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
Repair Mechanisms Photoreactivation Excision Repair Recombinational repair SOS Repair
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.
Excision Repair (Dark Repair) uvrA, uvrB, and uvrC genes are responsible for excision repair in E.coli
Recombinational Repair (Postreplicational Repair) recA gene is responsible for strand exchange for recombinational repair.
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.
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 dilution. DF=10 2
E.coli colonies on a petri plate
Calculation of colony forming units cfu/mL = number of colonies x Dilution factor x 1/V
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
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.
Next week Count colonies on plates. Calculate log number of survivors. Plot a curve for comparison of E.coli vs B. Subtilis bacterial strains.