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  1. May 2019
    1. Bacterial transformation
    2. E. colibacterial strain DH5α was taken out on LB-agar mediumfrom -80°C freezer and incubated at 37°C for 14-16 h. To obtain the starter culture,single bacterial colony was inoculated in 25 ml of SOB medium ina25 ml flask. The flask was incubatedfor 6-8 hat 37°C with continuous shaking at 200 rpm. Next, 2, 4 and 10 ml of the starter culture was inoculated in three different 1 litre flasks each containing 250 ml of SOB medium. Cultures were incubated overnightat 18°C with continuous shaking at 200 rpm. After overnight incubation, OD600 of allthree cultures were monitored after every 45 min interval till OD600of any of the three cultures reached 0.55. These cells werekept onice for 10 min and the other two cultures were discarded.Cells were harvested by centrifugation at 2,500g in a Sorvall GSA rotor for 10 min at 4°C. Supernatant was poured offcompletely andcells were gently resuspended in 80 ml of ice-cold Inoue transformation buffer by swirling the tubes (pipetting was avoided at this step). Followingresuspension, cells were spun down by centrifugation at 2,500g in a Sorvall GSA rotor for 10 min at 4°C and the supernatant was discarded completely. The cell pellet was resuspended gently in 20 ml of ice-cold Inoue transformation buffer by swirling.1.5 ml of DMSO was added to the cell suspension and incubated on ice for 10 min. 50 μl aliquotsof cell suspensionwere dispensed in pre-chilled 1.5 ml microfuge tubes, snap-frozen in liquid nitrogen and stored in -80°C freezer till further use
    3. Preparation of ultra-competent E. colicells
    4. ml YPD broth at an initial OD600of 0.1. Cultures were allowed to grow for 4-5 hin a shaker incubator setat 30°C, 200 rpm until the OD600of the cultures reached 0.4-0.6. Next,cells were harvested ina15 ml centrifuge tube by centrifugation, washed twice with 10 ml of sterile water, resuspended in 1 ml of sterile water and were transferred to a 1.5 ml microfuge tube. Cells were harvested by centrifugation at 4,000 rpm for 5 min,resuspended in 50 μl of100 mM lithium acetate solution and transformation mixture was added. Transformation mixture consisted of 240 μl polyethylene glycol (50%), 36 μl of lithium acetate (1 M), 5 μl of heat denatured single stranded carrier DNA (10 mg/ml), 500 ng to 1 μg of transforming DNA and final volume was made to 360 μl with sterile water. The tubes were incubated at 30°C for 45 min. To this, 43 μl of sterile DMSO was added and heat shock was given at 42°C for 15 min. Next, tubeswere transferred to ice for 10-15 sec, centrifuged at 4,000 rpm and transformation mixture reagents wereremoved completely by pipetting. Cells were resuspended in 200 μl of sterile water and spread-plated on appropriate selection medium. Plates were incubated at 30°C for 24-48 h
    5. Yeast transformation was performed as described previously (Gietz et al., 1992) with fewmodifications. Briefly, overnight grown C. glabratacultures were freshly inoculated in 10
    6. Yeast transformation
    7. To phenotypically characterize C. glabratamutants,serial dilution spot growth assays were performed. Briefly, the optical density of overnight-grown C. glabratacultures wasnormalized to OD600of 1.0andnormalized cultures were further diluted 10-fold in 1X sterile PBS five times. 3 μl of serially diluted culture were spotted on test plates. Plates were incubated at 30°C (unless mentioned otherwise) for 24-48hand growth was recorded by capturing plate images. For experiments involvingchecking theability of mutants to utilize non-fermentable carbon sources,growth was scoredafter 6-7 days of incubation
    8. Serial dilution spot growth assay
    9. For time course growth analysis,C. glabratacells were inoculated in appropriate medium and grown for 14-16 h. Followed by overnight growth,yeast cells were sub-cultured in test medium at an initial OD600of 0.1 and growth was monitored by recording the absorbance of the culture at 600 nm at regular time-intervals till 72h. Absorbance values were plotted with respect to time and generation time of yeast strains were calculated between 4-8 h of growthwhen cells were in logarithmic phase of growth using following equation.Generationtime(G)=T2−T1Xlog2logNf/NiG = Generation time (h)T1 = Initial time point taken for analysisT2 = Final time point taken for analysisNf= Number of cells at time T2 (1 OD600of C. glabratacorresponds to 2 X 107cells)Ni = Number of cells at time T1 (calculated from OD600 value as mentioned above)For CFU-based viability assays, appropriate dilutions of yeast cultures were prepared in 1X sterile PBS and suitable volume of diluted cultures wasplated on YPD-agar medium. Plates were incubated at 30°C for 24-48 h and viable colonies that appeared on YPD-agar plate were counted.To obtain logarithmic phase cells, overnight-grown C. glabratacell suspension was inoculated in appropriate medium at an OD600of 0.1 and grown ina shakerincubator set at 30°C, 200 rpm for approximately 4 htill the culture density reached OD600of 0.4-0.6
