Team:HKUST/Part3

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Attractant production and reporter Construct

The attractant production pathway contains three parts, which are FUS1 promoter, ARO9 gene and FUS1 terminator. These three genes are first derived through PCR from yeast genome and then cloned into PRS426 yeast expression vector for further functional assay.
The reporter construct also contains three parts, which are FUS1 promoter, EGFP gene and FUS1 terminator. These three genes are first derived through PCR and then cloned into PRS426 yeast expression vector for further functional assay.

Figure 5. Attractant production pathway and Reporter detailed design

I. Primer Design

We have designed several sets of primers for our construction, which are listed in Table 1. These mainly consist of primers for FUS1 promoter amplification, EGFP reporter amplification, ARO9 gene amplification and FUS1 terminator amplification. Primer statistics are calculated using NetPrimer.

Table 4 Primer sequences designed for the constructions
*Note: Restriction sites are highlighted in yellow.

II. PCR

Three out of four genes, FUS1 promoter, FUS1 terminator and ARO9 gene are directly amplified out from yeast genomic DNA (strain YPH501). The other gene, EGFP gene, is amplified from 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. Cloning

After the all the fragments are successfully derived through PCR, standard cloning procedures are followed to construct an integrated expression vector.
The PRS426 yeast expression vector is used in the expression of our construction. The FUS1 promoter is cloned into the multicloning site via Sac I and Not I digest. The ARO9 gene and the EGFP gene are cloned via blunt-end Sma I digest. FUS1 terminator is cloned via Hind III and Xho I digest.