | <img src="https://static.igem.org/mediawiki/2019/1/16/T--Humboldt_Berlin--GrowthModeling.jpeg" /> |
− | <p><b>Fig. Overview of the components of a growth model.</b>
| + | <figcaption> <b>Fig. 9: Overview of the components of a growth model.</b> 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> |
− | <p>
| + | |
− | <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> | | </figure> |
| </div> | | </div> |
| <div> | | <div> |
| <p> | | <p> |
− | 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>. | + | 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>. |
| </p> | | </p> |
| <p> | | <p> |
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| <!-----------------------------------------------------------------------> | | <!-----------------------------------------------------------------------> |
| <!------------------PET degradation-----------------------> | | <!------------------PET degradation-----------------------> |
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| </p> | | </p> |
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| <!--- IMAGE ---> | | <!--- IMAGE ---> |
− | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/b/be/T--Humboldt_Berlin--microplastic_icon_ablauf.png" alt="Bringing Chlamy to iGEM" /> | + | <img src="https://static.igem.org/mediawiki/2019/b/be/T--Humboldt_Berlin--microplastic_icon_ablauf.png" alt="Bringing Chlamy to iGEM" /> |
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| <!--- IMAGE ---> | | <!--- 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" /> | + | <img src="https://static.igem.org/mediawiki/2019/3/32/T--Humboldt_Berlin--modell_uebersicht.png" alt="PET degradation model" /> |
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| <div class="expandable-container" style="margin-bottom:0px;padding-bottom:0px;margin-top:0px;padding-top:0px"> | | <div class="expandable-container" style="margin-bottom:0px;padding-bottom:0px;margin-top:0px;padding-top:0px"> |
− | <div class="standardmarginleft" style="margin-bottom:0px;padding-bottom:0px" id="Transformation"> | + | <div class="standardmarginleft" id="Transformation"> |
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| <h3 class="headline3" :>2.2 Transformation of PETase and MHETase into <i>C. reinhardtii</i></h3> | | <h3 class="headline3" :>2.2 Transformation of PETase and MHETase into <i>C. reinhardtii</i></h3> |
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| <div class="expandable-preview"> | | <div class="expandable-preview"> |
| <p class="medium-sized"> | | <p class="medium-sized"> |
− | 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.
| + | Expressing and secreting the enzymes PETase and MHETase with <i>C. reinhardtii</i> could pose 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. |
| </p> | | </p> |
− | <img src="https://static.igem.org/mediawiki/2019/f/f1/T--Humboldt_Berlin--Chlamytrafo.png"/" /> | + | <img src="https://static.igem.org/mediawiki/2019/f/f1/T--Humboldt_Berlin--Chlamytrafo.png" /> |
| </div> | | </div> |
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| <UL class="medium-sized"> | | <UL class="medium-sized"> |
− | <LI class="medium-sized" > | + | <LI class="medium-sized"> |
| PETase with secretion signal GLE and fluorescent tag YFP <br> </br> | | PETase with secretion signal GLE and fluorescent tag YFP <br> </br> |
| </LI> | | </LI> |
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| <div> | | <div> |
| <p> | | <p> |
− | 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> | + | 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. | | 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 ---> | | <!--- IMAGE ---> |
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− | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/8/80/T--Humboldt_Berlin--ToxTest.png" /> | + | <img src="https://static.igem.org/mediawiki/2019/8/80/T--Humboldt_Berlin--ToxTest.png" /> |
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| We therefore decided to address this problem by designing and constructing an <a href="/Team:Humboldt_Berlin/Hardware">open source platform</a> that is capable of growing phototrophic organisms in turbidostat mode, not only monitoring growth but also making it possible to adjust environmental conditions.<br> </br> | | We therefore decided to address this problem by designing and constructing an <a href="/Team:Humboldt_Berlin/Hardware">open source platform</a> that is capable of growing phototrophic organisms in turbidostat mode, not only monitoring growth but also making it possible to adjust environmental conditions.<br> </br> |
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− | To help us in the process of design, we asked several sources with experience and knowledge in algae cultivation on different scales to get a first overview of problems and demands of cultivation (Joerg Ullmann, Gunnar Muehlstaedt, Ralf Steuer ). With this help and knowledge gathered from modeling, we were able to pin down necessary functions our platform has to fulfill and therefore components it has to be built of. We always keep one function important to the Synbio community in the back of our head: The ability to perform many experiments and/or screening events at once. This means that a general demand for our setup is to be easily affordable, reproducible and manufacturable with as little effort as possible. <br> </br> | + | To help us in the process of design, we asked several sources with experience and knowledge in algae cultivation on different scales to get a first overview of problems and demands of cultivation (Joerg Ullmann, Gunnar Muehlstaedt, Ralf Steuer). With this help and knowledge gathered from modeling, we were able to pin down necessary functions our platform has to fulfill and therefore components it has to be built of. We always keep one function important to the Synbio community in the back of our head: The ability to perform many experiments and/or screening events at once. This means that a general demand for our setup is to be easily affordable, reproducible and manufacturable with as little effort as possible. <br> </br> |