    10. Time course growth analysis and determination of generation time
    11. C. glabratastrains were routinely grown in rich YPD medium or synthetically defined YNB medium, or YNB medium supplemented with CAA, unlessstatedotherwise.To obtain overnight grown liquid cultures, C. glabratacells were inoculated in appropriate medium and incubated at 30°C under constant agitation (200 rpm) to maintain proper aeration.To revive the frozenstocks,about one tipfull of frozen culture was streaked either on YPD-agar or on CAA-agar medium. In general, frozen stocks of C. glabratastrains were revived on YPD-agar medium.However,C. glabratastrains harbouring plasmidscontainingURA3as a selectable marker were revived on CAA-agar medium. After streaking, plates were allowed to grow for 24-48 h at 30°C and were stored at 4°C for a maximum period of two weeks. For long term storage, freezer stocks of C. glabratastrainswere prepared in 15% glycerol and stored at -80° C.Escherichia colistrain DH5αwas revived on LB-agar medium from frozenstock and incubated at 37°C for 14-16 h. DH5α strainwas used for transformation purpose and maintaining plasmids. Bacterial strains harbouring plasmids containing selection markerswere revived on LB-agar medium supplemented with appropriate antibiotics.Bacterial liquid cultures were either grown in LB broth or LB broth containing suitable antibioticsand incubatedin a shakerincubator set at 37°C, 200 rpm for 14-16 h. For preparation of bacterial frozenstocks, 1 ml overnight grown bacterial culture was added to500 μl of 50% glycerolto obtain final concentration of ~16 % glyceroland stored at -80°Cuntil use
    12. Strains and culture conditions
    13. Microbiological methods
    14. For cryopreservation of THP-1 and Lec-2 cells, 5-6 million cells wereresuspendedin 0.5 ml of eithercommercially procuredcell preservation medium from GIBCO(12648010)or complete medium supplemented with 10 % fetal bovine serum and 10 % DMSO.Cells were initially kept inanisopropanol bath and werelatertransferred to -70°C freezer. After 2-3 days, frozencells were transferred to liquid nitrogen container till further use. To revive the cells, frozenstockswere taken out of the liquid nitrogen container and immediately transferred to water bath set at 37°Cfor thawing. When freezing medium has thawed completely, cells were transferred to a 100 mm cell culture dishcontaining 12 ml completemedium and incubated under tissue culture conditions at 37°C and 5% CO2for 12 h. Afterincubation, medium was replaced by 12 ml fresh pre-warmed medium and incubated under tissue culture conditions till they reached 70-80% of confluencebefore splitting
    15. Cryopreservation and revival of cell lines
    1. the selection marker.Knockdown was confirmed by immunoblot analysis with an IP6K1 specific antibody (Table 2.3) as described in Section 2.2.10
    2. lines were used for stable knockdown of IP6K1 expression. Viral particles harboring either non-targeting control or IP6K1directed shRNA were used to infect HeLa or HCT116 cell lines at 0.5 MOI, following treatment with polybrene (8 μg/mL)for 2 h.After 48 h, transduced cells were selected with 2 μg/mL puromycin. Medium was changed twice a week and observed for colony formation. After reaching the optimum growth, selected cells were maintained in DMEM supplemented with 10% FBS and 1 μg/mL puromycin as
    3. Generation of stable cell lines expressing shIP6K1-HeLa and HCT116 cell
    4. Cells were plated at a density of 30,000 cells per well in triplicates in a 24-well plate. After 24 h, cells at a confluency of 30-40%, were treated with different genotoxic agents such as hydroxyurea (0.5 mM), neocarzinostatin (0.25 μg/mL) and mitomycin-C (1 μg/mL) for 12 h. Spent media containing drug was removed and cells were washed gently twice with PBS. Cells were then incubated for different lengths of time ranging from 24 h to 120 h, in fresh complete DMEMto allow them to recover from genotoxic stress. At each time point cell survival was analyzed by MTT assay as described in 2.2.1. Cell survival was expressed as a fold increase in cell population relative to cells treated with drug for 12 h
    5. DNA damage and recovery assay