1 / 18

UV IRRADIATION, SURVIVAL CURVE AND REPAIR MECHANISM

UV IRRADIATION, SURVIVAL CURVE AND REPAIR MECHANISM. GENE367 MOLECULAR GENETICS LABORATORY BEGÜM TUNCER 07/10/2009. http://waterecotechnology.com. 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

connor
Download Presentation

UV IRRADIATION, SURVIVAL CURVE AND REPAIR MECHANISM

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

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

  2. http://waterecotechnology.com

  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

  4. Repair Mechanisms • Photoreactivation • Excision Repair • Recombinational repair • SOS Repair

  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.

  6. http://trishul.sci.gu.edu.au

  7. Excision Repair (Dark Repair) • uvrA, uvrB, and uvrC genes are responsible for excision repair in E.coli http://www.phys.ksu.edu/gene

  8. Recombinational Repair (Postreplicational Repair) • recA gene is responsible for strand exchange for recombinational repair.

  9. http://www.mun.ca/biochem

  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.

  11. Bacterial Growth • Aseptic techniques • Culture techniques: spread plate • Dilutions • Measurement of growth: viable count

  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=102

  13. E.coli colonies on a petri plate

  14. Calculation of colony forming units cfu/mL = number of colonies x Dilution factor x 1/V

  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

  16. 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.

  17. Next week • Count colonies on plates. • Calculate log number of survivors. • Plot a curve for comparison of E.coli vs B. Subtilis bacterial strains.

More Related