Composite

Part:BBa_K525551

Designed by: Anna Drong   Group: iGEM11_Bielefeld-Germany   (2011-09-11)

NADP+ Oxidoreductase and polycistronic BisdA and BisdB

Ferredoxin-NADP+ oxidoreductase and polycistronic bisdA and bisdB for characterization of intracellular bisphenol A degradation with E. coli.

Usage and Biology

Expressing this BioBrick in E. coli enables the bacterium to degrade the endocrine disruptor bisphenol A (BPA). The fusion protein works better than a polycistronic expression of the two BioBricks bisdA (BBa_K123000) and bisdB (BBa_K123001).

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+ oxidoreductase (FNR) Sasaki05a. The three gene products act together to reduce BPA while oxidizing NADH + H+. 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+ oxidoreductase consuming NADH + H+ so the BPA degradation can continue Sasaki05a. 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):

Fig. 1: Animation of proposed reaction mechanism of bisphenol A hydroxylation by the involved enzymes FNR (BBa_K525499), Fd (BisdA, BBa_K123000) and CYP (BisdB, BBa_K123001)


Important parameters

Tab. 1: Important parameters of BBa_K525551 under control of constitutive promoter BBa_J23110.

Experiment Characteristic Result
Expression in E. coli Compatibility E. coli KRX, TOP10, MACH1, BL21(DE3)
Expression Constitutive
Optimal temperature 30 °C
BPA working concentration 120 mg L-1 (0.53 mM)
Purification Molecular weight 28.0, 11.2 and 48.3 kDa
Theoretical pI 6.17, 4.31 and 5.27
High absorbtion 450 nm (due to CYP)
Degradation of BPA Completely degradation of 0.53 mM BPA > 36 h
Specific BPA degradation rate 5.67 10-11 mM cell-1


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1426
    Illegal BamHI site found at 2164
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 4
    Illegal NgoMIV site found at 789
    Illegal NgoMIV site found at 1145
    Illegal AgeI site found at 751
    Illegal AgeI site found at 1107
    Illegal AgeI site found at 2417
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 685


Bisphenol A degradation with E. coli

The bisphenol A degradation with the BioBricks BBa_K123000, BBa_K123001 and BBa_K525499 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 (BBa_J23110). 30 °C is in addition the cultivation temperature of S. bisphenolicum AO1. With this promoter upstream of a polycistronic bisdAB and FNR gene we were able to degrade a substantial amount (~80%) of BPA in about 36 h starting at 120 mg L-1 BPA . This data is shown in the following figure:

Figure 2: BPA degradation by E. coli KRX carrying the polycistronic bisdAB and FNR genes behind the medium strong constitutive promoter BBa_J23110 with RBS BBa_B0034. 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.


Modelling of intracellular bisphenol A degradation

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.

To model the BPA degradation by E. coli carrying BioBricks for BPA degradation (BBa_K123000 and BBa_K123001) the cell growth has to be described first to calculate a specific BPA degradation rate per cell. The observed growth of E. coli on (our) LB medium was [http://en.wikipedia.org/wiki/Diauxie diauxic] with two different growth phases. Cell growth is a [http://en.wikipedia.org/wiki/First_order_kinetics#First-order_reactions first-order reaction] and is mathematically described as


Bielefeld-Germany2011-growth.png
(1)


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


Bielefeld-Germany2011-growthrate.png
(2)


with the substrate concentration S, the Monod constant KS and the maximal specific growth rate µmax ([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. Due to the diauxic growth two different substrates with different Monod constants and consumption rates are necessary to model the cell growth. The amount of a substrate S can be modelled as follows


Bielefeld-Germany2011-substrate.png
(3)


with the specific substrate consumption rate per cell qS. The whole model for the diauxic growth of E. coli on LB medium with two not measurable (imaginary) substrates looks like:


Bielefeld-Germany2011-model-ecoligrowth.png
(4)


The specific BPA degradation rate per cell qD is modelled with an equation like eq. (3). In the beginning of the cultivations, when E. coli growths on the "good" imaginary substrate S1, no BPA degradation is observed. When this substrate is consumed, the BPA degradation starts. The model for this diauxic behavior is as follows:


Bielefeld-Germany2011-model-ecoliBPA.png
(5)


Fig. 3 shows a comparison between modelled and measured data for cultivations with BPA degrading E. coli. In Tab. 2 the parameters for the model are given, obtained by curve fitting the model to the data.


