Difference between revisions of "Part:BBa K5201002"
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| − | <partinfo><a href="http://localhost:5173/hongkong-uccke/parts">BBa_K5201002</a> short</partinfo> | + | <!--<partinfo><a href="http://localhost:5173/hongkong-uccke/parts">BBa_K5201002</a> short</partinfo>--> |
<h3>Characterization </h3> | <h3>Characterization </h3> | ||
<p>Colony PCR and Agarose gel electrophoresis (AGE) </p> | <p>Colony PCR and Agarose gel electrophoresis (AGE) </p> | ||
<img src="https://static.igem.wiki/teams/5201/parts-registry/part-registry-11.png"></img> | <img src="https://static.igem.wiki/teams/5201/parts-registry/part-registry-11.png"></img> | ||
| − | <p>Fig. 11 Agarose gel electrophoresis of the colony PCR products of composite part (BBa_K5201002)</p> | + | <p>Fig. 11 Agarose gel electrophoresis of the colony PCR products of composite part (<a href="https://parts.igem.org/Part:BBa_K5201002>BBa_K5201002</a>)</p> |
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| − | <p>In our project, we used <i>E. coli</i> DH5α as host. Within its plasmid backbone, we inserted Lac Promoter (BBa_R0010), <i>pmHAS*1-703</i> (BBa_K5201000), RBS (BBa_B0030), <i>kfiD </ | + | <p>In our project, we used <i>E. coli</i> DH5α as host. Within its plasmid backbone, we inserted Lac Promoter (BBa_R0010), <i>pmHAS*1-703</i> (<a href="https://parts.igem.org/Part:BBa_K5201000">BBa_K5201000</a>), RBS (BBa_B0030), <i>kfiD </I>(<a href="https://parts.igem.org/Part:BBa_K5201001">BBa_K5201001</a>) and a double terminator (BBa_B0015) into our gene circuit. <i>Lac Promoter </i>is chosen since it is an inducible promoter where it can be induced by lactose or isopropylthio-galactoside (IPTG). In order for stronger induction, a strong RBS (BBa_B0030) is chosen. Most importantly, we have inserted <i>pmHAS*1-703 </i>and <i>kfiD </i>gene to produce HA. <i>pmHAS*1-703 </i>encodes a class II hyaluronic acid synthase (HAS) which converts UDP-glucuronic acid and UDP-N-Acetylglucosamine into HA. The residues 1-703 and 704-972 encode the catalytic domain and the transmembrane domain respectively. By using only the first 703 amino acids, we can express a non-membrane bound but functional HAS. Meanwhile, <i>kfiD</i> is responsible for synthesis of UDP-glucose-6-dehydrogenase (UGDH) which converts UDP-glucose into UDP-glucuronic acid. This allows the transformed bacteria to synthesize HA more efficiently.</p> |
Latest revision as of 10:10, 2 October 2024
Characterisation of BBa_K5201002: HongKong-UCCKE
Characterization
Colony PCR and Agarose gel electrophoresis (AGE)
Fig. 11 Agarose gel electrophoresis of the colony PCR products of composite part (BBa_K5201000), RBS (BBa_B0030), kfiD (BBa_K5201001) and a double terminator (BBa_B0015) into our gene circuit. Lac Promoter is chosen since it is an inducible promoter where it can be induced by lactose or isopropylthio-galactoside (IPTG). In order for stronger induction, a strong RBS (BBa_B0030) is chosen. Most importantly, we have inserted pmHAS*1-703 and kfiD gene to produce HA. pmHAS*1-703 encodes a class II hyaluronic acid synthase (HAS) which converts UDP-glucuronic acid and UDP-N-Acetylglucosamine into HA. The residues 1-703 and 704-972 encode the catalytic domain and the transmembrane domain respectively. By using only the first 703 amino acids, we can express a non-membrane bound but functional HAS. Meanwhile, kfiD is responsible for synthesis of UDP-glucose-6-dehydrogenase (UGDH) which converts UDP-glucose into UDP-glucuronic acid. This allows the transformed bacteria to synthesize HA more efficiently.