Difference between revisions of "Part:BBa K3739008"
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===Biology===
 
===Biology===
−
Aly01
+
=====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.
 
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.
  
−
CBM
+
=====CBM=====
  
 
Cellulose enzymes have two domains, and the one that helps bind to cellulose is called cellulose binding module (CBM), and therefore it helps our fusion protein bind to cellulose-rich cell wall. Here we choose the CBM of CenA from ''Cellulomonas fimi'', which has been successfully expressed in ''Escherichia coli''.
 
Cellulose enzymes have two domains, and the one that helps bind to cellulose is called cellulose binding module (CBM), and therefore it helps our fusion protein bind to cellulose-rich cell wall. Here we choose the CBM of CenA from ''Cellulomonas fimi'', which has been successfully expressed in ''Escherichia coli''.
  
−
hutH
+
=====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.
 
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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−
===References===
+
===Reference===
 
1.https://parts.igem.org/Part:BBa_K3185005
 
1.https://parts.igem.org/Part:BBa_K3185005
 
<br/>2.Meng, Q. et al. Characterization and enhanced extracellular overexpression of a new salt-activated alginate lyase. Journal of the science of food and agriculture 101, 5154-5162, doi:10.1002/jsfa.11161 (2021).
 
<br/>2.Meng, Q. et al. Characterization and enhanced extracellular overexpression of a new salt-activated alginate lyase. Journal of the science of food and agriculture 101, 5154-5162, doi:10.1002/jsfa.11161 (2021).

Revision as of 00:35, 22 October 2021


Aly01-his-CBM-hutH

Aly01 here represents a signal peptide used to secrect the fusion protein outside the cell. The fusion protein CBM-hutH works on the surface of single cell Phaeocystis. globosa and the enzyme catalyzes the reaction of converting histidine to form toxic urocanic acid. His-tag is added to purify the protein. We use BBa_K3739008 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.

CBM

Cellulose enzymes have two domains, and the one that helps bind to cellulose is called cellulose binding module (CBM), and therefore it helps our fusion protein bind to cellulose-rich cell wall. Here we choose the CBM of CenA from Cellulomonas fimi, which has been successfully expressed in Escherichia coli.

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

Used to construct the composite part BBa_K3739036 and BBa_K3739106.


Reference

1.https://parts.igem.org/Part:BBa_K3185005
2.Meng, Q. et al. Characterization and enhanced extracellular overexpression of a new salt-activated alginate lyase. Journal of the science of food and agriculture 101, 5154-5162, doi:10.1002/jsfa.11161 (2021).


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 615
    Illegal NgoMIV site found at 1051
    Illegal NgoMIV site found at 1786
    Illegal NgoMIV site found at 2022
    Illegal AgeI site found at 73
    Illegal AgeI site found at 361
    Illegal AgeI site found at 451
    Illegal AgeI site found at 688
    Illegal AgeI site found at 1515
    Illegal AgeI site found at 2011
  • 1000
    COMPATIBLE WITH RFC[1000]