Difference between revisions of "Part:BBa K4081996"

Revision as of 07:24, 18 October 2021

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.