1 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, OriT, the Origin of Transfer. OriT can be knocked out to severely hinder the efficiency of conjugation, but the efficiency can be restored by a complementation in trans. Bacterial Conjugation

2 OriTKey OriT Key OriT Lock OnGFP OriT Complementation

3 Mutation and complementation of oriT for both F and R plasmids works! Conjugation Assays Matings with recipient cell ec100D::pir+, which is TriR, show if the F or R plasmid transfers by the TriK plate and if an OriT plasmid transfers by the TriA plate

4 Assay shows the key can be transferred efficiently to the lock! Sending the Key to the Lock Mating Results of sending the key plasmid to a cell holding the locked RFP plasmid, pJ23006-J23077 Mating Results of sending the key plasmid to a cell holding a OnRFP plasmid, pSB1A2-J01022

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