Building method and application of lactobacillus reuteri resistance-marker-free gene integration system
A Lactobacillus reuteri, no resistance marker technology, applied in the field of genetic engineering to avoid mutation
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Embodiment 1
[0047] Cloning of pheS gene and construction of its mutant gene
[0048] Design specific primers according to the pheS gene sequence of Lactobacillus reuteriXNY published in Genebank, then use Lactobacillus reuteriXNY genomic DNA as a template to amplify, connect to the cloning vector pMD19-T for sequencing, and obtain the correct L.reuteriXNYpheS gene; then Perform amino acid sequence alignment of pheS gene ( figure 1 ), determine the mutation site as the second base (C is mutated to G) of the 312th amino acid (Arg), and then utilize overlapping PCR technique to construct the point mutation gene pheSM( figure 2 ).
Embodiment 2
[0050] Construction of Lactobacillus reuteri Strain Recombined with pheSM Gene
[0051] Select the insertion site of the target gene, and use Lactobacillus reuteriXNY genomic DNA as a template for PCR amplification to obtain 700bp homology arm fragments (LR, RR) upstream and downstream of the insertion site; then amplify the promoter fragment (P), red The mycin resistance gene (EM) fragment was connected with the pheSM gene and the homology arm fragment by overlapping PCR technology to obtain the LR-P-pheSM-EM-RR integration framework, which was connected to the cloning vector pMD19-T for sequencing, and obtained After the correct transformants were obtained, the LR-P-pheSM-EM-RR integration box was obtained by double enzyme digestion, electrotransformed into L. reuteriXNY competent cells, coated with MRS plates containing erythromycin, and the genomic DNA of positive transformants was extracted and carried out PCR detection, obtained pheSM gene recombinant lactobacillus ( im...
Embodiment 3L
[0052] The construction of embodiment 3L.reuteri recombinant cellulase genetically engineered bacteria
[0053] The cellulase gene eel15 (including the promoter) was connected with the homology arm sequences at both ends of the insertion site by overlapping PCR technology to obtain the cel15 gene integration framework LR-cel15-RR, which was connected to the cloning vector pMD19-T for sequencing to obtain the correct After the transformant, the integrated frame was obtained by double enzyme digestion, electrotransformed into the pheSM gene recombinant Lactobacillus competent cell, spread on the MRS plate containing p-cl-phe, extracted the genomic DNA of the positive transformant, and obtained the integrated gene through PCR detection. L.reuteri recombinant bacteria of cellulase gene cel15 ( Figure 4 ).
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