Difference between revisions of "Part:BBa K4081996"

Latest revision as of 09:57, 13 October 2022

LAC12

Biology and Usage

Wild-type S. cerevisiae is incapable of transporting lactose into the cytosol. But lactose as a fucose acceptor in 2-FL production, needs to be transported into the cytosol of S. cerevisiae cells. Lactose permease, such as Lac12 from Kluyveromyces lactics needs to be introduced into S. cerevisiae for transporting lactose into the cytosol.

Figure1.Using TEF1 promoter to promote the expression of LAC12.

Sequence and Features

Design and Properties

In our project, we use TEF1 promoter(BBa_K4081995) to promote the expression of LAC12(BBa_K4081996), and use ADH1 terminator(BBa_K4081823) to terminate transcript in Saccharomyces cerevisiae BY4741 (figure 1).

We transform the gene "TEF1 promoter- LAC12 - ADH1 terminator” into Saccharomyces cerevisiae BY4741 by lithium acetate conversion method to integrate the genes into the genome of BY4741 for expression.The result of SDS-PAGE showed that we successfully expressed LAC12(figure2).

Figure 2. The result of SDS-PAGE. Line 1: Marker; line 2: Control; line 3: Express FKP(106KD), LAC12(65KD), FucT2(35KD).

Experimental approach

1.Construct recombinant plasmids. Get GAP promoter from vector PML104. Get TEF1 promoter from the genome of Saccharomyces cerevisiae BY4741. Get ADH1 promoter from vector pAUR123. Company synthetic genes of FKP, LAC12 and FucT2. Use vector pAUR123 to construct our plasimd “pAUR123-pGAP-FKP-pADH1-FucT2-pTEF1-LAC12”.

2.Transform the product (2.5μL) into DH5α competent cells (50μL), grow cells on agar plates (containing Ampicillin). Incubate plates at 37°C overnight. Colonies were screened by colony PCR and then grown at 37℃, 200rpm. Plasmids were extracted and sent for sequencing.

3.PCR the genes “pGAP-FKP-pADH1-FucT2-pTEF1-LAC12” and the resistance gene AurR from the plasmid with homology arms of BY4741. Transform it into BY4741 by lithium acetate conversion method to integrate genes into the genome of BY4741 for expression. Screen for transformants by AbA-YPD selection medium.

4.Extract yeast total protein. Use SDS-PAGE to test whether the three proteins(FKP, LAC12, FucT2) are successfully expressed.

References

[1]Yu, S. , Liu, J. J. , Yun, E. J. , Kwak, S. , Kim, K. H. , & Jin, Y. S. . (2018). Production of a human milk oligosaccharide 2′-fucosyllactose by metabolically engineered saccharomyces cerevisiae. Microbial Cell Factories, 17.

Contribution by 2022 iGEM Team Zhengjiang_United

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.