Part:BBa_K410000
Periplasmic Heat Generation
What it is:
This part consists of hybB-ompA-AOX1A.
HybB is a cold shock promoter. OmpA is a periplasmic targeting sequence, and AOX1A is a gene responsible for thermogenesis in the sacred lotus, a plant.
What it does:
Induces AOX1A expression in response to a cold shock. Here, we describe a "cold shock" as incubating E. coli between 20-30C for 10-12 hours. The part should produce a thermogenic response raising the temperature of the E. coli's immediate environment (plate or liquid cultures) above ambient temperature.
Usage and Biology
Characterization
Absolute Temperature Characterization
pSB1A3 containing biobrick part hybB-OmpA-AOX1a was transformed into E. coli BL21. Transformants were grown in 600 mL of LB media(+carbenicillin) at 37 degrees Celsius overnight. The solutions were centrifuged, the soft pellet collected, and resuspended in equivalent amount of fresh LB media(+carbenicillin). The cultures were then placed in a 10 degrees Celsius incubator for 1 hour. 75 mL were pipetted into three different insulated containers and placed in 15, 20 and 25 degrees Celsius incubators for 1 hour acclimation period. Temperatures were recorded at regular intervals using a thermistor thermometer. In addition to the pSB1A3 vector containing the biobrick part, a control (pSB1A3 vector with insert hybB-OmpA-mRFP) was also monitored. The graphs shown below illustrate absolute temperature change over time at different incubation temperatures.
The graph below illustrated absolute temperature difference from initial temperature, when t=0.
Cell Density and Growth Rate Monitoring
Two constructs (hybB -- ompA -- AOX1A, and hybB-ompA-mRFP)in vector pSB1A3 were transformed into E. coli BL21. The mRFP construct served as a control in this experiment, and an additional control of empty vector (psB1a3) was also transformed. Colonies were inoculated into LB and carbenicillin (100 mL) and incubated at 37 until the optical density reached 0.8. After the OD levels reached 0.8 for all cultures, flasks were transferred to 37, 30, 25, 20, or 15 degrees Celsius incubators (with agitation @ 210 rpm). Optical density was measured at regular intervals using a spectrometer. Data below is plotted on a logarithmic scale.
At 30 and 37 degrees Celsius, there is no substantive difference between the growth curves of AOX producing bacteria and control constructs. As temperature drops, the advantage of AOX becomes more apparent suggesting that a heating on the micro-environmental scale positively affects growth rate. Since growth rate is a function of temperature, this data suggests that AOX is being expressed, localized to the periplasm, and interfering with the electron transport chain.
Part Design
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 394
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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