Difference between revisions of "Part:BBa K3394000"

Latest revision as of 17:09, 25 October 2020

Coding Sequence of Endo5a cellulase (for E. coli)

This part consists oF the coding region of Endo5A, an endo-1,4-beta-glucanase. Endo5a breaks down cellulose by cleaving the internal 1-4 beta glucose linkages in the cellulose chain.

Figure 1: Cellulose chain. 1-4 beta glucose linkage is highlighted

The complete cellulose hydrolysis reaction involves this enzyme along with several other enzymes: endo-1,4-beta-glucanases, exo-1,4-beta-glucanases (or cellobiohydrolases) and beta-glucosidases.

Endo5a is a relatively small protein with an optimal operating temperature of 50o C and an optimal pH range of 6.0-7.0. The NCBI GenBank number for Endo5a is HQ657203.1 (DNA sequence), AEB00655.1 (amino acid sequence).

We learned about the cellulase from a past iGEM team – the British Columbia 2016 team which fused it to the surface layer of Caulobacter crescentus (BBa_K2139001). In our project, we have performed codon optimization for its expression in Escherichia coli, and the sequence was synthesized by IDT for us.

Endo5A was isolated from the bacterial flora found in the gut of a cotton bollworm (Helicoverpa armigera) which secretes a variety of plant-hydrolyzing enzymes.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 898
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 898
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 898
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 898
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 898
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3394000 short</partinfo>
-This part consists the coding region of Endo5A, an endo-1,4-beta-glucanases. Endo5a breaks down cellulose by making internal cuts in the cellulose chain. This enzyme is part of the complete cellulose hydrolysis reaction which involves several enzymes: endo-1,4-beta-glucanases, exo-1,4-beta-glucanases (or cellobiohydrolases) and beta-glucosidases.
+This part consists oF the coding region of Endo5A, an endo-1,4-beta-glucanase. Endo5a breaks down cellulose by cleaving the internal  1-4 beta glucose linkages in the cellulose chain.
+[[File:T--BGU-Israel--Celluloseimage.jpeg|300px|thumb|center|Figure 1: Cellulose chain. 1-4 beta glucose linkage is highlighted]]
-It is a relatively small protein with an optimal temperature of 50 Celsius degrees and an optimal pH range of 6-7.
+The complete cellulose hydrolysis reaction involves this enzyme along with several other enzymes: endo-1,4-beta-glucanases, exo-1,4-beta-glucanases (or cellobiohydrolases) and beta-glucosidases.
-The NCBI Genbank number for Endo5a is HQ657203.1 (DNA), AEB00655.1 (amino acid). This construct was synthesized by IDT for use in Escherichia coli.
+Endo5a is a relatively small protein with an optimal operating temperature of 50o C and an optimal pH range of 6.0-7.0. The NCBI GenBank number for Endo5a is HQ657203.1 (DNA sequence), AEB00655.1 (amino acid sequence).
+We learned about the cellulase from a past iGEM team – the British Columbia 2016 team which fused it to the surface layer of Caulobacter crescentus (BBa_K2139001). In our project, we have performed codon optimization for its expression in Escherichia coli, and the sequence was synthesized by IDT for us.
-We verified that the sequence is optimized for use in Escherichia coli. Furthermore, Endo5a was expressed with an N-terminal histidine tag (6-histidine tag) instead of the 17 first amino acids that serve as a signal peptide (51 nucleotides).
 Endo5A was isolated from the bacterial flora found in the gut of a cotton bollworm (Helicoverpa armigera) which secretes a variety of plant-hydrolyzing enzymes.