From 2010.igem.org

Revision as of 13:20, 27 October 2010

Characterization of <partinfo>K389015</partinfo>

On this page the experiments and results that lead to the <partinfo>K389015</partinfo> characterization data presented on our characterization page are shown in detail.

Growth functions and Luciferase expression for <partinfo>K389015</partinfo>

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 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 <partinfo>K389015</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml^-1 chloramphenicol.

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 <partinfo>K389015</partinfo> 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. The data was fitted with a dose response 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.

Fig. 3: Transfer function for the part <partinfo>K389015</partinfo> (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 [http://partsregistry.org/Switch_Point 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 <partinfo>K389015</partinfo> 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 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 <partinfo>K389015</partinfo> 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 <partinfo>K389015</partinfo> 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 <partinfo>K389015</partinfo> in <partinfo>pSB1C3</partinfo> 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]).

Fig. 6: Chromatogram of the CGE of the BioBrick <partinfo>K389015</partinfo> in <partinfo>pSB1C3</partinfo> performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] (Bielefeld).