Look Who’s Talking Constructs Rough Draft 3/26/2005

1. Look Who’s 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 cell’s address Transmit F-plasmid Activated when Transmit is complete pRM pRPOE ON TraJR YFP antiTx This tag expresses Antisense to transmitting cell’s 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 • 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 • 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: • Fred+Fred  Should do nothing (not even adhere) • Fred+Barney  Should do nothing (not even adhere) • Fred+Betty Should adhere and transmit but Betty should fail to turn Green or transmit her plasmid back to Fred • Barney+WilmaShould adhere and transmit to Wilman– but Wilman should not turn green or transmit her plasmid back to Barney • Wilma+Betty, 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. F+strain 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 don’t interfere • Mix strains. Image conjugal pairs Green cell attached to Red Cell. • Induce TraJ in F-cell. Show Green/Yellow cell. • Etc.