Team:Bielefeld-Germany/Results/Characterization/K389015

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Characterization of BBa_K389015

On this page the experiments and results that lead to the BBa_K389015 characterization data presented on our characterization page are shown in detail.


Growth functions and Luciferase expression for BBa_K389015

To characterize this part we performed several cultivations with different concentrations of acetosyringone as inducer and measured the luminescence emitted by the luciferasereaction 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 stocksolution 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.


Fig. 1: Growth curves for E. coli DB3.1 without plasmid and carrying BBa_K389015 with different acetosyringone concentrations in LB medium with 10 mg ml-1 chloramphenicol.


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


Bielefeld Specific growth rate.jpg
(1)


Bielefeld Doubling time.jpg
(2)


Table 1: Averaged specific growth rates and doubling times for cultivations of E. coli DB3.1 without plasmid and carrying BBa_K389015 with different acetosyringone concentrations in LB medium with 10 mg ml-1 chloramphenicol.

E. coli DB3.1 µ / h-1 td / h
without plasmid 0.35 1.98
carrying K389015 0.31 2.24
carrying K389015 with 400 µM acetosyringone 0.26 2.67


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 (Nelson et al., 2002). The data was fitted with a function of the form


Bielefeld Doseresponse fit.jpg
(3)


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 ɸI between induced (i) and uninduced (u) relative luminescence units (RLU) per OD600 plotted against the logarithm of the concentration of the inductor acetosyringone in µM. The fit has an R2 = 0.98.


Fig. 3: Transfer function for the part BBa_K389015 (R2 = 0.98).


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

Table 2: Data from the transfer function for the part BBa_K389015.

Parameter Value
Hill coefficient 1.092
Switch point 31.6 µM
Top asymptote 2.16


The fully induced VirA/G signaling system with luciferase readout has a 2.2 fold increased expression compared to the uninduced system. The Hill coefficient is > 1, so a positive cooperation can be observed (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.

Fig. 4: Cultivation of BBa_K389015 in Escherichia coli DB3.1. Induction with 200 µM acetosyringone during the exponential growth phase.

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 ɸI between induced (i) and uninduced (u) relative luminescence units (RLU) per OD600 is calculated:


Bielefeld Quotient RLU.jpg
(4)


As seen above, at least one hour is needed to separate the induced luminescence signal from the uninduced, so ɸI > 1. Within a cultivation ɸI 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 ɸI 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 ɸI 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 ɸI samples for analyzation should be taken at OD600 = 1 +/- 0.5. The highest ɸI 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 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 (PlasmidFactory).


Fig. 6: Chromatogram of the CGE of the BioBrick BBa_K389015 in pSB1C3 performed by the PlasmidFactory (Bielefeld).


Table 3: Data from the CGE of the BioBrick BBa_K389015 in pSB1C3 performed by the PlasmidFactory (Bielefeld).

Conformation Ratio / %
ccc monomer 91
ccc dimer 3.7
oc 5.3


References

Chu D, Zabet NR, Mitavskiy B (2009) 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) An amino-acid taste receptor , Nature 416: 199-202.

Promega Luciferase Assay System

PlasmidFactory Homepage