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| − | __NOTOC__
| + | 0605 http://manualss7iy.pp.ua/bnwrwn1.html http://manualss7iy.pp.ua/xpem2.html http://documentsoex.pp.ua/tsq1.html http://documentsoex.pp.ua/kvxtcu2.html http://instructions9rxg.pp.ua/iiejn2.html http://instructions9rxg.pp.ua/lddpim4.html http://instruktsiya721.pp.ua/olkkpx2.html http://instruktsiya721.pp.ua/vrol1.html http://rukovodstnnr.pp.ua/mwcfk2.html http://rukovodstnnr.pp.ua/xuykyh3.html http://rukovodstnnr.pp.ua/juqpxs2.html http://rukovodstnnr.pp.ua/wil3.html http://instruktsiya5nd.pp.ua/mvcgo2.html http://instruktsiya5nd.pp.ua/ytjmtg3.html http://rukovodstvqk.pp.ua/tqu2.html http://rukovodstvqk.pp.ua/kjanan3.html |
| − | <partinfo>BBa_K525562 short</partinfo>
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| − | Fusion protein of ferredoxin-NADP<sup>+</sup> oxidoreductase, BisdA and BisdB; RFC 25 (Freiburg BioBrick assembly standard); used for characterization of intracellular BPA degradation.
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| − | ===Usage and Biology===
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| − | Expressing this BioBrick in ''E. coli'' enables the bacterium to degrade the endocrine disruptor bisphenol A (BPA).
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| − | BPA is mainly hydroxylated into the products 1,2-Bis(4-hydroxyphenyl)-2-propanol and 2,2-Bis(4-hydroxyphenyl)-1-propanol. In ''S. bisphenolicum'' AO1, a total of three genes are responsible for this BPA hydroxylation: a cytochrome P450 (CYP, ''bisdB''), a ferredoxin (Fd, ''bisdA'') and a ferredoxin-NAD<sup>+</sup> oxidoreductase (FNR) <cite>Sasaki05a</cite>. The three gene products act together to reduce BPA while oxidizing NADH + H<sup>+</sup>. The cytochrome P450 (BisdB) reduces the BPA and is oxidized during this reaction. BisdB in its oxidized status is reduced by the ferredoxin (BisdA) so it can reduce BPA again. The oxidized BisdA is reduced by a ferredoxin-NAD<sup>+</sup> oxidoreductase consuming NADH + H<sup>+</sup> so the BPA degradation can continue <cite>Sasaki05a</cite>. This electron transport chain between the three enzymes involved in BPA degradation and the BioBricks needed to enable this reaction ''in vivo'' and ''in vitro'' are shown in the following figure (please have some patience, it's an animated .gif file):
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| − | [[Image:Bielefeld-Germany2011-BPAdegrad2.gif|center|700px|thumb|'''Fig. 1: Animation of proposed reaction mechanism of bisphenol A hydroxylation by the involved enzymes FNR (<partinfo>K525499</partinfo>), Fd (BisdA, <partinfo>K123000</partinfo>) and CYP (BisdB, <partinfo>K123001</partinfo>)''']]
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| − | ===Important parameters===
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| − | <center>
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| − | '''Tab. 1: Important parameters of <partinfo>K525562</partinfo>.'''
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| − | {|{{Table}}
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| − | !Experiment
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| − | !Characteristic
| + | |
| − | !Result
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| − | |-
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| − | |rowspan="4"|[[Part:BBa_K525562#Bisphenol_A_degradation_with_E._coli | Expression in ''E. coli'']]
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| − | |Compatibility
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| − | |''E. coli'' KRX, TOP10, MACH1, BL21(DE3)
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| − | |-
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| − | |Expression
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| − | |Constitutive
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| − | |-
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| − | |Optimal temperature
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| − | |30 °C
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| − | |-
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| − | |BPA working concentration
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| − | |120 mg L<sup>-1</sup> (0.53 mM)
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| − | |-
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| − | |rowspan="3"|Purification
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| − | |Molecular weight
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| − | |87.1 kDa
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| − | |-
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| − | |Theoretical pI
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| − | |5.26
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| − | |-
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| − | |High absorbtion
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| − | |450 nm (due to CYP)
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| − | |-
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| − | |rowspan="2"|Degradation of BPA
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| − | |[[Part:BBa_K525562#Bisphenol_A_degradation_with_E._coli | Completely degradation of 0.53 mM BPA]]
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| − | |> 36 h
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| − | |-
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| − | |[[Part:BBa_K525562#Modelling_of_intracellular_bisphenol_A_degradation | Maximal specific BPA degradation rate]]
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| − | |1.32 10<sup>-10</sup> mM cell<sup>-1</sup>
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| − | |-
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| − | |}
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| − | </center>
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| − | <!-- Add more about the biology of this part here
