Difference between revisions of "Part:BBa K5201002"

 
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===Characterisation of BBa_K5201002: HongKong-UCCKE===
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<partinfo>BBa_K5201002 short</partinfo>
 
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<h3>Characterization </h3>
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<p>Colony PCR and Agarose gel electrophoresis (AGE) </p>
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<img src="https://static.igem.wiki/teams/5201/parts-registry/part-registry-11.png"></img>
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<p>Fig. 11 Agarose gel electrophoresis of the colony PCR products of composite part (BBa_K5201002)</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 </i>(BBa_K5201001) 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>
  
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Characterization
 
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Colony PCR and Agarose gel electrophoresis (AGE)
 
  
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In our project, we used E. coli DH5&#945; as host. Within its plasmid backbone, we inserted Lac Promoter (BBa_R0010), pmHAS*1-703 (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, a non-membrane bound but functional HAS is produced.  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.
 
  
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Revision as of 15:11, 1 October 2024


Characterisation of BBa_K5201002: HongKong-UCCKE

Document BBa_K5201002 short

Characterization

Colony PCR and Agarose gel electrophoresis (AGE)

Fig. 11 Agarose gel electrophoresis of the colony PCR products of composite part (BBa_K5201002)



In our project, we used E. coli DH5α as host. Within its plasmid backbone, we inserted Lac Promoter (BBa_R0010), pmHAS*1-703 (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.