Method for multi-copy integration of target gene, preparation method for recombinant bacteria, resveratrol and recombinant human serum albumin
A gene and purpose technology, applied in the field of preparation of recombinant bacteria, resveratrol and recombinant human serum albumin, can solve the problems of unsatisfactory expression efficiency and expression scale, and achieve the effect of avoiding potential risks and increasing production
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Embodiment 1
[0070] Example 1 Establishment of Hansenula yeast scarless multi-copy gene integration method
[0071] 1. Obtaining a linearized vector expressing Cas9
[0072] Using the Hansenula HP001 genome as a template, using HP230 and HP231 as primers to amplify the downstream homology arm of the gene MET2 to obtain a PCR product (sequence 1) with a length of 1570 bp; using HP232 and HP233 as primers to amplify the upstream homology arm of the gene MET2, Obtain the long 1535bp PCR product (sequence 2); Use HP234 and HP235 as primer amplification methanol inducible promoter P MOX , to obtain a 1528bp long PCR product (SEQ ID NO: 3). Using the plasmid pCRCT as a template and using HP236 and HP237 as primers to amplify the gene Cas9, a 4255bp PCR product (sequence 4) was obtained. The above four PCR products were purified. Plasmid pWYE3200 was double digested with restriction endonucleases BglII and XbaI to obtain a fragment with a length of 2325 bp, and the fragment was purified. The ...
Embodiment 2
[0090] Embodiment 2, efficient synthesis of resveratrol in Hansenula yeast
[0091] 1. Integrate fusion expression cassette P scTEF1 -TAL-P scTPI1 -4CL-P scTEF2 -Acquisition of STS repair template
[0092] Sequence 9 was amplified by referring to the method in Example 1; using HP324 and HP211 as primers, the downstream homology arm of the rDNA site was amplified to obtain a 1057bp PCR product (Sequence 10). Using Saccharomyces cerevisiae SC001 genome as a template and using HP164 and SC31 as primers to amplify the promoter P scTEF1 , to obtain a long 732bp PCR product (sequence 11); using HP398 and SC23 as primers to amplify the promoter P scTPI1 , obtain a long 450bp PCR product (sequence 15); use SC26 and SC27 as primers to amplify the promoter P scTEF2 , a 707bp long PCR product (SEQ ID NO: 16) was obtained. Using the synthesized gene TAL as a template, using SC20 and HP397 as primers to amplify the gene TAL fragment to obtain a 1738bp long PCR product (SEQ ID NO: 17)...
Embodiment 3
[0107] Example 3, Biosynthesis of Recombinant Human Serum Albumin in Hansenula
[0108] 1. Acquisition of integrated HSA gene repair template
[0109] Refer to the method in Example 1 to amplify the upstream homology arm of the rDNA site (SEQ ID NO: 9); refer to the method in Example 2 to amplify the downstream homology arm of the rDNA site (SEQ ID NO: 10). Using Saccharomyces cerevisiae SC001 genome as template and HP164 and HP321 as primers to amplify promoter P scTEF1 , to obtain a 622bp long PCR product (SEQ ID NO: 11). Using the synthetic HSA gene as a template and using HP325 and HP326 as primers to amplify the HSA gene, a 1892bp long PCR product (SEQ ID NO: 20) was obtained. Plasmid pWYE3200 was double digested with restriction endonucleases BglII and BamHI to obtain a 1920bp fragment. The above PCR products and restriction fragments were purified and then subjected to Gibson assembly reaction. The reaction product was transformed into Escherichia coli DH5α by chemi...
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