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| − | <h2>Cloning strategies and the Yeast Toolbox for multipart-assembly</h2>
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| − | <p>Describing our cloning strategies we mentioned several <i>levels</i>, which stand for different stages of our plasmids. They are further described in the work of J.M. Dueber and colleagues, who designed the well established yeast toolkit we used in this project
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| − | <abbr title="2015, Michael E. Lee et al., - A highly characterized yeast toolkit for modular, multipart assembly.">
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| − | Michael E. Lee (2015)
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| − | </abbr>
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| − | </a>.
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| − | The toolkit offers the possibility to design plasmids with desired antibiotic resistances, promoters as well as terminators from standardized parts. It also provides fluorescence proteins, protein-tags and many more useful components as part plasmids. These part plasmids are distinguished in different part types due to their specific overhangs to ensure their combination in the correct order (e.g. promoter - gene of interest - terminator) all in a versatile one-pot Golden Gate reaction without time-consuming conventional cloning steps.</p>
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| − | <figure>
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| − | <img src=<i>https://static.igem.org/mediawiki/2017/3/3d/T--cologne-duesseldorf--dueber_toolbox.png <i>>
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| − | <figcaption>The yeast toolkit starter set comprises of 96 parts and vectors. The eight primary part types can be further divided into subtypes. Lee, Michael E., et al. "A highly characterized yeast toolkit for modular, multipart assembly." ACS synthetic biology 4.9 (2015): 975-986.</figcaption>
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| − | </figure>
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| − | <p>The cloning steps regarding the plasmid <i>levels</i> are implemented in <i>E.coli</i> in order to reduce the required time to generate the final plasmids. The different <i>levels</i> are therefore defined by their part content and their antibiotic resistances. </p>
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| − | <p>To generate a <i>level 0</i> plasmid, the gene of interest is ligated into the provided <i>level 0</i> backbone via golden gate assembly using the enzyme BsmBI. The backbone contains a resistance to Chloramphenicol, as well as an origin of replication, creating a very basic yet functional plasmid.</p>
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| − | <p>The <i>level 1</i> plasmid contains a promoter and terminator suited for <i>S. cerevisiae</i>. There is the possibility of including a polyhistidine-tag if there is a need for Western blot analysis. The antibiotic resistance contained in the <i>level 1</i> plasmid changes from chloramphenicol to ampicillin which enables filtering out residual <i>level 0</i> plasmids contained in the Golden Gate product. Furthermore, the <i>Dueber toolbox</i> includes the possibility of designing GFP-Dropout cassettes. These are custom-built <i>level 1</i> backbones whose inserts are sfGFP as well as promoter and terminator suited for <i>E. coli</i>. Upon a successful cloning step the GFP is replaced by the part(s) of interest, and correct colony shows a white colour. In case of a wrong ligation event colonies show a green fluorescence. This provides a very useful tool to detect unsuccessful cloned colonies. The enzyme used for <i>level 1</i> changes from BsmBI to BsaI to avoid any interference between different steps. </p>
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| − | <p>The <i>level 2</i> plasmid combines two or more genes of interest with their respective promoters, terminators and tags. The resistance changes from ampicillin to kanamycin. The enzyme of this step is BsmBI again. This level is useful, if the construct you are designing requires multiple genes to be transformed into one yeast strain.</p>
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