Team:Bielefeld-Germany/Results/Characterization/K389016

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=Characterization of <partinfo>K389016</partinfo>=

On this page the experiments and results that lead to the <partinfo>K389016</partinfo> characterization data presented on our [[Team:Bielefeld-Germany/Results/Characterization | 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 ([[Team:Bielefeld-Germany/Project/Protocols#Measuring of 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 growthrates (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::K389016 plasmid growths nearly linear. 


[[Image:K389016growth.jpg|600px|thumb|center|'''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<sup>-1</sup> chloramphenicol.''']]


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

[[Image:Bielefeld_Specific_growth_rate.jpg|175px|center]] <div align="right">(1)</div>


[[Image:Bielefeld_Doubling_time.jpg|175px|center]] <div align="right">(2)</div>


<center>Table 1: Averaged specific growth rates and doubling times for cultivations of  ''E. coli'' DB3.1 without plasmid and carrying <partinfo>K389016</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol.

{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"
|-
!style="border-style: solid; border-width: 1px"| ''E. coli'' DB3.1
!style="border-style: solid; border-width: 1px"| µ / h<sup>-1</sup>
!style="border-style: solid; border-width: 1px"| t<sub>d</sub> / h
|-
|style="border-style: solid; border-width: 1px"| without plasmid
|style="border-style: solid; border-width: 1px"| 0.35
|style="border-style: solid; border-width: 1px"| 1.98
|-
|style="border-style: solid; border-width: 1px"| carrying K389016
|style="border-style: solid; border-width: 1px"| 0.27
|style="border-style: solid; border-width: 1px"| 2.57
|-
|style="border-style: solid; border-width: 1px"| carrying K389016 with 150 µM acetosyringone
|style="border-style: solid; border-width: 1px"| 0.25
|style="border-style: solid; border-width: 1px"| 2.77
|-
|style="border-style: solid; border-width: 1px"| carrying K389016 with 1000 µM acetosyringone
|style="border-style: solid; border-width: 1px"| 0.23
|style="border-style: solid; border-width: 1px"| 3.01
|}

</center>


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 for BBa_K389016 | data analysis]] and [[#Transfer function of BBa_K389016 | transfer function]] is described below. 


[[Image:K389016induction.jpg|600px|thumb|center|'''Fig. 2: Induction Curves for ''E. coli'' DB3.1 carrying <partinfo>K389016</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> 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 [[Team:Bielefeld-Germany/Results/Characterization/K389016#Data analysis for BBa_K389016 | described below]]. Nelson ''et al.'' (2002) suggest 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


[[Image:Bielefeld_Doseresponse_fit.jpg|175px|center]] <div align="right">(3)</div>


with the Hill coefficient p, the bottom asymptote A1, the top asymptote A2 and the switch point log(x<sub>0</sub>). Figure 3 shows the measured normalized specific production rates q<sub>P,n</sub> (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 R<sup>2</sup> = 0.99. 


[[Image:Bielefeld_Final_RFP_fit.jpg|600px|thumb|center|'''Fig. 3: Transfer function for the part <partinfo>K389016</partinfo> (R<sup>2</sup> = 0.99).''']]


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


<center>Table 2: Data from the transfer function for the part <partinfo>K389016</partinfo>.

{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"
|-
!style="border-style: solid; border-width: 1px"| Parameter
!style="border-style: solid; border-width: 1px"| Value
|-
|style="border-style: solid; border-width: 1px"| Hill coefficient
|style="border-style: solid; border-width: 1px"| 1.673
|-
|style="border-style: solid; border-width: 1px"| [http://partsregistry.org/Switch_Point Switch point]
|style="border-style: solid; border-width: 1px"| 26.5 µM
|-
|style="border-style: solid; border-width: 1px"| Top asymptote
|style="border-style: solid; border-width: 1px"| 2.62
|}

</center>


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: 


[[Image:Bielefeld_RFU_corrected.jpg|250px|center]] <div align="right">(4)</div>


In the second step the RFU<sub>corrected</sub> of every sample is plotted against the cultivation time it was drawn. The data is fitted by an exponential fit of the following style: 


[[Image:Bielefeld_Expfit.jpg|100px|center]] <div align="right">(5)</div>


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


[[Image:Bielefeld_ExpFit_auf_RFU.jpg|500px|thumb|center|'''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<sup>-1</sup> chloramphenicol and 150 µM acetosyringone. ''']]


The product accumulation in a cultivation can be described as: 


[[Image:Bielefeld_Produktbildung.jpg|100px|center]] <div align="right">(6)</div>


with the amount of product P, the cell count X and the specific production rate q<sub>P</sub>. 

