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<h4>Achievements:</h4> | <h4>Achievements:</h4> | ||
− | <p>(I) The SpyTag-SpyCatcher system, originally developed by the 2013 team of <a target="_blank"href="http://parts.igem.org/Part:BBa_K1159200">TU-Munich</a> was improved and adapted to secretion in <i>Bacillus subtilis</i>. (II) Secretion and interaction of SpyTag-SpyCatcher system was demonstrated. (III) 8 <a href=" | + | <p>(I) The SpyTag-SpyCatcher system, originally developed by the 2013 team of <a target="_blank"href="http://parts.igem.org/Part:BBa_K1159200">TU-Munich</a> was improved and adapted to secretion in <i>Bacillus subtilis</i>. (II) Secretion and interaction of SpyTag-SpyCatcher system was <a href="#results" class="hashlink">demonstrated</a>. (III) 8 <a href="#basic" class="hashlink">basic BioBrick parts</a> were improved and 8 <a href="#composite" class="hashlink">composite parts</a> were generated and evaluated.</p> |
</figure> | </figure> | ||
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<h1 id="secretion" class="box-heading">Short Description</h1> | <h1 id="secretion" class="box-heading">Short Description</h1> | ||
− | In combining <i>Bacillus subtilis</i> powerful secretion capacity with Peptidosomes as a new platform for functional co-cultivation we aim to produce multiprotein complexes. Various strains - each secreting distinct proteins of interest - can be cultivated in one reaction hub while being physically separated. In this part of EncaBcillus we study extracellular protein interaction mediated by the SpyTag/SpyCatcher system. This set-up bears the potential for an effective production of customizable biomaterials or enzyme complexes.</p> | + | <p>In combining <i>Bacillus subtilis</i> powerful secretion capacity with Peptidosomes as a new platform for functional co-cultivation we aim to produce multiprotein complexes. Various strains - each secreting distinct proteins of interest - can be cultivated in one reaction hub while being physically separated. In this part of EncaBcillus we study extracellular protein interaction mediated by the SpyTag/SpyCatcher system. This set-up bears the potential for an effective production of customizable biomaterials or enzyme complexes.</p> |
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<div class="contentbox"> | <div class="contentbox"> | ||
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<h1 id="background" class="box-heading">Background</h1> | <h1 id="background" class="box-heading">Background</h1> | ||
<p>Efficient and low cost production of valuable natural compounds, like proteins, has developed into a leading industry. Starting from the choice of a suitable production host to the establishment of a profitable downstream process, every step is constantly optimized to increase overall yields. </p> | <p>Efficient and low cost production of valuable natural compounds, like proteins, has developed into a leading industry. Starting from the choice of a suitable production host to the establishment of a profitable downstream process, every step is constantly optimized to increase overall yields. </p> | ||
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<p>To ensure the assembly of proteins outside of the Peptidosomes we further characterized the SpyTag/SpyCatcher system. Theses functional units can be attached to any protein of interest and upon secretion will result in a covalent isopeptide bond between the SpyTag/SpyCatcher partners.<a target="_blank" href =" https://www.ncbi.nlm.nih.gov/pubmed/28230977">[2]</a> | <p>To ensure the assembly of proteins outside of the Peptidosomes we further characterized the SpyTag/SpyCatcher system. Theses functional units can be attached to any protein of interest and upon secretion will result in a covalent isopeptide bond between the SpyTag/SpyCatcher partners.<a target="_blank" href =" https://www.ncbi.nlm.nih.gov/pubmed/28230977">[2]</a> | ||
− | The system originates from <i>Streptococcus pyogenes</i> and still remains under constant developments.<a target="_blank" href =" https://www.ncbi.nlm.nih.gov/pubmed/ | + | The system originates from <i>Streptococcus pyogenes</i> and still remains under constant developments.<a target="_blank" href =" https://www.ncbi.nlm.nih.gov/pubmed/22366317>[3]</a> In our project we applied codon-adapted <i> B. subtilis</i> specific tags and a SpyCatcher that is reduced in length to enhance it´s usability when translationally fused to a protein of interest. Thus, decreasing the chances of the tag interfering with overall protein folding.<a target="_blank" href =" https://www.ncbi.nlm.nih.gov/pubmed/24161952">[4]</a></p> |
<p>To demonstrate the applicability of both tags we fused them to a green (sfGFP) and a red (mCherry) fluorescent protein, enabling an easy detectable output. (For more details please check our Design section) | <p>To demonstrate the applicability of both tags we fused them to a green (sfGFP) and a red (mCherry) fluorescent protein, enabling an easy detectable output. (For more details please check our Design section) | ||
Since a core part of this project involves secretion, we included a signal peptide in front of all our constructs. (click <a href =" https://2017.igem.org/Team:TU_Dresden/Measurement">here</a> to learn more about our Signal Peptide Toolbox).</p> | Since a core part of this project involves secretion, we included a signal peptide in front of all our constructs. (click <a href =" https://2017.igem.org/Team:TU_Dresden/Measurement">here</a> to learn more about our Signal Peptide Toolbox).</p> | ||
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− | <h1 class="box-heading">Results</h1> | + | <h1 id="results" class="box-heading">Results</h1> |
<h3>Fluorescence assay of supernatants derived from strains with single copy genes</h3> | <h3>Fluorescence assay of supernatants derived from strains with single copy genes</h3> | ||
