Bacterial Conjugation

1
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, 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.

2
OriT Complementation
3
Conjugation Assays
Matings with recipient cell ec100Dpir, 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
Mutation and complementation of oriT for both F
and R plasmids works!
4
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
Assay shows the key can be transferred
efficiently to the lock!
5
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
Graph will NOT BE USED