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| Taking these demands and information into account we decided on the following functions and components performing them: | | Taking these demands and information into account we decided on the following functions and components performing them: |
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| We will create a library of step by step tutorials showing how to build all of these different functions and make it possible and motivate other iGEM participants and scientists to engage in do it yourself (DIY) setups and electronics. <br> </br> | | We will create a library of step by step tutorials showing how to build all of these different functions and make it possible and motivate other iGEM participants and scientists to engage in do it yourself (DIY) setups and electronics. <br> </br> |
| </p></br></br> | | </p></br></br> |
− | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/3/3f/T--Humboldt_Berlin--3drender.jpeg" alt="Our Bioreactor" style="width=50%"/> | + | <img src="https://static.igem.org/mediawiki/2019/3/3f/T--Humboldt_Berlin--3drender.jpeg" alt="Our Bioreactor" style="width=50%"/> |
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| </div> | | </div> |
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− | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/f/fc/T--Humboldt_Berlin--Algae_cult.png" alt="Usage of a Bioreactor" /> | + | <img src="https://static.igem.org/mediawiki/2019/f/fc/T--Humboldt_Berlin--Algae_cult.png" alt="Usage of a Bioreactor" /> |
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| + | <section class="learn-more"> |
| + | <h3>Learn more...</h3> |
| + | <div class="learn-more-links"> |
| + | <a href="/Team:Humboldt_Berlin/Results"> |
| + | <img src="https://static.igem.org/mediawiki/2019/9/9f/T--Humboldt_Berlin--results.jpg" alt="results preview" /> |
| + | <h4>Results</h4> |
| + | <p class="block-text"> |
| + | This is the data we collected with our experimental set-up. Click here to see what we found out. |
| + | </p> |
| + | </a> |
| + | <a href="/Team:Humboldt_Berlin/Experiments" class="white-text"> |
| + | <img src="https://static.igem.org/mediawiki/2019/4/43/T--Humboldt_Berlin--design.jpg" alt="experiments preview" /> |
| + | <h4>Experiments</h4> |
| + | <p class="block-text"> |
| + | If you are interested in our day-to-day lab protocols or standard workflows - our Experiments page lists all resources you might need. |
| + | </p> |
| + | </a> |
| + | </div> |
| + | </section> |
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| <a href="#" class="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 | | <img src="https://static.igem.org/mediawiki/2019/3/3e/T--Humboldt_Berlin--ArrowDown.jpg" /> Go to top |
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| 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://doi.org/10.1111/nph.13532. doi:10.1111/nph.13532 | | 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://doi.org/10.1111/nph.13532. doi:10.1111/nph.13532 |
− | </p>
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− | <p>
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− | 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
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| </p> | | </p> |
| <p> | | <p> |
| Palm, G. J., Reisky, L., Böttcher, D., Müller, H., Michels, E. A., Walczak, M. C., ... & Weber, G. (2019). Structure of the plastic-degrading Ideonella sakaiensis MHETase bound to a substrate. Nature communications, 10(1), 1717. | | Palm, G. J., Reisky, L., Böttcher, D., Müller, H., Michels, E. A., Walczak, M. C., ... & Weber, G. (2019). Structure of the plastic-degrading Ideonella sakaiensis MHETase bound to a substrate. Nature communications, 10(1), 1717. |
| + | </p> |
| + | <p> |
| + | Ramos-Martinez, E. M., Fimognari, L., & Sakuragi, Y. (2017). High-yield secretion of recombinant proteins from the microalga Chlamydomonas reinhardtii. Plant Biotechnol J, 15(9), 1214-1224. doi:10.1111/pbi.12710. |
| + | </p> |
| + | <p> |
| + | Rasala, B. A., Lee, P. A., Shen, Z., Briggs, S. P., Mendez, M., & Mayfield, S. P. (2012). Robust Expression and Secretion of Xylanase1 in Chlamydomonas reinhardtii by Fusion to a Selection Gene and Processing with the FMDV 2A Peptide. PLOS ONE, 7(8), e43349. |
| </p> | | </p> |
| <p> | | <p> |
| Strenkert, S., Schmollinger, S., Gallaher, S. D., Salomé, P. A., Purvine, S. O., Nicora, C. D., Mettler-Altmann, T., Soubeyrand, E., Weber, A. P. M., Lipton, M. S., Basset, G. J., Merchant, S. S. Proceedings of the National Academy of Sciences Feb 2019, 116 (6) 2374-2383; DOI:10.1073/pnas.1815238116 | | Strenkert, S., Schmollinger, S., Gallaher, S. D., Salomé, P. A., Purvine, S. O., Nicora, C. D., Mettler-Altmann, T., Soubeyrand, E., Weber, A. P. M., Lipton, M. S., Basset, G. J., Merchant, S. S. Proceedings of the National Academy of Sciences Feb 2019, 116 (6) 2374-2383; DOI:10.1073/pnas.1815238116 |
| </p> | | </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> |
| + | White, A. K., & Metcalf, W. W. (2007). Microbial metabolism of reduced phosphorus compounds. Annu Rev Microbiol, 61, 379-400. doi:10.1146/annurev.micro.61.080706.093357. |
| + | </p> |
| <p> | | <p> |
| </div> | | </div> |