Difference between revisions of "Part:BBa K3739037"
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<partinfo>BBa_K3739037 short</partinfo>
 
<partinfo>BBa_K3739037 short</partinfo>
  
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Aly01 is a signal peptide from the alginate lyase of V.natriegens and GFP can show the secretion effciency of  Aly01
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Aly01 here represents a signal peptide used to secrete the fusion protein outside the cell. The enzyme catalyzes the reaction of converting histidine to form toxic urocanic acid and his-tag is added to purify the protein. We use BBa_xxxx to construct the expression system and to express and to purify the protein.
  
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===Biology===
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Aly01
  
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Aly01 is alginate lyase from ''Vibrio natriegens'' SK42.001, which is secreted out and able to digest alginate to unsaturated alginate oligosaccharide. Its signal peptide (named Aly01 in our parts), which is fused with heterogenous protein, is performed well in ''E. coli''. It is implied that the heterogenous protein fused with Aly01 signal peptide may be also secreted efficiently.
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hutH
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The HutH comes from ''Pseudomonas putida''. Under natural conditions, many microorganisms can use the histidine ammonia-lyase (HutH) to change L-histidine into urocanic acid. HutH catalyzes the first step in the degradation of histidine, and the product urocanic acid is further metabolized to glutamate. This enzyme could be found in the liver of vertebrates and in bacteria such as ''Escherichia coli'', ''Salmonella'' and ''Pseudomonas''. It is specific for L-histidine and can be inhibited by D-histidine or imidazole. The active center of the enzyme is thought to be dehydroalanine.
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===Usage===
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In order to easily purify HutH, we added a his-tag (6*His) at the C-terminal. We ligased the strong promoter (BBa_J23100) and the parts (RBS-Aly01-his-hutH-Terminator) on the expression vector pET-28a (+) by standard assembly. Then the ligation mixture was transformed into E. coli DH5α, and the correct recombinant one was confirmed by kanamycin, colony PCR and sequencing.
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We used BBa_xxxx to construct the expression system and demonstrated of the effect of secretory peptides and CBM-hutH together with BBa_xxxx and BBa_xxxx
  
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===Usage and Biology===
 
  
 
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Revision as of 17:49, 21 October 2021


J23100-B0030-Aly01-his-hutH-B0010

Aly01 here represents a signal peptide used to secrete the fusion protein outside the cell. The enzyme catalyzes the reaction of converting histidine to form toxic urocanic acid and his-tag is added to purify the protein. We use BBa_xxxx to construct the expression system and to express and to purify the protein.

Biology

Aly01

Aly01 is alginate lyase from Vibrio natriegens SK42.001, which is secreted out and able to digest alginate to unsaturated alginate oligosaccharide. Its signal peptide (named Aly01 in our parts), which is fused with heterogenous protein, is performed well in E. coli. It is implied that the heterogenous protein fused with Aly01 signal peptide may be also secreted efficiently.

hutH

The HutH comes from Pseudomonas putida. Under natural conditions, many microorganisms can use the histidine ammonia-lyase (HutH) to change L-histidine into urocanic acid. HutH catalyzes the first step in the degradation of histidine, and the product urocanic acid is further metabolized to glutamate. This enzyme could be found in the liver of vertebrates and in bacteria such as Escherichia coli, Salmonella and Pseudomonas. It is specific for L-histidine and can be inhibited by D-histidine or imidazole. The active center of the enzyme is thought to be dehydroalanine.

Usage

In order to easily purify HutH, we added a his-tag (6*His) at the C-terminal. We ligased the strong promoter (BBa_J23100) and the parts (RBS-Aly01-his-hutH-Terminator) on the expression vector pET-28a (+) by standard assembly. Then the ligation mixture was transformed into E. coli DH5α, and the correct recombinant one was confirmed by kanamycin, colony PCR and sequencing. We used BBa_xxxx to construct the expression system and demonstrated of the effect of secretory peptides and CBM-hutH together with BBa_xxxx and BBa_xxxx


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 232
    Illegal NgoMIV site found at 668
    Illegal NgoMIV site found at 1403
    Illegal NgoMIV site found at 1639
    Illegal AgeI site found at 305
    Illegal AgeI site found at 1132
    Illegal AgeI site found at 1628
  • 1000
    COMPATIBLE WITH RFC[1000]