To characterize and test the leakiness of the LuxPr promoter, we created a construct that placed the LuxPr promoter in front of RFP on a 1C3 backbone. When induced with (N-B-ketocaproyl)-L-homoserine lactone, a specific AHL molecule, the LuxPr promoter will allow transcription of RFP.
To characterize the promoter activity, we measured the RFP fluorescence with a flow cytometer. We tested cells grown in 5 mL LB or 5mL M9 media with 25uL chloramphenicol at different concentrations of AHL starting at 0.1nM, increasing by a factor of 10 (0.1nM, 1.0nM, 10nM, 100nM, 1uM, 10uM). We also included a negative control with no AHL added (0nM). These overnights were incubated for 18 hours, then centrifuged so the pellets could be compared in red color by eye.
The first trial using LB media yielded all samples except 100nM turning a similar shade of deep red. This included the negative control turning red, giving us no baseline for comparison.
Another trial was done in LB. The 100nM sample did not grow.
There was a gradient of increasing red color between 0nM through 10nM, which appeared the reddest to the eye. The samples from trial 5 were suspended in PBS and then run through a flow cytometer to measure the relative fluorescence. Below is the cytometer data. 1H (pink) is the 0nm negative control. 2H (blue) is the 0.1nm sample. 3H (orange) is the 1.0 nm sample. 4H (green) is the 10nm sample.
All three of the samples with AHL added produced relatively more fluorescence than the negative control. A concentration of 10nm produced the most RFP, so would be the ideal concentration to use according to this data. However, the upper concentrations we aimed to test failed to grow, so the experiment should be redone to determine if a higher concentration of AHL would be more optimal.
We had planned to test the promoter on a 4C5 backbone instead of the 1C3 used in this experiment. We hypothesized the lower copy number plasmid would provide more distinction between the concentrations of AHL, especially after our first result in which all concentrations including the negative control turned a deep red. The experience page with the promoter also described achieving good results using EZ media, an additional component we were not able to complete either.
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.
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.
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.