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| − | ===Usage and Biology===
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| − | <!-- -->
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| − | <span class='h3bb'>Sequence and Features</span>
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| − | <partinfo>BBa_K525562 SequenceAndFeatures</partinfo>
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| − | <!-- Uncomment this to enable Functional Parameter display
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| − | ===Functional Parameters===
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| − | <partinfo>BBa_K525562 parameters</partinfo>
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| − | <!-- -->
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| − | ===Bisphenol A degradation with ''E. coli''===
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| − | The bisphenol A degradation with the BioBricks <partinfo>K123000</partinfo>, <partinfo>K123001</partinfo> and <partinfo>K525499</partinfo> works in ''E. coli'' KRX in general. Because [http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2008.03843.x/full Sasaki ''et al.'' (2008)] reported problems with protein folding in ''E. coli'' which seem to avoid a complete BPA degradation, we did not cultivate at 37 °C and we did not use the strong T7 promoter as [http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2008.03843.x/full Sasaki ''et al.'' (2008)] did for expressing these BioBricks but we cultivated at 30 °C and we used a medium strong constitutive promoter (<partinfo>J23110</partinfo>). 30 °C is in addition the cultivation temperature of ''S. bisphenolicum'' AO1. With this promoter upstream of the gene expressing the bisdA | bisdB |FNR fusion protein we were able to degrade a substantial amount (~85%) of BPA in about 36 h starting at 120 mg L<sup>-1</sup> BPA . This data is shown in the following figure and indicates that the fusion protein of all three enzymes that are involved in the degradation of BPA is functional:
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| − | [[Image:Bielefeld_2011_562_BPA_Degradation.png|650px|center|thumb| '''Figure 2: BPA degradation by ''E. coli'' KRX carrying genes for the fusion protein of BisdA, BisdB and FNR behind the medium strong constitutive promoter <partinfo>J23110</partinfo> with RBS <partinfo>B0034</partinfo>. [[Part:BBa_K525517#Methods | Cultivations]] were carried out at 30 °C in LB + Amp + BPA medium for 36 h with automatic sampling every three hours in 300 mL shaking flasks without baffles with silicon plugs. Two biological replicates were analyzed.''']]
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| − | ===Modelling of intracellular bisphenol A degradation===
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| − | The modelling was done with the software [http://www.berkeleymadonna.com/ Berkeley Madonna] using the [http://en.wikipedia.org/wiki/Runge–Kutta_methods#Common_fourth-order_Runge.E2.80.93Kutta_method common fourth-order Runge-Kutta] method to solve the equations. The model was fitted to the measured data shown above by the function "curve fit" in Berkeley Madonna to calculate the parameters, constants ''etc''.
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| − | To model the BPA degradation by ''E. coli'' carrying BioBricks for BPA degradation (<partinfo>K525499</partinfo>, <partinfo>K123000</partinfo> and <partinfo>K123001</partinfo>) the cell growth has to be described first to calculate a specific BPA degradation rate per cell. Cell growth is a [http://en.wikipedia.org/wiki/First_order_kinetics#First-order_reactions first-order reaction] and is mathematically described as
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| − | [[Image:Bielefeld-Germany2011-growth.png|center|75px]] <div align="right">(1)</div>
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| − | with the '''specific growth rate''' '''µ''' and the '''cell count''' '''X'''. The specific growth rate is dependent on the concentration of the growth limiting substrate (e.g. glucose) and can be described as
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| − | [[Image:Bielefeld-Germany2011-growthrate.png|center|110px]] <div align="right">(2)</div>
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| − | with the '''substrate concentration''' '''S''', the '''Monod constant''' '''K<sub>S</sub>''' and the '''maximal specific growth rate''' '''µ<sub>max</sub>''' ([http://www.annualreviews.org/doi/abs/10.1146/annurev.mi.03.100149.002103 Monod, 1949]). Because LB medium is a complex medium we cannot measure the substrate concentration so we have to assume an imaginary substrate concentration. The amount of a substrate S can be modelled as follows
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| − | [[Image:Bielefeld-Germany2011-substrate.png|center|75px]] <div align="right">(3)</div>
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| − | with the '''specific substrate consumption rate per cell''' '''q<sub>S</sub>'''. The whole model for the diauxic growth of ''E. coli'' on LB medium with two not measurable (imaginary) substrates looks like:
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| − | [[Image:IGEM-Bielefeld2011-ecoligrowthsimple.jpg|center|220px]] <div align="right">(4)</div>
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| − | The '''specific BPA degradation rate per cell''' '''q<sub>D</sub>''' is modelled with an equation like eq. (2) because it is dependent from the BPA concentration. The BPA degradation starts in the stationary growth phase when the imaginary substrate is consumed. The model for this behavior is as follows:
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| − | [[Image:IGEM-Bielefeld2011-BPAdegradcomp.jpg|center|220px]] <div align="right">(5)</div>
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| − | with the '''maximal specific BPA degradation rate per cell q<sub>D,max</sub>''' and the '''constant K<sub>D</sub>'''.
