Team:SoundBio/Characterization
Characterization
Background
We characterized FixK2 (BBa_K592006), an inducible promoter that’s part of our blue light system. FixK2 is activated when phosphorylated FixJ binds to it, which happens under dark conditions. We wanted to test the reliability of this promoter -- is there leaky expression? And if so, how much is there?Leaky expression is when a promoter initiates transcription of a downstream region of DNA when it’s not supposed to. In this case, FixK2 is only supposed to initiate transcription when FixJ-p binds to it. So to test for leaky expression, the genetic circuit shouldn’t contain FixJ. Thus, any expression of the chosen marker is a result of leaky expression. In our experiments, we used GFP as an indicator of FixK2’s activity. We designed a positive control that constitutively expresses GFP and a negative control that doesn’t contain GFP. Below are our genetic circuits:
- Experimental
- FixK2, RBS, GFP, Double Terminator
- Positive Control
- Constitutive Promoter, RBS, GFP, Double Terminator
- Negative Control
- FixK2
Because YF1/FixJ is sensitive to blue light, we also exposed our three genetic constructs to blue light or dark conditions. Since our circuits do not contain YF1/FixJ, we don’t expect the light condition to affect FixK2 promoter activity. However, we chose to incorporate the light conditions to mimic FixK2’s actual function.
Methods
After making 2mL overnight cultures of E. coli Top10 containing these genetic constructs, we made a 1:100 backdilution into M9 media. We then exposed them to their separate light conditions for 3 or 5 hours. From there, we extracted 150uL from each sample and put them into a 96-well microplate and analyzed its fluorescence via flow cytometry. FL1-A values were determined by selecting FSC and SSC between the 25th and 75th percentiles. We used this python package to analyze the fsc data from the flow cytometer.Results
Click here to see all raw data3 Hour Light Exposure
Raw Data
Processed Data
5 Hour Light Exposure
Raw Data
Processed Data
*Data from trials 1 and 2 were removed in calculating the average and standard deviation. Those values were extreme outliers and skewed the results too much.
