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Applications of BBa_K389015

Growth functions and luciferase expression for BBa_K389015

To characterize this part we performed several cultivations with different concentrations of [http://www.chemblink.com/products/2478-38-8.htm acetosyringone] as inducer and measured the luminescence emitted by the luciferase reaction with luciferin (Protocol). We used Escherichia coli DB3.1 carrying the pSB1C3::K389015 plasmid. Even without inducer the bacteria carrying the plasmid showed decelerated growth. In addition acetosyringone affected the growth rates (we used a stock solution of 20 mM acetosyringone solved in 10 % (v/v) DMSO). Growth curves, averaged specific growth rates and doubling times are shown below. It can be observed, that E. coli carrying the pSB1C3::K389015 plasmid growths nearly linear.

The specific growth rates µ and doubling times t_d are calculated with the OD₆₀₀ and following formulas:

Exemplary induction curves with the luminescence normalized to OD₆₀₀ are shown in Fig. 2. We observed a basal transcription, but the induction with acetosyringone is undoubtedly. The detailed data analysis and transfer function is described below.

Fig. 2: Induction curves for E. coli DB3.1 carrying BBa_K389015 with different acetosyringone concentrations in LB medium with 10 mg ml^-1 chloramphenicol. The relative luminescence units (RLU) are normalized to OD₆₀₀ and plotted against the cultivation time in h

Transfer function

The data for the transfer function was measured and analyzed as described below. Nelson et al. suggests using a dose response function and fitting it with a logistical equation for the data analysis of receptor systems ([http://www.nature.com/nature/journal/v416/n6877/abs/nature726.html Nelson et al., 2002]). The data was fitted with a function of the form

with the Hill coefficient p, the bottom asymptote A1, the top asymptote A2 and the switch point log(x₀). Figure 3 shows the measured ratio ɸ_I between induced (i) and uninduced (u) relative luminescence units (RLU) per OD₆₀₀ plotted against the logarithm of the concentration of the inductor [http://www.chemblink.com/products/2478-38-8.htm acetosyringone] in µM. The fit has an R² = 0.98.

The important data from the transfer function is summarized in table 1:

The fully induced VirA/G signaling system with luciferase read-out has a 2.2 fold increased expression compared to the uninduced system. The Hill coefficient is > 1, so a positive cooperation can be observed ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 D Chu et al., 2009]). The switch point of the system is at about 32 µM, so this is the concentration at which the device output is 50% of the maximum output.

Response time

The system needs at least one hour to show a measurable reaction to an induction with acetosyringone. In the following illustration the reaction of the system to induction with 200 µM acetosyringone in the exponential growth phase is shown. For a good separation of the induced system from the uninduced system at least two hours are needed.

Data Analysis

Because the luciferase accumulation is very different in different cultivations, the uninduced negative control was used as internal standard. To show the behaviour of the VirA/G signaling system when induced, the ratio ɸ_I between induced (i) and uninduced (u) relative luminescence units (RLU) per OD₆₀₀ is calculated:

As seen above, at least one hour is needed to separate the induced luminescence signal from the uninduced, so ɸ_I > 1. Within a cultivation ɸ_I is rising during the first hours and is decreasing after it reached a maximum at OD₆₀₀ ~ 1. This is shown in figure 3:

Fig. 5A: Typical development of ɸ_I plotted to OD₆₀₀ of a cultivation with BBa_K389015 in E. coli DB3.1 with polynomial fit (5th order, R² = 0.75, induced with 200 µM acetosyringone). Fig. 5B: Another typical development of ɸ_I plotted to OD₆₀₀ of a cultivation with BBa_K389015 in E. coli DB3.1 with polynomial fit (5th order, R² = 0.87, induced with 400 µM acetosyringone).

To measure the ratio of increasing promoter activity by inducing the system ɸ_I samples for analyzation should be taken at OD₆₀₀ = 1 +/- 0.5. The highest ɸ_I in this range of the cultivation is taken for the calculation of the transfer function.

Plasmid conformation analysis

A plasmid conformation analysis for the BioBrick BBa_K389015 in pSB1C3 was performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] by Capillary Gel Electrophoresis (CGE). The chromatogram is shown in fig. 6 and the results in tab. 3. The data shows a high percentage of covalently closed circular (ccc) plasmid DNA. This is the biological active shape of plasmids so a high percentage of ccc plasmid DNA indicates a high quality of plasmid DNA ([http://web.plasmidfactory.com/en/service_CGE.html PlasmidFactory]).

Protocols

Cultivation

• Inoculate 10 mL LB containing desired Antibiotic with glycerol stock

• Cultivate over night at 37 °C and 175 rpm

• Measure the OD₆₀₀

• Prepare shake flasks with LB, antibiotic and inductor
  • For luciferase measurement at least 10 mL starting volume

• Inoculate the main culture with a starting OD₆₀₀ of 0.1

• Cultivate at 37 °C and 175 rpm

• Take a sample at least every hour and measure the OD₆₀₀

Measurement

• 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^-1 lysozyme, 2.5 mg mL^-1 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 s
  • Integration: 3 s

References

Chu D, Zabet NR, Mitavskiy B (2009) [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 Models of transcription factor binding: Sensitivity of activation functions to model assumptions], J Theor Biol 257(3):419-429.

Greg Nelson, Jayaram Chandrashekar, Mark A. Hoon, Luxin Feng, Grace Zhao, Nicholas J. P. Ryba & Charles S. Zuker (2002) [http://www.nature.com/nature/journal/v416/n6877/abs/nature726.html An amino-acid taste receptor ], Nature 416: 199-202.

[http://www.promega.com/tbs/tb281/tb281.pdf Promega Luciferase Assay System]

[http://web.plasmidfactory.com/en/service_CGE.html PlasmidFactory Homepage]

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