Genetically engineered bacterium of high-yield gibberellin GA3, construction method and application
A technology of genetically engineered bacteria and gibberellins, applied in genetic engineering, microorganism-based methods, applications, etc., can solve problems such as difficulty in improving, and achieve the effects of improving production capacity, improving synthesis capacity, and weakening transcriptional inhibition.
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Embodiment 1
[0029] Example 1: F. fujikuroi -OE: Acquisition of AreA
[0030] Amplified by PCR using primers 1 and 2 using the pAN7-1 plasmid as a template wxya For the promoter fragment, the PCR reaction conditions are as follows: 98°C for 5min; 98°C for 30s, 60°C for 30s, 72°C for 1min, repeating 30 cycles; 72°C for 5min. Using the same method using primers 3 and 4 to amplify trpC Terminator fragment, amplified using primers 5 and 6 to give hygromycin hyg fragment. The PCR products were detected by 1.0% agarose gel electrophoresis and the gel was cut to recover the purified fragments ( wxya promoter fragment, trpC terminator fragment and hygromycin hyg1 The nucleotide sequences of the fragments are respectively shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.7).
[0031] by PCR using primers 7 and 8 to Gibberella fujikura ( Fusarium fujikuroi ) CCTCC NO: M2019378 (CN110527630A) genome was amplified as a template areA For gene fragments, the PCR reaction conditions are a...
Embodiment 2
[0037] Example 2: F. fujikuroi -OE: Acquisition of Lae1
[0038] Amplified by PCR using primers 9 and 10 using the Aspergillus nidulans genome as a template tef1 For the promoter fragment, the PCR reaction conditions are as follows: 98°C for 5min; 98°C for 30s, 60°C for 30s, 72°C for 1min, repeating 30 cycles; 72°C for 5min. Using the same method using primers 11 and 12 to amplify tef1 The terminator fragment, using the pAN7-1 plasmid as a template, was amplified using primers 13 and 14 to obtain hygromycin hyg2 fragment. The PCR products were detected by 1.0% agarose gel electrophoresis and the gel was cut to recover the purified fragments ( tef1 promoter fragment, tef1 terminator fragment and hygromycin hyg2 The nucleotide sequences of the fragments are respectively shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.8).
[0039] Amplified by PCR using primers 15 and 16 using the genome of Gibberella fujikuroi (Fusarium fujikuroi) CCTCC NO:M2019378 as a template ...
Embodiment 3
[0045] Example 3: F. fujikuroi -OE: AreA OE: Acquisition of Lae1
[0046] Using primers 17 and 18 by PCR, the pUC19-Hyg2- Ptef1 -Lae1- Ttef1 The vector is used as a template to amplify the complete Lae1 expression cassette. The PCR reaction conditions were as follows: 98°C for 8 minutes; 30 cycles of 98°C for 30s, 60°C for 30s, and 72°C for 4 minutes; 72°C for 8 minutes. The PCR products were detected by 1.0% agarose gel electrophoresis and the purified fragments were recovered by cutting the gel.
[0047] With pUC19-Hyg1- PgpA -Are A- TrpC The carrier vector was used as a template, and was connected with the ClonExpress II One Step Cloning Kit (purchased from Nanjing Weizan vazyme) to construct pUC19-Hyg1-P wxya -Are A-T trpC-Ptef1 -Lae1- Ttef1 The vector and the ligated product were transformed into E. coli DH5α recipient bacteria, spread on LB solid plates containing a final concentration of 100 mg / L ampicillin resistance, and cultured at 37°C for 12 hours....
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