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.

1
 OriT Complementation

Lock
OriT

OnGFP

Key Key

OriT OriT

2
 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

OriT # o f Co lo nie s o n TriK # o f Co lo nie s o n TriA

Wild typ e F p la sm id p OX38 c is 396800 n/a
p OX38 x p SB1A2 - J01064 c is&tra n s 288000 176000
J23015 none 0 0
J23015 x p SB1A2 - J01064 tra n s 1000 126400

Wild typ e R p la sm id RP1 c is 112000 n/a

Rla m b d a none 1 0
Rla m b d a x p SB1A2 - J01003 tra n s 0 1664

Mutation and complementation of oriT 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,
pJ23006-J23077
OriT Ke y # o f Co lo nie s o n Am p Ca m
p J23006-J23086 x BW20767 tra n s ye s 109600
p J23006-J23066 x BW20767 none ye s 3
p SB1A2 - J01003 x BW20767 tra n s no 140800
TG1 none no 0

Mating Results of sending the key plasmid to a cell holding a OnRFP plasmid, pSB1A2-
J01022
p J23006-J23086 x BW20767 tra n s ye s 160000
p J23006-J23066 x BW20767 none ye s 4
p SB1A2 - J01003 x BW20767 tra n s no 147200
TG1 none no 0

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
m a te d with J23077 m a te d with J01022
p J23006-J23086 x BW20767 503 4745
p SB1A2 - J01003 x BW20767 74 5913

The controls show that conjugation is equivalent to a cotransformation of the key and lock
plasmids into a cell
p J23006-J23086 x BW20767 (tra nsfo rm e d in TGIs) 473
TGI 61
p J23006 - J23077 63

7000
6000
5000

RFP 4000 Series1

Graph will NOT BE USED

3000 Series2

Flurorescence 2000
1000

Graph 0

pJ23006 -
(transformed
(mated with
(mated with
BW20767

J23077
pJ23006-

pJ23006-

BW20767
J23086 x

J23086 x

BW20767
pJ23006-
J23086 x