Bacterial Conjugation Conjugation will be used as a

Bacterial Conjugation • Conjugation will be used as a means to transfer genetic information from one bacteria to another • Cells with a F+ plasmid or a R plasmid can transfer their DNA to other bacteria • On the F and R plasmids, DNA transfer begins at a specific sequence, Ori. T, the Origin of Transfer. • Ori. T can be knocked out to severely hinder the efficiency of conjugation, but the efficiency can be restored by a complementation in trans. 1

Ori. T Complementation Lock Ori. T On. GFP Key Ori. T 2

Conjugation Assays Matings with recipient cell ec 100 D: : pir+, which is Tri. R, show if the F or R plasmid transfers by the Tri. K plate and if an Ori. T plasmid transfers by the Tri. A plate Mutation and complementation of ori. T for both F and R plasmids works! 3

Sending the Key to the Lock Mating Results of sending the key plasmid to a cell holding the locked RFP plasmid, p. J 23006 -J 23077 Mating Results of sending the key plasmid to a cell holding a On. RFP plasmid, p. SB 1 A 2 -J 01022 Assay shows the key can be transferred efficiently to the lock! 4

Tecan Fluorescence Assay The Tecan test shows that the key plasmid results in the recovery of fluorescence The controls show that conjugation is equivalent to a cotransformation of the key and lock plasmids into a cell RFP Flurorescence Graph will NOT BE USED 5