Glucoamylase producing method
A technology of glucoamylase and gene expression, which is applied in the field of constructing microbial engineering bacteria to produce recombinant protein and producing recombinant glucoamylase, which can solve the problem of narrow reaction temperature range and suitable reaction pH range, and cannot satisfy the reaction temperature and suitable reaction pH range Demand and other issues to achieve the effect of saving raw materials
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Embodiment 1
[0022] 1.1 Pichia pastoris expression vector containing the glucoamylase gene expression framework of Aspergillus, the glucoamylase gene expression framework of Chaetomium and the glucoamylase gene expression framework of yeast
[0023] 1.1.1 Construction of cloning vector
[0024] A professional DNA sequence synthesis company synthesizes two base complementary double strands containing ampicillin (AMP) gene sequence, polyclonal adapter and E. coli replication origin, and forms cohesive ends at both ends of each DNA strand sequence. It is circularized by the action of DNA ligase to form a DNA cloning vector. The cloning vector was named pPA.
[0025] 1.1.2 Acquiring genes
[0026] ① Amplification of Aspergillus glucoamylase gene by reverse transcription PCR
[0027] Primer 1: 5'CT GGATCC atgtcgttccgatctcttct3'[Description: The 8 bases of 5' are the restriction enzyme protection bases (2 bases) and the enzyme recognition site (6 underlined bases)]
[0028] Primer 2: 5'aa ...
Embodiment 2
[0075] 2.1 Saccharomyces cerevisiae expression vector containing the glucoamylase gene expression framework of Aspergillus, the glucoamylase gene expression framework of Chaetomium and the glucoamylase gene expression framework of yeast
[0076] 2.1.1 Construction of cloning vector
[0077] A professional DNA sequence synthesis company synthesizes two base complementary double strands containing ampicillin (AMP) gene sequence, polyclonal adapter and E. coli replication origin, and forms cohesive ends at both ends of each DNA strand sequence. It is circularized by the action of DNA ligase to form a DNA cloning vector. The cloning vector was named pSA.
[0078] 2.1.2 Acquiring genes
[0079] ④ Amplification of Aspergillus glucoamylase gene by reverse transcription PCR
[0080] Primer 1: 5'CT GGATCC atgtcgttccgatctcttct3'[Description: The 8 bases of 5' are the restriction enzyme protection bases (2 bases) and the enzyme recognition site (6 underlined bases)]
[0081] Primer...
Embodiment 3
[0134] 3.1 Bacillus subtilis expression vector containing the glucoamylase gene expression framework of Aspergillus, the glucoamylase gene expression framework of Chaetomium and the glucoamylase gene expression framework of yeast
[0135] 3.1.1 Construction of cloning vector
[0136] A professional DNA sequence synthesis company synthesizes two base complementary double strands containing ampicillin (AMP) gene sequence, polyclonal adapter and E. coli replication origin, and forms cohesive ends at both ends of each DNA strand sequence. It is circularized by the action of DNA ligase to form a DNA cloning vector. The cloning vector was named pBA.
[0137] 3.1.2 Acquiring genes
[0138] Amplification of Aspergillus Glucoamylase Gene by Reverse Transcription PCR
[0139] Primer 1: 5'CT GGATCC atgtcgttccgatctcttct3'[Description: The 8 bases of 5' are the restriction enzyme protection bases (2 bases) and the enzyme recognition site (6 underlined bases)]
[0140] Primer 2: 5'aa ...
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