Team:Cornell/Contribution
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In implementing an inducible expression system, we chose to incorporate pDawn and pDusk as optogenetic promoters to affect the expression of superoxide dismutase.
We can characterize pDawn and pDusk by Western blot. By growing E. coli under different intensities of visible light, we can compare the relative expression levels of a FLAG-tagged superoxide dismutase under optogenetic control.
We first inserted superoxide dismutase into the pDusk circuit. We then proceeded to incorporate a cI repressor (BBa_K2296045) and a pR promoter into the pDusk system followed by insertion of superoxide dismutase to create a light-inducible pDawn circuit.
We assessed the transcriptional activity of pDawn and pDusk in response to three different levels of light intensity from a standard laboratory fluorescent light:
Our initial characterization revealed a dose-dependence of pDusk. However, pDawn did not display a dose-dependent response in response to stimulation from fluorescent light. We suspect this to be the result of low light intensity.
Our subsequent characterization uses a significantly greater intensity of LED light:
Our results suggest a light-intensity dependence of pDusk and pDawn. Our results also show that our cI repressor biobrick successfully converted the light-repressible pDusk into a light-inducible pDawn.
In addition to dose-dependent characterization, we also conducted time-dependence characterization of pDawn following stimulation by 470 nm LED light (LTL3H3TBPADS1) with the following emission spectrum [12]:
Our initial characterization was conducted with 1 second pulses and revealed a significant lag between initial exposure and translational response:
For our subsequent characterization, we increased the intensity and pulse duration of the LED to parse out more subtle effects at earlier time points:
Our results suggest a light intensity and time-dependence of the pDawn and pDusk optogenetic circuit.
We initially pursued this part as part of a of detector for toxins by cigarette smoke. Upon failure to observe an appreciable difference in expression as well as a transition to a different overarching application, we abandoned this aspect of the project and no further testing was conducted.
Characterization was conducted on this part to determine the plausibility of using it for detection of low concentrations of lead. To this effect, we characterized expression at three different concentrations of lead and documented this on a gel as seen below. The concentrations are as follows: 0, 0.012, 0.024, 0.072 glL Pb(NO3)2.
As can be seen from the gel, no appreciable difference was observed in any of the samples. While we did not conduct further testing, preliminary data suggests that this part does not function as described.
Furthermore, it should be noted that the construct contains mRFP and not sfGFP as the name might suggest, which was confirmed by BLAST.
