We designed 3 pairs of primers whose templates were the upstream homologous arm, the marker and the downstream homologous arm respectively. The marker was Uracil synthesis gene abbreviated as ura.Initially, we got 3 fragments by PCR and they had their own overlapping areas with corresponding to each homologous arms.Then, we got the recombination fragment from OE-PCR that has been transformed to the CEN.PK2-1C which has been already knocked out the endogenous genes ura. Additionally, the colony was chosen by the ura nutritional deficiency medium.
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We cloned the upstream homologous arm and the downstream homologous arm of yca1 from the genome of CEN.PK2-1C and cloned Ura marker from pESC-Ura.Then we ligased Ura marker and the complete fragment by Overlap Extension PCR then transformed them into CEN.PK2-1C(△yca1).as shown in Figure 2.
After sequencing verification,we tested the survival rate of Cenpk-2-1c which has been knocked out yca1 in order to verify the function of the part
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1.we take Δyca1 and wild type (or gal1-yno1-Δyca1, gal1-yno1) cenpk 2-1c glycerol, inoculate in 5ml YPD medium, culture them until plateau (OD600 greater than 10, UV spectrophotometer), as Seed liquid.
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2. The seed solution was inoculated into 50 ml of YPD medium, and the OD600 of the inoculum was determined to be 0.6 by calculation.
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3. 2 ml of YPD medium containing 100 mM hydrogen peroxide was preliminarily placed. Prepackage into 12 tubes (6 of which are connected to 50 μl and 6 to 100μl).
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4. Put the bacterial solution into a test tube with or without hydrogen peroxide, make the total volume 5ml, and shake it quickly. The concentration of hydrogen peroxide is 0., 1, 2 mM, 3 parallel controls in each group. Incubate with a shaker at 30 °C.
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5. After culturing for 2 h and 4 h, take 1-3 ml of the bacterial solution (according to the concentration of the bacterial solution), measure each group of OD600 with an ultraviolet spectrophotometer, and record the data.
This part consists of the upstream homologous arm and the downstream homologous arm of yca1 and ura marker .yca1 is the yeast endogenous gene，it can control the apoptosis of yeast cell .In order to improve the tolerance of yeast cells to high levels of ROS and construct a biological detection system to complete the function of antioxidant detection and screening, we knocked out the yca1 gene.as shown in Fig.1.
Sequence and Features
Assembly Compatibility:
10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 2416
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 1825 Illegal BsaI.rc site found at 277 Illegal SapI.rc site found at 559 Illegal SapI.rc site found at 580 Illegal SapI.rc site found at 1672
