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From 2012.igem.org

Double Population Toggle Switch Results

Tests of our dual population toggling mechanism were met with some success, though not to the extent of our single population toggle. Although inducing the mechanism with IPTG and ATc did have an effect on the amount of GFP and RFP expressed by the cells, background noise was high. Looking at only the microscope photos does not reveal a significant difference in RFP or GFP levels.

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Both populations were inoculated together into three tubes with 3mL of LB broth. One tube was induced with 800ng ATc. .2M IPTG was diffused 300X into another tube to make .67mM. The third tube was a control. After ~24 hours, samples from the tubes were checked under a microscope. Next, the cells were spun down in a centrifuge and resuspended in 350uL of LBS. Data was collected with FACS.

The two populations were tested individually to check responsiveness of the populations to quorum signalling molecules. These tests showed that the two populations did respond to quorum signalling molecules as expected. The next step would be to determine whether the cells are able to produce quorum signalling molecules at a high enough rate.

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Populations 1 and 2 were inoculated from glycerol stock separately into two tubes, with 3mL of LB broth. Two other populations capable of producing AHL and AI-1 were also inoculated from glycerol stock into two separate tubes, with 3mL of LB broth. The cells were allowed to grow overnight (~12hrs). The tubes containing AHL and AI-1 were spun down to separate the cells and the quorum signalling molecules. 490uL of the broth containg AHL was pipetted into a 1.5mL tube (T1). 490uL of the broth containing AI-1 was also pipetted into a 1.5mL tube (T2). Two 1.5mL tubes were filled with 490uL of LB broth each (T3 & T4). 10uL of Population 1 cells were pipetted into T1 and T3. The same amount of Population 2 cells were pipetted into T2 and T4. The cells were incubated for another 24 hours. After incubation, samples from each of the tubes were checked under the microscope for fluorescence.