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UV IRRADIATION, SURVIVAL CURVE AND REPAIR MECHANISM GENE367 MOLECULAR GENETICS LABORATORY BEGÜM TUNCER 07/10/2009

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UV IRRADIATION, SURVIVAL CURVE AND REPAIR MECHANISM GENE367 MOLECULAR GENETICS LABORATORY BEGM TUNCER 07/10/2009 Slide 2 http://waterecotechnology.com Slide 3 Cells and nucleic acids absorb UV 260 nm is absoption peak of DNA Pyrimidine dimers (T-T, C-C, T-C) can be formed after UV exposure Repair mechanisms may remove these dimers Mutations and/or DNA replication inhibition are possible Slide 4 Repair Mechanisms Photoreactivation Excision Repair Recombinational repair SOS Repair Slide 5 Photoreactivation (Light Repair) In E.coli an enzyme called photoreactivating enzyme (PRE) is produced Binds to dimers, to seperate them to monomers. Only active after immediate exposure to visible light after UV light. Slide 6 http://trishul.sci.gu.edu.au Slide 7 Excision Repair (Dark Repair) uvrA, uvrB, and uvrC genes are responsible for excision repair in E.coli http://www.phys.ksu.edu/gene Slide 8 Recombinational Repair (Postreplicational Repair) recA gene is responsible for strand exchange for recombinational repair. Slide 9 http://www.mun.ca/biochem Slide 10 SOS Repair The SOS repair system is induced in response to major damage to the bacterial DNA or in response to agents which inhibit DNA replication. The system is a complex one with over 20 genes involved. Two of these are the important regulator genes: lexA and recA. Slide 11 Bacterial Growth Aseptic techniques Culture techniques: spread plate Dilutions Measurement of growth: viable count Slide 12 Dilutions To count the individual cells/colonies dilution is necessary. Serial dilutions can be performed either tenfold or hundred fold. Ex: 1 mL of bacterial culture in 100 mL solution makes 10 -2 dilution. DF=10 2 Slide 13 E.coli colonies on a petri plate Slide 14 Calculation of colony forming units cfu/mL = number of colonies x Dilution factor x 1/V Slide 15 Procedure Grow 10 mL of E.coli/ B. subtilis cells o/n at 37 C Centrifuge at 5000 rpm for 15 min. Discard supernatant and resuspend the pellet in 10 mL 0.85% saline solution. Pour it into a sterile petri plate. Take 0.1 mL aliquot from sampling plate and dilute according to the table Slide 16 Slide 17 Procedure Label LB petri plates: Group, Date, Time, Strain, Dilution. Spread the diluted samples onto LB agar plates (only 0 min). Put the Sample plate under UV light and expose to UV for 30 seconds. Dilute the samples according to table. Spread the diluted samples on LB agar plates (only 30 sec). Repeat the UV exposure for 1, 3, 5, and 10 min exposures by adding up the time required. Repeat dilutions and spreading. Incubate your plates at 37 C for 24 hours. Slide 18 Next week Count colonies on plates. Calculate log number of survivors. Plot a curve for comparison of E.coli vs B. Subtilis bacterial strains.