Part:BBa_J45181:Experience
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Applications of BBa_J45181
Exponential phase dependent wintergreen odor production from the exogenous precursor salicylic acid.
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The exponential phase wintergreen odor generator (BBa_J45181) produces a wintergreen odor in culture when supplemented with salicylic acid. However, the exponential phase wintergreen odor generator produced levels of methyl salicylate indistinguishable from that of the constitutive wintergreen odor generator (BBa_J45120). |
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Characterization
Failure analysis
We were unable to regulate wintergreen odor production by combining the exponential phase transcriptional control device (BBa_J45994) with the wintergreen odor enzyme generator (BBa_J45119). The exponential phase wintergreen odor generator (BBa_J45181) did produce wintergreen odor when cultures were supplemented with salicylic acid. However, the exponential phase wintergreen odor generator produced levels of methyl salicylate indistinguishable from that of the constitutive wintergreen odor generator (BBa_J45120). We were surprised by the gas chromatography results because we were able to demonstrate that the exponential phase device successfully regulated a GFP generator (Part:BBa_J45994:Experience#Characterization). To better understand why the exponential phase wintergreen odor generator failed to function as intended, we consider in detail the device's component parts and operation as compared to the constitutive device. The constitutive wintergreen odor generator is composed of a promoter (BBa_R0040) and the wintergreen odor enzyme generator (BBa_J45119). The exponential phase wintergreen odor generator is composed of a stationary phase promoter (BBa_J45992), a transcriptional inverter device (BBa_Q04401) and the wintergreen odor enzyme generator (BBa_J45119). The transcriptional inverter device is composed of four parts: a ribosome binding site (BBa_B0064), a tetR repressor (BBa_C0040), a terminator (BBa_B0015), and a cognate promoter (BBa_R0040). Thus, the promoter BBa_R0040 is used in both the constitutive and exponential phase odor generators. In the absence of the cognate TetR repressor, the promoter BBa_R0040 operates as a constitutive promoter as in the constitutive wintergreen odor generator. In the presence of the cognate TetR repressor, the promoter BBa_R0040 operates as a repressible promoter as in the exponential wintergreen odor generator. During exponential growth, cultures of the constitutive and exponential phase wintergreen odor generators should produce similar levels of methyl salicylate since both devices include the same promoter. In stationary phase, the constitutive wintergreen odor generator should continue to produce methyl salicylate. In contrast, for the exponential phase wintergreen odor generator, the osmY stationary phase promoter should turn on and provide a high input into the transcriptional inverter device. The transcriptional inverter will produce a low transcriptional output leading to a drop in expression of BSMT1 and, by extension, a drop in methyl salicylate production. If, however, the BSMT1 enzyme is very stable or there is some leaky expression from the inverter device, then there will continue to be methyl salicylate production in stationary phase as we observed in the gas chromatography analysis.
Growth dependent wintergreen odor production
The BSMT1 enzyme generator (BBa_J45119) is used in both wintergreen odor generators (BBa_J45120 and BBa_J45181).
To demonstrate growth phase dependent wintergreen odor production, we compared the behavior of constitutive and exponential phase dependent wintergreen odor generators (A and B, respectively). We measured methyl salicylate concentration (relative to the pentachloronitrobenzene internal standard) of cultures of the constitutive and exponential phase wintergreen odor generators at different culture cell densities (OD600nm) (C). The constitutive and exponential phase wintergreen odor generators produced similar levels of methyl salicylate at all cell densities examined. Thus, the exponential phase wintergreen odor generator does not work as intended.