Transcript ConjugationSlides

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
OriT
Key
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
Wildtype F plasmid pOX38
pOX38 x pSB1A2 - J01064
J23015
J23015 x pSB1A2 - J01064
Wildtype R plasmid RP1
Rlambda
Rlambda x pSB1A2 - J01003
OriT
cis
cis&trans
none
trans
# of Colonies on TriK
396800
288000
0
1000
# of Colonies on TriA
n/a
176000
0
126400
cis
none
trans
112000
1
0
n/a
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
pJ23006-J23086 x BW20767
pJ23006-J23066 x BW20767
pSB1A2 - J01003 x BW20767
TG1
OriT
trans
none
trans
none
Key
yes
yes
no
no
# of Colonies on AmpCam
109600
3
140800
0
Mating Results of sending the key plasmid to a cell holding a OnRFP plasmid,
pSB1A2-J01022
pJ23006-J23086 x BW20767
pJ23006-J23066 x BW20767
pSB1A2 - J01003 x BW20767
TG1
trans
none
trans
none
yes
yes
no
no
160000
4
147200
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
mated with J23077
503
74
pJ23006-J23086 x BW20767
pSB1A2 - J01003 x BW20767
mated with J01022
4745
5913
The controls show that conjugation is equivalent to a cotransformation of the
key and lock plasmids into a cell
pJ23006-J23086 x BW20767 (transformed in TGIs)
TGI
pJ23006 - J23077
473
61
63
7000
6000
5000
4000
3000
2000
Series1
Graph will NOT BE USED
Series2
1000
pJ23006 J23077
pJ23006J23086 x
BW20767
(transformed
pJ23006J23086 x
BW20767
(mated with
0
pJ23006J23086 x
BW20767
(mated with
RFP
Flurorescence
Graph
5