Part:BBa_K2368017
Introduction
500 bp of homologous arm＋His
Sst2 protein is an important negative regulatory factor of the pheromone GPSTP. In order to improve the detection sensitivity, we designed this part to knock out the sst2 gene.
Similarly, we designed 3 pairs of primers and the marker was Histone synthesis gene, short termed His. We got the 3 fragments by PCR and they had the overlap areas with each other as shown in the Fig.1. Then, the complete fragment observed by OE-PCR was transformed to the yeast. Additionally, the positive clones were screened on the relevant nutritional deficiency medium.
Fig1. The schematic diagram of knocking out sst2 gene.
We cloned the upstream homologous arm and the downstream homologous arm from the genome of CEN.PK2-1C. Meanwhile, we cloned his from the pESC-His. The agarose gel electrophoresis analysis of homologous arms, his and the complete fragment observed by OE-PCR are shown in Fig.2.
Fig2. The positive clones of homologous arms, his 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 sst2 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.
After knocking out sst2, we tested the growth curve of the yeast as shown in the Fig.5.
Fig5. The growth curve of CEN.PK2-1C and Δsst2 strain
Sst2 protein is an important negative regulatory factor of GPSTP. When we knocked out sst2 gene, compared with CEN.PK2-1C, the sensitivity of the Δsst2 strain GPSTP could be improved. And the inhibition of cell growth will be enhanced.
We also tested the function of Δsst2 strain by the inhibitive effects acted by the α factor to the growth of CEN.PK2-1C.
Fig6. The antibacterial circle of CEN.PK2-1C and Δsst2 strain
As we can see, Δsst2 strain is much more sensitive to α pheromone. Compared with CEN.PK2-1C, less amount of α pheromone can cause Δsst2 strain growth arrest.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1240
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1131
Illegal BglII site found at 1191 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1801
- 1000COMPATIBLE WITH RFC[1000]
| None |