Latest revision as of 16:05, 28 October 2017

Introduction

500 bp of homologous arm＋Trp

The GPCR of yeast pheromone (α factor) GPSTP is encoded by ste2 gene, the presence of endogenous Ste2 protein can cause signal interference. In order to avoid the signal interference, we designed this part to knock out the ste2 gene.

We designed 3 pairs of primers cleverly whose templates were the upstream homologous arm(500bp), marker and the downstream homologous arm(500bp) respectively. The marker was Trptophan synthesis gene, short termed trp. trp would replace ste2. Initially, we got the 3 fragments by PCR and they had the overlap areas with each other as shown in the Fig. 1 Secondly, the complete fragment observed by OE-PCR was transformed to the yeast. Additionally, the positive clones were screened on the relevant nutritional deficiency medium(SD-Trp), so that only the positive cloning could survival on it.

Fig. 1 The schematic diagram of knocking out ste2 gene.

We cloned the upstream homologous arm and the downstream homologous arm from the genome of CEN.PK2-1C. Meanwhile, we cloned trp from the pESC-Trp. The agarose gel electrophoresis analysis of homologous arms, trp and the complete fragment observed by OE-PCR are shown in Fig. 2.

Fig. 2 The positive clones of homologous arms, trp and the complete fragment observed by OE-PCR.

To verify whether the gene was actually knocked out and avoided the false positive clones, we designed the primer 1,2,3 and 4 for each gene, as shown in the Fig. 3. The primer 1 and primer 4 were on the yeast genome. The primer 2 was on the marker and primer 3 was on the gene which would be knocked out.

Fig. 3 Schematic diagram of the primer which is used to verify the result of knocking out genes.

Fig. 4 The result of knocking out ste2 gene.

We got correct results by the primer 1 and 2 as well as nothing from primer 1 and 3, demonstrated that the gene was knocked out. Then we sequenced the PCR product using primer 1 and 4 to make sure the sequence was right.

Sequence and Features