Part:BBa_K389004:Experience

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Applications of BBa_K389004

Accumulation of luciferase

The production of the reporter gene luciferase by Escherichia coli is dependent from the specific growth rate µ (fig. 1A, eq. (2)) and the growth phase (fig. 1B). Fig. 1A shows the specific production rate q_P which was determined with equation (1)

(1)

plotted against the specific growth rate µ which was determined with equation (2)

File:....

(2)

With decreasing growth rate the luciferase production slows down and with stopping growth the luciferase is degraded. Especially in the stationary growth phase the luciferase degradation is high (fig. 1B).

Fig. 1A: Specific production rates q_P of many different cultivations of BBa_K389015 in Escherichia coli DB3.1, some induced with acetosyringone, some uninduced. Fig. 1B: Cultivation of BBa_K389307 in E. coli TOP10.

Measurement protocol

• For the luciferase detection we used a [http://www.promega.com/tbs/tb281/tb281.pdf Promega Luciferase Assay System], containing a Cell Culture Lysis Reagent, Luciferase Assay Substrate and Luciferase Assay Buffer

• Prepare reaction tubes with 10 µL of high salt buffer (1M K₂HPO₄, 20mM EDTA, pH 7.8)

• Add 90 µL sample, mix and freeze at -80 °C

• For the measurement thaw by placing the tubes in room temperature water

• Add 300 µL of freshly prepared lysis mix ( 1X Cell Culture Lysis Reagent, 1.25 mg/mL lysozyme, 2.5 mg/mL BSA, add water for desired volume)

• Mix and incubate the cells for 10 minutes at room temperature

• Prepare the Luciferase Assay Reagent, by adding 10 mL of Luciferase Assay Buffer to the vial containing the Luciferase Assay Substrate

• Fill each well of a white, flat bottom 96 well microtiter plate with 20 µL of cell lysate

• For the detection of luciferase use a plate reading luminometer with injector for the Luciferase Assay Reagent and following settings (we used a Promega GloMax®-Multi Detection System with dual injector):
  • Injection volume of Luciferase Assay Reagent: 100 µL
  • Delay: 20 secs
  • Integration: 3 secs

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