Part:BBa_K4292000

k. lactis lac12

k. lactis lac12

The lac12 and wbgl gene fragments were amplified by PCR. And the DNA fragment wbgL-lac12, as well as XI-2 integration plasmids were digested with NotI and XhoI to form the cohesive ends, respectively. Then, the DNA fragments and vector were joined together by the ligase. In the recombinant XI-2-wbgL-lac12 plasmid, the wbgL and lac12 gene expression cassettes are inserted between the XI-2 homology arm, in different orientations. Next, the recombinant plasmid was transformed into the E.coli DH5α and verified by colony PCR (figure 2) and Sanger sequencing (figure 3).

Figure 1. Gene integration plasmid construction: Wbgl and lac12.

There were ten transformants extracted and verified by Xho1+BamH1 double-enzyme digestion (Figure 2). The positive transformant band was 6007+2635 bp, and the correct NO.11 was selected for sequencing. The results were shown as Figure 3. The sequence alignment results showed well matched, indicating that the XI-2-wbgL-lac12 plasmid was constructed successfully.

Figure 2. Validation of XI-2-wbgL-lac12 plasmid BamH1+Xho1 digestion.

After that, we used CRISPR-Cas9 technology to integrate the target genes WbgL and lac12 into the genome of S. cerevisiae. The corrected XI-2-wbgL-lac12 plasmid, which was digested with NotI, and gRNA were introduced into the yeast that already contains the Cas9 expression plasmid using lithium acetate transformation method. These yeast colonies were used colony PCR to verify whether the colony’s genome carried wbgL-lac12 expression cassettes.After that, we used CRISPR-Cas9 technology to integrate the target genes WbgL and lac12 into the genome of S. cerevisiae. The corrected XI-2-wbgL-lac12 plasmid, which was digested with NotI, and gRNA were introduced into the yeast that already contains the Cas9 expression plasmid using lithium acetate transformation method. These yeast colonies were used colony PCR to verify whether the colony’s genome carried wbgL-lac12 expression cassettes.

Figure 3. The sequencing blast result of XI-2-wbgL-lac12 plasmid.

Colony PCR was used to verify whether the XI-2 loci gene fragments were integrated into S. cerevisiae strains. The results are shown in Figure 4. The integrated copy number of the four transformants was verified using different primer pairs. If the internal primers (XI-2 inner-primer pairs) can amplify the target band, and the outer primers (XI-2 outer-primer pairs) cannot amplify the target band with the size of the integration homology arm ( 4880bp), it means that two copies have been integrated. If the primers outside the site amplify the target band of the size of the integration homology arm, it means that a copy of the target gene has been integrated. Based on the above analysis, we judged that the middle transformants No.2 and No.3 have clearly integrated a copy of the target gene successfully.

Figure 4. Validation of exogenous gene integration by colony PCR From left to right, No.1, 2, 3, 4 transformants.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 202
  Illegal BglII site found at 376
  Illegal BglII site found at 793
  Illegal BglII site found at 811
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]