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| − | Fig. 2 shows a comparison between modelled and measured data for cultivations with <partinfo>K525562</partinfo> in ''E. coli'' KRX. In Tab. 2 the parameters for the model are given, obtained by curve fitting the model to the data.
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| − | [[Image:IGEM-Bielefeld2011-ModellK525562.png|center|600px|thumb|'''Figure 2: Comparison between modelled (lines) and measured (dots) data for [http://2011.igem.org/Team:Bielefeld-Germany/Protocols/Downstream-processing#Expression_of_bisphenol_A_degrading_BioBricks_in_E._coli cultivations] of ''E. coli'' KRX carrying BPA degrading BioBrick <partinfo>K525562</partinfo>. The BioBrick <partinfo>K525562</partinfo> (fusion protein between FNR, BisdA and BisdB behind medium strong promoter) was cultivated four times in ''E. coli'' KRX in LB + Amp + BPA medium at 30 °C, using 300 mL shaking flasks without baffles with silicon plugs. The BPA concentration (closed dots) and the cell density (open dots) is plotted against the cultivation time. ''']]
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| − | <center>
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| − | '''Tab. 2: Parameters of the model. '''
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| − | {| class="wikitable" style="text-align:left"
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| − | ! Parameter !! style="padding-left:5px;" |<partinfo>K525562</partinfo>
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| − | | X<sub>0</sub> || style="padding-left:5px;" |0.115 10<sup>8</sup> mL<sup>-1</sup>
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| − | | µ<sub>max</sub> || style="padding-left:5px;" |1.730 h<sup>-1</sup>
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| − | | K<sub>S,1</sub> || style="padding-left:5px;" |13.87 AU<sup>-1</sup>
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| − | |-
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| − | | S<sub>1,0</sub> || style="padding-left:5px;" |3.003 AU
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| − | |-
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| − | | q<sub>S,1</sub> || style="padding-left:5px;" |0.240 AU 10<sup>-8</sup> cell<sup>-1</sup>
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| − | |-
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| − | | BPA<sub>0</sub> || style="padding-left:5px;" |0.45 mM
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| − | | q<sub>D,max</sub> || style="padding-left:5px;" |1.32 10<sup>-10</sup> mM cell<sup>-1</sup>
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| − | | K<sub>D</sub> || style="padding-left:5px;" |0.121 mM cell<sup>-1</sup>
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| − | |-
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| − | |}
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| − | </center>
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| − | ===Methods===
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| − | '''Cultivations'''
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| − | <html>
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| − | <div style="float:right; width:400px; text-align:center;">
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| − | <img src="https://static.igem.org/mediawiki/2011/thumb/4/40/Bielefeld-Germany2011-automaticsampling.jpg/800px-Bielefeld-Germany2011-automaticsampling.jpg" width="90%" height="90%" />
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| − | <br/>
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| − | </div>
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| − | </html>
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| − | * Chassis: Promega's [http://www.promega.com/products/cloning-and-dna-markers/cloning-tools-and-competent-cells/bacterial-strains-and-competent-cells/single-step-_krx_-competent-cells/ ''E. coli'' KRX]
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| − | * Medium: LB medium supplemented with 100 mg L<sup>-1</sup> Ampicillin and 120 mg L<sup>-1</sup> bisphenol A (Sigma, 97 %)
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| − | ** BPA is thermally stable -> you can autoclave it together with the medium
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| − | * 100 mL culture in 300 mL shaking flask without baffles (Schott) with silicon plugs
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| − | * Cultivation temperature: 24 °C, 30 °C or 37 °C, tempered with Infors AG AQUATRON at 120 rpm
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| − | * for characterizations: automatic sampling every three hours with Gilson fraction controller F2XX cooled (< 4 °C) with Julabo F10 water bath
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| − | ** the characterization experiment setup is shown on the picture on the right
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| − | '''Extraction with ethylacetate'''
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| − | * mix 100 µL culture supernatant with 100 µL internal standard bisphenol F (Alfa Aesar, 98 %) , 100 µg L<sup>-1</sup>)
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| − | * add 200 µL ethylacetate (VWR, HPLC grade) for extraction
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| − | * vortex (30 s)
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| − | * centrifuge for phase separation (5 min, 5000 g)
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| − | * take a bit from upper phase and put it in a clean eppi
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| − | * SpeedVac at 40 °C to remove ethlyacetate
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| − | * solve remaining BPA in water (HPLC grade), vortex (30 s)
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| − | * solubility of BPA in water only 300 mg L<sup>-1</sup>
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| − | ** for LC-MS analysis of BPA, 300 mg BPA L<sup>-1</sup> is rather too much
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| − | ** if you want to detect or expect higher concentrations of BPA, solve it in an acetonitrile-water-mix
