Part:BBa_K2765030:Experience
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：
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.
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.
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).
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.
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.
As Figure.4~ Figure.6 shown, comparing the growth curves of WT and ∆yca1 under H₂O₂ stress with 0mM, 1mM and 2mM, we can see that distinction of them are small. It maybe because that H₂O₂ stress at that level was not enough to distinguish growth differences.
Besides,comparing the growth curves of yno1 and yno1-∆yca1 under H₂O₂ stress with 0mM, 1mM and 2mM, we can see that the growth of ∆yca1- average was less inhibited by H₂O₂ stress than yno1-average.
According to the experimental results, we still can’t rigorously conclude that knock out yca1 can improve the yeast tolerance to H₂O₂.
References：
[1]Mao Shaochun, Li Zhuying, Li Cong. Determination of antioxidant activity by biospectrophotometry [J]. Analytical Laboratory, 2008 27 (4): 36-39.
[2]Wolfe KL , Liu RH. Cellular antioxidant activity (CAA) assay for assessing antioxidants, foods, and dietary supplements[J]. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 2007,55(22): 8896–8907.
[3]AgataŚwięciło,KamilaRybczyńska-Tkaczyk,AgnieszkaNajda,AnnaKrzepiłko,RomanPrażak,GrażynaZawiślak. Application of growth tests employing a Δsod1 mutant of Saccharomyces cerevisiae to study the antioxidant activity of berry fruit extracts[J]. LWT-FOOD SCIENCE AND TECHNOLOGY,2018,94:96-102.
[4]Robin E. C. Lee, Steve Brunette, Lawrence G. Puente, and Lynn A. Megeney. Metacaspase Yca1 is required for clearance of insoluble protein aggregates[J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2010,107 (30):13348-13353.
[5]Maša Ždralević,Valentina Longo,Nicoletta Guaragnella,Sergio Giannattasio,Anna Maria Timperio,Lello Zolla. Differential proteome–metabolome profiling of YCA1-knock-out and wild type cells reveals novel metabolic pathways and cellular processes dependent on the yeast metacaspase[J]. MOLECULAR BIOSYSTEMS,2015,11(6):1573-1583.
[6]Sandra Malmgren Hill,Thomas Nyström. The dual role of a yeast metacaspase: What doesn't kill you makes you stronger[J]. BIOESSAYS,2015,37(5):525-531.
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