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From 2012.igem.org

Results

Our testing consisted mostly of microscopy experiments. Cells with our designed plasmid were inoculated into a tube of broth along with varying amounts of inducer chemicals. Although our first series of experiments were for the most part unsuccessful, we were able to find concentrations of chemicals that allowed for optimal performance of our mechanism. 1-2 mM of IPTG and 800ng/uL of ATc were used in our most successful experiments. Some of our test results are given below. Although noise was found to be quite high, our toggling mechanism was successful in that it did lock into one state or the other depending on the chemical it was induced with. Microscopy photos generally show higher levels of red fluorescence when the cells are induced with IPTG and green when induced with Atc. We were not able to successfully test its capability of switching to the opposite state after it had already been induced once.

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The mechanism was inoculated into 3 separate 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 the broth was discarded. The cells were resuspended in 350uL LBS. Data was collected with FACS.

LacO Binding Sites

In order to remove some of the noise inherent in our design, we tried to make the binding of LacI to pLac more effective by adding extra LacO sites to the end of the pLac sequence. This was accomplished with PCR. Ultimately, we were unable to show that the extra LacO sites added to the effectiveness of LacI on the pLac promoter.