©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Practicals : Aims 1. Phage restriction –Observe phage restriction and consider the implications of this phenomenon in nature. –Acquire the skills needed to cultivate and titre phage in practice 2. Phage co-transduction –Demonstrate the close linkage of two bacterial genes by transductional mapping –Observe the connection between RecA function and DNA repair 3. Conjugation and transposition –Demonstrate plasmid conjugation –Demonstrate Insertion Sequence transposition/cointegration

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. IS1 mediated excisions in the gal operon gal::IS1 in this sector reverts as red WT (gal+) colonies on McConkey Agar. Note the high frequency  gal (deletion). No revertants possible gal point (amber) mutation. Supression revertants at low frequency

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Effect of UV irradiation on the growth of E.coli recA mutants on an agar plate UV this side This side covered E.coli WT recA mutant another recA mutant

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Demonstration of CI857 phage restriction in E.coli K12. CI857 titred on C600 (restriction minus)after growth on DP990 dcm methylase_+ CI857 titred on C600 (pN3 EcoRII restriction plus) after growth on DP990 dcm methylase+ CI857 titred on C600 (restriction minus) after growth on DP1060 dcm methylase minus CI857 titred on C600 (pN3 EcoRII restriction plus) after growth on DP1060 dcm methylase minus CCA/TGG Dcm methylase recoginises here EcoRII from pN3 cuts here

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Plasmid cointegration mediated by IS1. See practical exercise Crosses of: A. E.coli DP990 (pOXKm, pKPG16 (pBR322::IS1)) with C600 nal R B. E.coli DP990 (pOXKm, pBR322 (Control)) with C600 nal R A. 100µl undiluted and plated on Km,Tet and Nal agar plates. Cointegrates grow B. 100µl undiluted and plated on Km,Tet and Nal agar plates. No cointegrates detected 100µl of 10-3 dilution plated on Km, Nal plates. Indicates the pOXKm transconjugants

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Deletion of adjacent genes due to activity of IS1 inserted in the gal operon gal IS1 chlD locus mutations confer resistance to chlorate Chlorate resistant colonies on chlorate/McConkey agar. A. IS1 has transposed to chlD locus but still reverts as papillae to gal+ B. IS1 has transposed to chlD locus then gal has been deleted; hence no papillae

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Deletion of adjacent genes due to activity of IS1 inserted in the gal operon Chlorate resistant colonies on chlorate/McConkey agar. A. IS1 has transposed to chlD locus but still reverts as papillae to gal+ B. IS1 has transposed to chlD locus then gal has been deleted; hence no papillae Close-up of colonies with gal+ papillae