Plasmid

Part:BBa_K5419003

Designed by: MEIJING SU   Group: iGEM24_Foshan-GreatBay   (2024-08-17)


pX-3-PagCrtB


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 3656
    Illegal NotI site found at 4104
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 4938
    Illegal BglII site found at 5517
    Illegal BglII site found at 5553
    Illegal BglII site found at 5733
    Illegal XhoI site found at 3877
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2651
    Illegal BsaI.rc site found at 132
    Illegal SapI site found at 284
    Illegal SapI.rc site found at 1629


BBa_K5419003 (pX-3-PagCrtB)

BBa_K5419003 (pX-3-PagCrtB)

Summary

To increase the yield of β-carotene in yeast cells, we added a new composite part, BBa_K5419003 (pX-3-PagCrtB), which was used together with other composite parts, BBa_K5419000 (pX-2-PaCrtE), BBa_K5419005 (pXII-5-BtCrtI), and BBa_K5419008 (pXI-2-PaCrtY), for the construction of yeast strains with high β-carotene production.

Construction Design

We constructed the plasmid by placing the gene under the regulation of a strong constitutive GAP promoter and a CYC terminator, respectively. Integration sequences were added upstream and downstream of the expression cassette to integrate the target gene into the genome of S. cerevisiae using the CRISPR/Cas9 system (Figure 1).

Figure 1: Design diagrams of pX-3-PagCrtB
Figure 1 Design diagrams of pX-3-PagCrtB.

Engineering Principle

In the S. cerevisiae, CrtE gene encodes GGPP synthase, in the presence of which FPP forms GGPP. The two GGPP molecules then form octahydrodicarbons via the CrtB gene-encoded octahydrodicarbon synthase. Then, octahydro lycopene dehydrogenase encoded by CrtI gene converts octahydro lycopene to lycopene. Finally, the CrtY-encoded lycopene β-cyclase will catalyze lycopene and eventually form β-carotene [1].

Experimental Approach

(1) Construction of integration plasmids

Firstly, we obtained the target gene expression frame (GAP promotor-gene-CYC terminator) by PCR amplification, and agarose gel electrophoresis results showed that we succeeded in obtaining the fragment. Next, we double-digested the target fragment and the vector (containing the S. cerevisiae X-3 integration site genes) and obtained the plasmid by enzymatic ligation. Finally, we transformed the enzyme-ligation product into E. coli DH5α competent cells and the colony PCR and sequencing results showed that we successfully constructed the plasmid (Figure 2).

Figure 2: The construction results of the pX-3-PagCrtB plasmid.
Figure 2 The construction results of the pX-3-PagCrtB plasmid.

(2) Integration of target genes into the yeast genome

After successfully obtaining the integration plasmids (pX-3-PagCrtB, pX-2-PaCrtE, pXII-5-BtCrtI, and pXI-2-PaCrtY), we used NotI restriction endonuclease to obtain the complete destination fragment (containing the sequence upstream of the integration site, the GAP promoter, the target gene, the CYC terminator and the sequence downstream of the integration site). Subsequently, we recovered these integration fragments in combination with the corresponding gRNA plasmids (X-2-XII-5-gRNA-HYG and X-3-XI-2-gRNA-HYG) and introduced them into the modified S. cerevisiae 1974 strain (which had pre-integrated the Cas9 gene) by a modified lithium acetate transformation method. After two rounds of integration, we used yeast colony PCR to verify that the target fragments were successfully integrated into the yeast genome (Figure 3).

Figure 3: Construction results of yeast strain
Figure 3 Construction results of yeast strain. (A) Strategy for constructing yeast strain. (B) Transformation plate and (C) colony PCR results of the yeast strain.

Measurement: Quantitative analysis

We used the quantitative PCR (qPCR) technique to determine the transcript levels of target genes integrated into the yeast genome. The primer amplification efficiency standard curves showed that this qPCR amplification has great reproducibility and accuracy (Figure 4). Subsequently, we analyzed the expression of the target genes in the recombinant yeast strains. The results showed that the expression of the target gene was increased 0.771~1.709-fold in this group compared to the control. These results confirmed that the target genes had been successfully integrated into the yeast genome and could be efficiently transcribed (Table 1).

Figure 4: Standard curves for primer amplification efficiency
Figure 4 Standard curves for primer amplification efficiency of (A) PaCrtE, (B) BtCrtI, (C) PaCrtY, and (D) PagCrtB.
Table 1: Gene expression analysis of yeast strain
Table 1 Gene expression analysis of yeast strain.

Finally, we quantified the β-carotene production of the recombinant yeast strain. We plotted a standard curve using a series of β-carotene standards with concentration gradients and established a linear regression equation for calculating β-carotene concentration. After extraction and analysis, we found that the strain produced β-carotene at a concentration of 13.37 mg/L (Figure 5).

Figure 5: β-carotene production in yeast strain
Figure 5 β-carotene production in yeast strain.

Reference

[1] WANG Rui-zhao, PAN Cai-hui, WANG Ying, XIAO Wen-hai, YUAN Ying-jin. Design and Construction of high β-carotene Producing Saccharomyces cerevisiae[J]. China Biotechnology, 2016, 36(7): 83-91.

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