Part:BBa_K2368018
Introduction
500 bp of homologous arm＋Ura
α factor can cause the yeast cell cycle arrest in the G1 phase through Far1 protein, resulting in that cell growth is inhibited. In order to short the detection time, we designed this part to knock out the far1 gene
Similarly, we designed 3 pairs of primers and the marker was Uracil synthesis gene, short termed Ura. 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(SD-Ura), so that only the positive cloning could survival on it.
Fig. 1 The schematic diagram of knocking out far1 gene.
We cloned the upstream homologous arm and the downstream homologous arm from the genome of CEN.PK2-1C. Meanwhile, we cloned ura from the pESC-Ura. The agarose gel electrophoresis analysis of homologous arms, ura and the complete fragment observed by OE-PCR are shown in Fig. 2.
Fig. 2 The positive clones of homologous arms, ura 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 far1 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.
As we know, α factor can inhibit the growth of CEN.PK2-1C. So, we tested the function of △far1 strain by the α factor.
Fig. 5 The antibacterial circle of CEN.PK2-1C and Δfar1
Far1 protein is a cyclin-dependent kinase inhibitor. Deletion far1 gene can relieve inhibition. As you can see in Fig. 5, △far1 strain will still keep growing under the effects acted by α factor and antibacterial circle will not be shown either.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1400
Illegal BsaI site found at 2010
Illegal SapI.rc site found at 1247
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