Attractant Production and Reporter ConstructThe attractant production pathway consists of three parts: FUS1 promoter, ARO9 gene and FUS1 terminator. These three genes are first derived through PCR from the yeast genome and then cloned into the PRS426 yeast expression vector for further functional assay.
The reporter construct also contains three parts: FUS1 promoter, EGFP gene and FUS1 terminator. These three genes are first derived through PCR and then cloned into the PRS426 yeast expression vector for further functional assay.
I. Primer DesignWe have designed several sets of primers for our construction, which are listed in Table 4. These primers are mainly designed for amplification of the FUS1 promoter, EGFP reporter, ARO9 gene and FUS1 terminator. Primer statistics are calculated using NetPrimer.
II. PCRThree of the four desired genes, FUS1 promoter, FUS1 terminator and ARO9 gene are directly amplified from yeast genomic DNA (strain YPH501). The other gene, EGFP gene, is amplified from the plasmid YTK-6/Tpi/EGFP.
For optimized PCR efficiency and accuracy, Taq polymerase is chosen for amplification PCR reactions. Gradient PCR is carried out for each step to optimize the reaction.
III. CloningAfter all the genes are successfully derived through PCR, standard cloning procedures are followed to construct an integrated expression vector.
The PRS426 yeast expression vector is used in our construction. The FUS1 promoter is cloned into the multiple cloning site by Sac I and Not I digest. The ARO9 gene and the EGFP gene are cloned by blunt-end Sma I digest. FUS1 terminator is cloned by Hind III and Xho I digest.