1. Microbial Genetics (Micr340) Lecture 7 Plasmids

2. DNA molecules other than chromosomes Widely present in most bacteria Roles: adaptation, evolution, pathogenesis Usually encode genes not essential Mostly circular Sizes: a few kb to >100 kb

3. Naming Plasmids Many name after genes they carry or functions of the plasmids In the beginning: ColE1, producing Bacteriocin to kill E. coli Tol, degradation of toluene Ti, Tumor initiation in plants RK2, resistance to amp, tet, kanamycin Now: pXXNNNN. eg, pHX200

4. Plasmid structure

5. Plasmid purification

7. Plasmid replication Replicons: DNA molecules that can replicate autonomously in cell (chromosome, plasmid) All replicons have at least one origin of replication (ori) For plasmid ori, it is called oriV (vegetative) Two mechanisms of replication: Theta replication Rolling-circle replication

8. Theta replication

9. Rolling-circle replication

10. Function of ori region Replication related genes close to ori. Host range determination Narrow host range Broad host range Regulation of copy-number Relaxed plasmids – high copy-number Stringent plasmids – low copy-number

11. Regulation of replication: ColE1 plasmids

12. Random plasmid curing

13. Plasmid Partition Plasmid avoid being lost from dividing cells by carrying partitioning systems The functions involved in these systems are called par functions cis-acting site: parS trans-acting site: parA and parB

14. Incompatibility Many bacteria contain multiple types of plasmids and can coexist for many generations Not all types of plasmids can stably coexist in same cell; some interfere with each other’s replication or partition and one of them will be lost This is called plasmid incompatibility

15. Incompatibility Two plasmids that cannot stably coexist are members of the same incompatibility (Inc) group If two plasmids can stably coexist, they belong to different Inc group

16. Coexistence of different Inc groups

17. Coexistence of same Inc groups

18. Measuring curing of a plasmid

19. Finding the ori of a plasmid