Construction method and application of engineering probiotics
A construction method and probiotic technology are applied in the fields of genetic engineering and microbial fermentation to achieve the effects of low cost of raw materials, simple process operation and low nutritional requirements
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Embodiment 1
[0173] Example 1: Construction of yeast strains inserted into Hbd, Crt foreign genes
[0174] The Hbd and Crt genes were simultaneously inserted into the yeast chromosome using the CRISPR / Cas9 system. Firstly, the YCplac33-Cas9 carrier plasmid of Cas9 protein was transformed into yeast competent cells by lithium acetate method, and positive yeast transformants with Cas9 plasmid were screened out with URA3 as the selection marker. Next, construct pRS42H-gHbd / Crt for guiding Cas9 protein to cut at a specific site and a donor fragment for repairing DNA strands, transform the gRNA expression plasmid and the donor fragment into yeast competent cells, and treat them with URA3 and hygromycin. The positive transformants screened by the double selection marker were verified by PCR, and the yeast strain BY4741-Hbd-Crt with homologous recombination was screened out.
[0175] The primers used for construction are shown in Table 1, and the schematic diagram is shown in Table 1. Figure 1...
Embodiment 2
[0188] Example 2: Construction of yeast strain inserted into Ter, BCoAT exogenous gene
[0189] 1: Construction of pRS42H-gTer / BCoAT plasmid
[0190] Ter, the construction of the plasmid pRS42H-gTer / BCoAT for the insertion of the BCoAT exogenous gene, the construction process is consistent with the construction of the pRS42H-gHbd / Crt plasmid in Example 1, and the primer pairs used are IPCR-Chr V HIS1-UP in Table 1, IPCR -Chr VHIS1-DW, the PCR product is a 6600bp DNA fragment. The plasmid pRS42H-gTer / BcoAT was obtained. The structure of the gRNA sequence is shown in SEQ ID NO:78.
[0191] Two: Ter / BCoAT Donor Fragment Construction
[0192] The construction of the donor fragment Ter / BCoAT for the insertion of Ter, BCoAT exogenous genes is basically the same as the construction of the Hbd / Crt donor fragment in Example 1. The nucleotide sequences of the Ter gene from Treponema denticola and the BCoAT gene from Faecalibacterium prausnitzii were queried at NCBI, and codon optimi...
Embodiment 3
[0198] Example 3: Construction of yeast strains overexpressing ERG10 endogenous genes
[0199] 1: Construction of pRS42H-gERG10 plasmid
[0200] The insertion of the ERG10 endogenous gene uses the construction of the plasmid pRS42H-gERG10. The construction process is consistent with the construction of the pRS42H-gHbd / Crt plasmid in Example 1. The primer pairs used are IPCR-Chr IV site1-UP, IPCR-Chr IVsite1- DW, the PCR product was a 6600bp DNA fragment. The plasmid pRS42H-gERG10 was obtained. The structure of the gRNA sequence is shown in SEQ ID NO:79.
[0201] Two: Construction of ERG10 Donor Fragments
[0202] Construction of the donor fragment ERG10 for insertion of the ERG10 endogenous gene. The synthesis system is shown in Table 4.
[0203] Refer to Table 4 for the construction system of the ERG10 donor fragment:
[0204] Table 4: ERG10 Donor Fragment Construction System
[0205]
[0206]
[0207]In the vector, the structure of the H1 left homology arm is sh...
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