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Latest revision as of 00:38, 28 October 2010

Characterization of <partinfo>K389016</partinfo>

On this page the experiments and results that lead to the <partinfo>K389016</partinfo> characterization data presented on our characterization page are shown in detail.

Growth functions and mRFP expression for <partinfo>K389016</partinfo>

To characterize this part we performed several cultivations with different concentrations of acetosyringone as inducer and measured the fluorescence emitted by mRFP ( Protocol). We used Escherichia Coli DB3.1 carrying the pSB1C3::K389016 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::K389016 plasmid growths nearly linear.

Fig. 1: Growth curves for E. coli DB3.1 without plasmid and carrying <partinfo>K389016</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 fluorescence 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>K389016</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml^-1 chloramphenicol. The relative fluorescence units are normalized to OD₆₀₀ and plotted against the ime in h

Transfer function of <partinfo>K389016</partinfo>

The data for the transfer function was measured and analyzed as described below. [http://www.nature.com/nature/journal/v416/n6877/abs/nature726.html Nelson et al. (2002)] suggest using a dose response function and fitting it with a logistical equation for the data analysis of receptor systems. 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 normalized specific production rates q_P,n (eq. 8) 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 = 0.99.

Fig. 3: Transfer function for the part <partinfo>K389016</partinfo> (R² = 0.99).

The important data from the transfer function is summarized in table 2:

So the fully induced VirA/G signaling system has a 2.6 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 25 µM, so this is the concentration at which the device output is 50% of the maximum output.

Data analysis for <partinfo>K389016</partinfo>

The data analysis is made in three steps. First step is the processing of the fluorescence raw data gained by the fluorescence plate reader for every sample:

In the second step the RFU_corrected of every sample is plotted against the cultivation time it was drawn. The data is fitted by an exponential fit of the following style:

The accumulation of mRFP in the cells is always exponential. A typical fitted product accumulation curve is shown below:

Fig. 4: Exponential fit on the measured RFU plotted against cultivation time of a cultivation of <partinfo>K389016</partinfo> in Escherichia coli DB3.1 in LB medium with 10 mg ml^-1 chloramphenicol and 150 µM acetosyringone.

The product accumulation in a cultivation can be described as:

with the amount of product P, the cell count X and the specific production rate q_P.

RFU is commensurate to the concentration of mRFP (P) and the OD₆₀₀ is commensurate to the cell count (X) ([http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Canton and Labno, 2004]):

With these assumptions it is possible to calculate the specific production rate of mRFP q_P in the third step: the specific production rate for every sample of a cultivation is calculated by the derivation of the exponential fit line which describes the accumulation of product in the culture (dRFU/dt) and the measured OD₆₀₀ data:

The specific production rates q_P of all samples of all cultivations made with a specific inductor concentration c are averaged and normalized against the specific production rate of the uninduced system q_P,0:

This normalized specific production rate we calculated is commensurate to relative promotor units (RPU) which is commensurate to PoPS (polymerase per seconds) ([http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Canton and Labno, 2004]; [http://partsregistry.org/Part:BBa_J23101:Experience Pasotti et al., 2009]):

Plasmid conformation analysis

A plasmid conformation analysis for the BioBrick <partinfo>K389016</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. 5 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. 5: Chromatogram of the CGE of the BioBrick <partinfo>K389016</partinfo> in <partinfo>pSB1C3</partinfo> performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] (Bielefeld).

Different possible inducers

A list of tested possible inducers for a VirA/G signaling system is shown in tab. 4. These inducers where tested as a mix. The specific production rate of mRFP q_P measured as described above for the mix did not significantly differ from the synthesis rate of the uninduced system (t-Test, p < 0.005). So none of the possible inducers listet in tab. 4 induce the VirA/G signaling system significantly in the measured concentration range. In tab. 4 the chemical structures of the testet possible inducers are shown, too. Acetosyringone is also in tab. 4 although it was not testet in the inducer mix to show the chemical similarity of the tested possible inducers to the natural inducer of the VirA/G signaling system.

For more information about the possible inducers click here.

Table 4: Tested possible inducers for a VirA/G signaling system and acetosyringone with concentrations and chemical structure that were tested.

Inducer	Concentration / µM	Chemical structure
Capsaicin	200
Dopamine	200
Homovanillic acid	200
3-Methoxytyramine	200
Acetosyringone	200

References

• Canton B and Labno A (2004) [http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Data processing of Part BBa_F2620].

• 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.

• Pasotti L, Zucca S, Del Fabbro E (2009) Characterization experiment on BBa_J23100, BBa_J23101, BBa_J23118, http://partsregistry.org/Part:BBa_J23101:Experience.

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