Part:BBa_K5409711:Experience

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Constructing

Different promoters and terminators were assembled onto the ends of the ndps1 and ls target fragments. Since there are multiple PTPT combinations, here we take PTPT = (CCW12p, RPL3t, TDH3p, FBA1t) as an example. Primers were designed based on the PTPT combination (with correct homologous arms). Six fragments were amplified using PrimeStar Mix polymerase. CCW12p, ndps1, and RPL3t were assembled by homologous recombination, as were TDH3p, ls, and FBA1t, resulting in two fragments. The two fragments were then recombined to form one.

Next, we need to obtain the backbone of plasmid 352. Using YEp352 as a template, the Backbone-F/R primers and PrimeStar Mix polymerase were used to perform PCR, yielding a linearized YEp352 backbone.

The CCW12p-ndps1-RPL3t-TDH3p-ls-FBA1t fragment was recombined with the linearized YEp352 backbone through homologous recombination, and the resulting product was transformed into E. coli. Positive transformants from the Patch (A+) plate were selected and cultured overnight in LB medium(A+), after which the plasmid was extracted. The plasmid was then transformed into yeast, and successfully transformed yeast was activated in SDΔU. Activated yeast was preserved in 60% glycerol. (Refer to the protocol for transformation, LB (A+) medium preparation, plasmid extraction, PCR amplification, PCR product purification and homologous recombination).

Testing

After transforming them into yeast, shake flask fermentation tests were conducted. Since limonene is sparingly soluble in water (13.8 mg/L) and highly volatile, we adopted a two-phase fermentation system consisting of 10 mL SDΔU and 2 mL IPM (isopropyl myristate). IPM is a colorless, transparent oily liquid, insoluble in water, and capable of absorbing volatile limonene, which facilitates subsequent gas-phase detection. The culture conditions were 30°C, 220 rpm for 48 hours with an initial OD of 0.05. (Refer to the protocol for SDΔU medium preparation, gas-phase detection methods, and the creation of a standard limonene content curve).

The strain we used for fermentation is (S. cerevisiae CEN.PK2-1C ΔROX1::tHMG1,IDI1)

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