Look Who

1
Look Whos Talking ConstructsRough Draft
3/26/2005
atc
ON
pTet
CFP
cI
spo0A
Genomic TxControl
Tx
This is DNA Tag which Is the address Of this
cell
Identifies Cell as F-type
Activates Received Message
When induced This activates F-plasmid transmit
F-bearing cell
pRPOE
pspoIIE
ON
F-Plasmid TxPlasmid
TraJF
GFP
antiRv
This tag expresses Antisense to Receiving
cells address
Transmit F-plasmid
Activated when Transmit is complete
pRPOE
pRM
ON
TraJR
YFP
antiTx
This tag expresses Antisense to transmitting
cells address
R-bearing cell
Transmit F-plasmid
Activated when Transmit is complete
RP4-Plasmid RcPlasmid
pR
ON
ON
Genomic RcControl
RFP
cro
rpoE
Rc
This Is the address of This cell
Identifies Cell as R-type
Activates Acknowledge And Transmit
When repressed Activates RPOE
2
F-type cell
TxPlasmid consitutively makes F-pili and a
surface exclusion protein. F-pili allow conjugal
attachment in solution. The surface exclusion
protein prevents F/F mating. It might also
prevent R/F mating but this can be remedied
genetically, Without TraJ being expressed,
however, there can be no transmittal of the
TxPlasmid. Addition of atc activates TraJ only
in F-type cell. If there is no attachment to
another cell Rxplasmid turns off F-plasmid
transmission and TxControl turns ON the RxPlasmid
acknowledge fluorophore.
TxPlasmid
atc
Activate TraJ
TxControl
3
RP4-type cell
RxPlasmid constitutively makes R-pili and a
surface exclusion protein. R-pili allow conjugal
attachment only on hard media since they are so
much shorter among other things. The surface
exclusion protein prevents R/R mating. Without
TraJ being expressed, however, there can be no
transmittal of the RxPlasmid. TraJ in R-type
cells is only activated by the TxPlasmid and only
then if the address on the TxPlasmid is
complementary to the RxControl address.
RxControl activates TxPlasmid Acknowledge
fluorophor
RxPlasmid
txPlasmid
Activate TraJ
RxControl
4
The desired result

1. Mix F and R cells with complementary addresses
   together. The addresses would normally be 25-mer
   tags but here I use names Fred for the
   transmitting cell, Wilma for the receiving cell.

Wilma
Fred
Fred
Wilma
Fred
Wilma
Fred
Wilma
Cyan
Red
Cyan
Red
2. Add atc to media. .Transfer of TxPlasmid to
Wilma activates GFP in Wilma and transfer of
RxPlasmid
Fred
Wilma
Fred
Wilma
atc
Fred
Wilma
Fred
Wilma
Red Green
Cyan
Red
Cyan
5
The desired result
3. Eventually, the Rxplasmid is transferred back
to the transmitting cell. In the current design
this will automatically turn Fred Yellow with the
Rxplasmid bearing the right address and in fact
there is little interesting about the address
control for RxPlasmid on Fred. Ideally, if the
message is incorrect it should be degraded (there
are specific restriction systems for this)- and
Fred should not turn Yellow. There are many ways
to do this but you get the idea.
Fred
Wilma
Fred
Wilma
Time
Fred
Wilma
Fred
Wilma
Red Green
Cyan
Red Green
Cyan yellow
6
Controls

1. Imaging we have two other cells Barney and
   Betty. Barney is F-type but has a different
   address tag than Fred and Betty is RP4 type but
   has a different address than Betty. Then we could
   do the following experiments
2. FredFred ? Should do nothing (not even adhere)
3. FredBarney ? Should do nothing (not even adhere)
4. FredBetty ?Should adhere and transmit but Betty
   should fail to turn Green or transmit her plasmid
   back to Fred
5. BarneyWilma?Should adhere and transmit to
   Wilman but Wilman should not turn green or
   transmit her plasmid back to Barney
6. WilmaBetty, etc.

7
Staged Construction I.

• It would be worth simply making two strains of
  bacteria. Both have integrated constructs of the
  following sort
• Promoter?Tag?Fluorescent Protein
• Make two Plasmids each with
• Promoter?antiTag
• Demonstrate that you need the right
  Plasmid/Strain combination to turn off
  fluorescence.
• This would demonstrate antisense based addressing
  of signals and allow a short paper outlining the
  whole strategy and demonstrating the addressing
  mechanism.

8
Staged Construction II.

• Demonstrate induced transmission. Fstrain
  bearing GFP and TraJ under an inducible promoter.
• Mix F/F- cells image conjugal pairs with one
  green cell and one dark cell.
• Induce TraJ and then image two green cells.
• Do the same for RP4 strains bearing RFP.
• Show F/RP4 dont interfere
• Mix strains. Image conjugal pairs Green cell
  attached to Red Cell.
• Induce TraJ in F-cell. Show Green/Yellow cell.
• Etc.