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The system originates from Streptococcus pyogenes and since it’s discovery it has been under constant development <a target="_blank" href =" https://www.ncbi.nlm.nih.gov/pubmed/28230977">[3]</a>. In our project we applied codon-adapted B. subtilis specific tags and reduced the SpyCatcher 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> | The system originates from Streptococcus pyogenes and since it’s discovery it has been under constant development <a target="_blank" href =" https://www.ncbi.nlm.nih.gov/pubmed/28230977">[3]</a>. In our project we applied codon-adapted B. subtilis specific tags and reduced the SpyCatcher 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 target="_blank" href =" https://2017.igem.org/Team:TU_Dresden/Measurement">here</a> | + | Since a core part of this project involves secretion, we included a signal peptide in front of all our constructs. (click <a target="_blank" href =" https://2017.igem.org/Team:TU_Dresden/Measurement">here</a> to learn more about our Signal Peptide Toolbox). |
To evaluate the efficiency of the secretion process we monitored the fluorescence of B. subtilis strains carrying our constructs and compared them to the wild type. In order to prove the functionality of the SpyTag/SpyCatcher system we performed an SDS-Page demonstrating the formation of a fusion protein derived from co-incubated supernatants. </p> | To evaluate the efficiency of the secretion process we monitored the fluorescence of B. subtilis strains carrying our constructs and compared them to the wild type. In order to prove the functionality of the SpyTag/SpyCatcher system we performed an SDS-Page demonstrating the formation of a fusion protein derived from co-incubated supernatants. </p> | ||
</div> | </div> |
Revision as of 15:06, 26 October 2017