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| − | '''HPLC method'''
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| − | * C18 reverse phase column
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| − | * Isocratic method: 45 % Acetonitrile
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| − | * Flow = 0.6 mL min<sup>-1</sup>
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| − | * UV-detection at 227 nm
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| − | * Internal standard: 100 mg L<sup>-1</sup> bisphenol F (BPF)
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| − | * Column:
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| − | ** Eurospher II 100-5 C18p by [http://www.knauer.net/ Knauer]
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| − | ** Dimensions: 150 x 4.6 mm with precolumn
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| − | ** Particle size: 5 µm
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| − | ** Pore size: 100 Å
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| − | ** Material: silica gel
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| − | * Software:
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| − | ** Clarity (Version 3.0.5.505) by [http://www.dataapex.com/ Data Apex]
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| − | * Autosampler:
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| − | ** Midas by [http://www.spark.nl/ Spark Holland]
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| − | ** Tray cooling: 10 °C
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| − | * Pump:
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| − | ** L-6200A Intelligent Pump by [http://www.hitachi.com/ Hitachi]
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| − | * UV-Detector:
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| − | ** Series 1050 by [http://www.hp.com/ Hewlett Packard]
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| − | '''LC-ESI-qTOF-MS(-MS)'''
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| − | '''HPLC method'''
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| − | * Column: C18 reverse phase column (Knauer [http://beta.knauer.net/products/column-detail-view/productdetail/vertex_plus_column_50_x_2_mm_blueorchid_175_18_c18-1.html Blue Orchid])
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| − | ** dimension: 50 x 2 mm
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| − | ** Pore size: 175 Å
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| − | ** Particle size: 1.8 µm
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| − | * Flow: 0.4 mL min<sup>-1</sup>
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| − | * Column temperature: 30 °C
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| − | * Gradient:
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| − | ** 0 - 1.05 min: 45 % acetonitrile
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| − | ** 2.55 min: 95 % acetonitrile
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| − | ** 6.00 min: 95 % acetonitrile
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| − | ** 6.15 min: 45 % acetonitrile
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| − | ** 12.00 min: 45 % acetonitrile
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| − | * VWR Hitachi LaChrom ULTRA HPLC equipment
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| − | * Software: HyStar 3.2, HyStarPP, mircrOTOF Control
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| − | '''Ionization method'''
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| − | * Using Bruker Daltonics micrOTOF<sub>Q</sub>
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| − | * ESI in negative mode
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| − | * Mass range: 50 - 1500 m/z
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| − | * End plate offset: - 500 V, 107 nA
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| − | * Capillary: 2500 V, 4 nA
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| − | * Nebulizer: 3 bar
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| − | * Dry gas: 8 L min<sup>-1</sup>
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| − | * Quadrupole
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| − | ** Ion energy: 5 eV
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| − | ** Low mass: 100 m/z
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| − | * Collision energy: 10 eV
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| − | * Collision RF: 150 Vpp
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| − | * Transfer time: 70 µs
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| − | * Pre puls storage: 7 µs
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| − | '''MS-MS'''
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| − | * Isolated mass: 243.1 +/- 0.1
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| − | * Collision energy: 30 eV
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| − | ===References===
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| − | Monod J (1949) The growth of bacterial cultures, ''Annu Rev Microbiol'' [http://www.annualreviews.org/doi/abs/10.1146/annurev.mi.03.100149.002103 3:371-394].
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| − | <biblio>
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| − | #Sasaki pmid=18492046
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| − | #Sasaki05a pmid=16332782
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| − | </biblio>
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0605 http://manualss7iy.pp.ua/bnwrwn1.html http://manualss7iy.pp.ua/xpem2.html http://documentsoex.pp.ua/tsq1.html http://documentsoex.pp.ua/kvxtcu2.html http://instructions9rxg.pp.ua/iiejn2.html http://instructions9rxg.pp.ua/lddpim4.html http://instruktsiya721.pp.ua/olkkpx2.html http://instruktsiya721.pp.ua/vrol1.html http://rukovodstnnr.pp.ua/mwcfk2.html http://rukovodstnnr.pp.ua/xuykyh3.html http://rukovodstnnr.pp.ua/juqpxs2.html http://rukovodstnnr.pp.ua/wil3.html http://instruktsiya5nd.pp.ua/mvcgo2.html http://instruktsiya5nd.pp.ua/ytjmtg3.html http://rukovodstvqk.pp.ua/tqu2.html http://rukovodstvqk.pp.ua/kjanan3.html