RFU is commensurate to the concentration of mRFP (P) and the OD<sub>600</sub> is commensurate to the cell count (X) ([[http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis | Canton and Labno, 2004]]): 


[[Image:Bielefeld_RFUpropP.jpg|100px|center]] <div align="right">(7)</div>

[[Image:Bielefeld_ODpropX.jpg|80px|center]] <div align="right">(8)</div>


With these assumptions it is possible to calculate the specific production rate of mRFP q<sub>P</sub> 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 OD<sub>600</sub> data: 


[[Image:Bielefeld_specific_production_rate.jpg|150px|center]] <div align="right">(9)</div>


The specific production rates q<sub>P</sub> 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<sub>P,0</sub>: 


[[Image:Bielefeld_QPN.jpg|100px|center]] <div align="right">(10)</div>


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]): 


[[Image:Bielefeld_QPNRPUPoPS.jpg|150px|center]] <div align="right">(11)</div>

==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]).  


[[Image:Bielefeld_CGE_K389016_2.jpg|600px|thumb|center|'''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).''']]



<center> Table 3: Data from the CGE of the BioBrick <partinfo>K389016</partinfo> in <partinfo>pSB1C3</partinfo> performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] (Bielefeld).

{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"
|-
!style="border-style: solid; border-width: 1px"| Conformation
!style="border-style: solid; border-width: 1px"| Ratio / %
|-
|style="border-style: solid; border-width: 1px"| ccc monomer
|style="border-style: solid; border-width: 1px"| 91.2
|-
|style="border-style: solid; border-width: 1px"| ccc dimer
|style="border-style: solid; border-width: 1px"| 3.2
|-
|style="border-style: solid; border-width: 1px"| oc
|style="border-style: solid; border-width: 1px"| 5.6
|}

</center>

==Different possible inducers==
A list of testet possible inducers for a VirA/G signaling system is shown in tab. 4. These inducers where testet as a mix. The specific production rate of mRFP q<sub>P</sub> measured as described [[Team:Bielefeld-Germany/Results/Characterization/K389016#Data analysis for BBa_K389016 | 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 testet possible inducers to the natural inducer of the VirA/G signaling system. 

[[Team:Bielefeld-Germany/Project/Outlook | For more information about the possible inducers click here. ]]



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

{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"
|-
!style="border-style: solid; border-width: 1px"| Inducer
!style="border-style: solid; border-width: 1px"| Concentration / µM
!style="border-style: solid; border-width: 1px"| Chemical structure
|-
|style="border-style: solid; border-width: 1px"| '''Capsaicin'''
|style="border-style: solid; border-width: 1px"| 200
|style="border-style: solid; border-width: 1px"|<html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=1548942" width="200px" /></html>
|-
|style="border-style: solid; border-width: 1px"| '''Dopamine'''
|style="border-style: solid; border-width: 1px"| 200
|style="border-style: solid; border-width: 1px"| <html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=681" width="200"></html>
|-
|style="border-style: solid; border-width: 1px"| '''Homovanillic acid'''
|style="border-style: solid; border-width: 1px"| 200
|style="border-style: solid; border-width: 1px"| <html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=1738" width="200"></html>
|-
|style="border-style: solid; border-width: 1px"| '''3-Methoxytyramine'''
|style="border-style: solid; border-width: 1px"| 200
|style="border-style: solid; border-width: 1px"| <html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=11957621" width="200"></html>
|-
|style="border-style: solid; border-width: 1px"| '''Acetosyringone'''
|style="border-style: solid; border-width: 1px"| 200
|style="border-style: solid; border-width: 1px"| <html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=17198" width="200px" /></html>
|}

</center>

=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]