Results
Characterization of Switch with LuxI-GFP
Preliminary results with RFP and GFP showed that the switch was leaky when on a pSB1C3 backbone. We believe that because of the high copy number of pSB1C3, there was a high number of switches in each cell so if only a small proportion of switches were to leak, this effect would be visible. In order to investigate this issue, we transfered the gate with a GFP reporter onto a low copy plasmid. As expected, we saw much less expression of our reporter.
To test the responsiveness of our integrase in tandem with the leakiness of the gate on a low copy plasmid we co-transformed the Bxb1 integrase behind the arabinose inducible promoter on a kanamycinbackbone with BBa_K1718005 (switch-LuxI-GFP) on 4C5.
It was tested by growing cells in overnight cultures with varying concentrations of arabinose, from 0% to .2%increasing by .04% for a total of 6 cultures. The cultures were made of 5mL of LB, 5ul of both chloramphenicol and kanamycin, and cells taken from a glycerol stock of cells with the desired plasmids. The 4th culture had low growth.
Flow cytometry was performed on the cells after being resuspended in PBS which returned the resulting data.
With the exception of the fourth sample, which was the culture with low growth, we see a general rightward trend in the peaks, indicating that higher concentrations of arabinose are more effective at inducing the production of the integrase. More tests should be done to confirm this trend and find the ideal concentration.
Testing response of pBAD promoter with Arabinose
Because we are using pBad as our promoter to start the cascade of responses in our system, we needed to test the leakiness of the promoter. To do this, we put pBad on a 1C3 plasmid with Red Fluorescent Protein (RFP).
The promoter, pBad, turns on in the presence of arabinose and in doing so, allows RFP to be transcribed. Therefore, once arabinose is present, our cells will fluoresce red.
The amount that our cells fluoresce depends on the amount of arabinose. To further characterize this observation, we grew up overnights with varying concentrations of arabinose and measured their fluorescence.
Initially, we documented with pictures to illustrate the change in fluorescence and we observed a relatively smooth increase in brightness as the arabinose concentration increased.
Varied concentrations of arabinose from 0% to 0.2% by 0.04% increments (7/14/15)
Then we used flow cytometry to more accurately characterize the fluorescence:
As illustrated in the flow cytometry characterization, we do see some cells fluorescing even when no arabinose is present, therefore the pBad promoter is leaky.
Although the promoter is leaky, it is sufficient for our proof of concept at this stage of our project. If the leakiness needed to be addressed, the promoter could be transferred to a lower copy plasmid or one could use a weaker pBAD promoter such as BBa_K206001, thereby reducing the effects of leakiness.
