A kind of aspergillus niger bacterial strain that can be used for protein production and application thereof
A technology of Aspergillus niger strains, Aspergillus niger, applied in the directions of microorganism-based methods, introduction of foreign genetic material, fungi, etc. using vectors
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Embodiment 1
[0039] Example 1 Construction and effect research of the Aspergillus niger genetically engineered strain that knocked out the fluG gene
[0040] 1. FluG upstream and downstream sequences are amplified with the A.niger N402 genomic DNA template, and primers are used to introduce NotI and XbaI / XhoI restriction endonuclease sites to the 5' and 3' ends of the upstream sequence, while XhoI and KpnI was then introduced into the 5' and 3' ends of the downstream sequence; the amplified primers were blunt-ended connected to the pJET 1.2 (Fermentas, Thermoscientific, Waltham, USA) vector; the resulting upstream and downstream fragment vectors were subjected to NotI / Digested with XhoI and XhoI / KpnI, the resulting fragments were recovered for later use; next, the vector pBluescriptIISK(+) was used as the backbone to link the three fragments; the upstream and downstream sequences of fluG were inserted into the PAN7-1XhoI / XbaI enzyme fragment, which contained hygromycine resistance Express...
Embodiment 2
[0042] Embodiment 2 Aspergillus niger mutant strain FY17 (ΔfluGamyR + ) construction
[0043] On the basis of the Aspergillus niger ΔfluG mutant prepared in Example 1, the amyR gene was overexpressed, and the specific implementation was as follows, using a 4.3 kb DNA fragment (SEQ ID NO.2) containing the amyR gene to be introduced into the pGEM11 vector and combined with pIM2101 (the The vector contains the argB gene) co-transformation, the mutants obtained after transformation and screening are carried out on a starch substrate for a series of further screening work, the obtained strains are subjected to molecular detection, screening and confirmation of the inserted copy number, and finally the FY17 strain is obtained. The FY17 engineering strain of the present invention exhibits the ability of hypersecretion of glucoamylase in whole hyphae after adding 8-12 copies of amyR gene to the fluG mutant strain (ΔfluG).
[0044] After introducing multiple copies of the glucoseamyla...
Embodiment 3
[0048] Embodiment 3 Aspergillus niger mutant strain (ΔfluGamyR + Construction of ΔpepA)
[0049] 1. Construction method
[0050] pepA upstream and downstream sequences were amplified with the A.niger N402 genomic DNA template, and primers introduced NotI and XbaI / XhoI restriction endonuclease sites to the 5' and 3' ends of the upstream sequence, while XhoI and salI were Introduced into the 5' and 3' ends of the downstream sequence; the amplified primers were blunt-ended connected to the pJET 1.2 (Fermentas, Thermoscientific, Waltham, USA) vector; the resulting upstream and downstream fragment vectors were carried out for NotI / XhoI and Digest with XhoI / salI, and recover the resulting fragments for later use; then use the vector pBluescriptIISK(+) as the backbone to link the three fragments; insert pXDRFP4 (Yang et al. Contains the pryG resistance expression gene; finally completes the construction of the pepA gene knockout plasmid pepAKO. FY17 was used as the host strain for...
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