<p>The first obstacle that we had to overcome was establishing a suitable protocol to boost the secretion capacities of our generated <i>Bacillus subitilis</i> producer strains. After initially testing various media and incubation periods we performed all experiments using 2xYT medium and an incubation time of 16 h.</p> | <p>The first obstacle that we had to overcome was establishing a suitable protocol to boost the secretion capacities of our generated <i>Bacillus subitilis</i> producer strains. After initially testing various media and incubation periods we performed all experiments using 2xYT medium and an incubation time of 16 h.</p> | ||
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alt="Figure 4: Endpoint measurement of the fluorescence from supernatants carrying our constructs and the wild type. " class="zoom"> | alt="Figure 4: Endpoint measurement of the fluorescence from supernatants carrying our constructs and the wild type. " class="zoom"> | ||
<figcaption><b>Figure 4: Endpoint measurement of the fluorescence from supernatants carrying our constructs and the wild type. </b> | <figcaption><b>Figure 4: Endpoint measurement of the fluorescence from supernatants carrying our constructs and the wild type. </b> | ||
− | Expression of the single copy mCherry or sfGFP fusion SpyTag/SpyCather constructs (purple) was induced with 1% xylose and the supernatants were harvested after 16 h of incubation. Wild type supernatant is shown as a control (pink). Excitation wavelength for sfGFP was set to 480 nm and emission was recorded at 510 nm and for mCherry excitation wavelength was set to 585 nm and emission was recorded at 615 nm. The fluorescence was normalized by the optical density ( | + | Expression of the single copy mCherry or sfGFP fusion SpyTag/SpyCather constructs (purple) was induced with 1% xylose and the supernatants were harvested after 16 h of incubation. Wild type supernatant is shown as a control (pink). Excitation wavelength for sfGFP was set to 480 nm and emission was recorded at 510 nm and for mCherry excitation wavelength was set to 585 nm and emission was recorded at 615 nm. The fluorescence was normalized by the optical density (OD<sub>600</sub>). Graph shows mean values and standard deviations of at least two biological and three technical replicates. |
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
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alt="Figure 6: Endpoint measurement of the fluorescence from supernatants. " class="zoom"> | alt="Figure 6: Endpoint measurement of the fluorescence from supernatants. " class="zoom"> | ||
<figcaption><b>Figure 6: Endpoint measurement of the fluorescence from supernatants. </b> | <figcaption><b>Figure 6: Endpoint measurement of the fluorescence from supernatants. </b> | ||
− | Expression of the multi copy mCherry or sfGFP fusion constructs (purple) was induced with 1% Xylose and the supernatants were harvested after 16 h of incubation. Wild type supernatant is shown as a control (pink). Excitation wavelength for sfGFP was set to 480 nm and emission was recorded at 510 nm and for mCherry excitation wavelength was set to 585 nm and emission was recorded at 615 nm . The fluorescence was normalized over the optical density ( | + | Expression of the multi copy mCherry or sfGFP fusion constructs (purple) was induced with 1% Xylose and the supernatants were harvested after 16 h of incubation. Wild type supernatant is shown as a control (pink). Excitation wavelength for sfGFP was set to 480 nm and emission was recorded at 510 nm and for mCherry excitation wavelength was set to 585 nm and emission was recorded at 615 nm . The fluorescence was normalized over the optical density (OD<sub>600</sub>). Graphs show mean values and standard deviations of at least two biological and three technical replicates. |
</figcaption> | </figcaption> | ||
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alt="Figure 7: Supernatants of <i>B. subtilis</i> cultures excited with blue light." | alt="Figure 7: Supernatants of <i>B. subtilis</i> cultures excited with blue light." | ||
class="makeresponsive zoom"> | class="makeresponsive zoom"> | ||
− | <figcaption><b>Figure 7: Supernatants of <i>B. subtilis</i> cultures excited with blue light.</b> | + | <figcaption><b>Figure 7: Supernatants of <i><b>B. subtilis</b></i> cultures excited with blue light.</b> |
Wild-type supernatant (left) and a SpyTag-sfGFP secreting strain (middle and right). The expression of the multi-copy sfGFP was induced with 1% Xylose and the supernatant was harvested after 16 h of incubation. The fluorescence was induced with a “Dark Reader Transilluminator”. | Wild-type supernatant (left) and a SpyTag-sfGFP secreting strain (middle and right). The expression of the multi-copy sfGFP was induced with 1% Xylose and the supernatant was harvested after 16 h of incubation. The fluorescence was induced with a “Dark Reader Transilluminator”. | ||
</figcaption> | </figcaption> | ||
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alt="Figure 8: Supernatants of <i>B. subtilis</i> cultures." | alt="Figure 8: Supernatants of <i>B. subtilis</i> cultures." | ||
class="makeresponsive zoom"> | class="makeresponsive zoom"> | ||
− | <figcaption><b>Figure 8: Supernatants of <i>B. subtilis </i>cultures.</b> | + | <figcaption><b>Figure 8: Supernatants of <i><b>B. subtilis</b></i> cultures.</b> |
Wild-type supernatant (left) and a mCherry-mini. SpyCatcher secreting strain (right). The expression of the multi-copy mCherry was induced with 1% Xylose and the supernatant was harvested after 16 h of incubation. | Wild-type supernatant (left) and a mCherry-mini. SpyCatcher secreting strain (right). The expression of the multi-copy mCherry was induced with 1% Xylose and the supernatant was harvested after 16 h of incubation. | ||
</figcaption> | </figcaption> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td><a target="_blank" href =" https://www.ncbi.nlm.nih.gov/pubmed/ | + | <td><a target="_blank" href =" https://www.ncbi.nlm.nih.gov/pubmed/22366317">[3]</a></td> |
<td>Zakeri et. All (2012) Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. <i>Applied Microbiology and Biotechnology</i>.</td> | <td>Zakeri et. All (2012) Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. <i>Applied Microbiology and Biotechnology</i>.</td> | ||
</tr> | </tr> |
Latest revision as of 15:50, 13 December 2017