Figure 3: 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 BBa_K525552. The BioBrick BBa_K525552 (polycistronic fnr : bisdA : bisdB genes 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.


Tab. 2: Parameters of the model.

Parameter BBa_K525552
X0 0.109 108 mL-1
µmax 0.963 h-1
KS,1 5.35 AU-1
KS,2 82.6 AU-1
S1,0 4.679 AU
qS,1 0.883 AU 10-8 cell-1
S2,0 3.873 AU
qS,2 0.082 AU 10-8 cell-1
BPA0 0.41 mM
qD 5.67 10-11 mM cell-1


Methods

Cultivations


  • 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]
  • Medium: LB medium supplemented with 100 mg L-1 Ampicillin and 120 mg L-1 bisphenol A (Sigma, 97 %)
    • BPA is thermally stable -> you can autoclave it together with the medium
  • 100 mL culture in 300 mL shaking flask without baffles (Schott) with silicon plugs
  • Cultivation temperature: 24 °C, 30 °C or 37 °C, tempered with Infors AG AQUATRON at 120 rpm
  • for characterizations: automatic sampling every three hours with Gilson fraction controller F2XX cooled (< 4 °C) with Julabo F10 water bath
    • the characterization experiment setup is shown on the picture on the right


Extraction with ethylacetate

  • mix 100 µL culture supernatant with 100 µL internal standard bisphenol F (Alfa Aesar, 98 %) , 100 µg L-1)
  • add 200 µL ethylacetate (VWR, HPLC grade) for extraction
  • vortex (30 s)
  • centrifuge for phase separation (5 min, 5000 g)
  • take a bit from upper phase and put it in a clean eppi
  • SpeedVac at 40 °C to remove ethlyacetate
  • solve remaining BPA in water (HPLC grade), vortex (30 s)
  • solubility of BPA in water only 300 mg L-1
    • for LC-MS analysis of BPA, 300 mg BPA L-1 is rather too much
    • if you want to detect or expect higher concentrations of BPA, solve it in an acetonitrile-water-mix


HPLC method

  • C18 reverse phase column
  • Isocratic method: 45 % Acetonitrile
  • Flow = 0.6 mL min-1
  • UV-detection at 227 nm
  • Internal standard: 100 mg L-1 bisphenol F (BPF)
  • Column:
    • Eurospher II 100-5 C18p by [http://www.knauer.net/ Knauer]
    • Dimensions: 150 x 4.6 mm with precolumn
    • Particle size: 5 µm
    • Pore size: 100 Å
    • Material: silica gel
  • Software:
    • Clarity (Version 3.0.5.505) by [http://www.dataapex.com/ Data Apex]
  • Autosampler:
    • Midas by [http://www.spark.nl/ Spark Holland]
    • Tray cooling: 10 °C
  • Pump:
    • L-6200A Intelligent Pump by [http://www.hitachi.com/ Hitachi]
  • UV-Detector:
    • Series 1050 by [http://www.hp.com/ Hewlett Packard]


LC-ESI-qTOF-MS(-MS)

HPLC method

  • 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])
    • dimension: 50 x 2 mm
    • Pore size: 175 Å
    • Particle size: 1.8 µm
  • Flow: 0.4 mL min-1
  • Column temperature: 30 °C
  • Gradient:
    • 0 - 1.05 min: 45 % acetonitrile
    • 2.55 min: 95 % acetonitrile
    • 6.00 min: 95 % acetonitrile
    • 6.15 min: 45 % acetonitrile
    • 12.00 min: 45 % acetonitrile
  • VWR Hitachi LaChrom ULTRA HPLC equipment
  • Software: HyStar 3.2, HyStarPP, mircrOTOF Control

Ionization method

  • Using Bruker Daltonics micrOTOFQ
  • ESI in negative mode
  • Mass range: 50 - 1500 m/z
  • End plate offset: - 500 V, 107 nA
  • Capillary: 2500 V, 4 nA
  • Nebulizer: 3 bar
  • Dry gas: 8 L min-1
  • Quadrupole
    • Ion energy: 5 eV
    • Low mass: 100 m/z
  • Collision energy: 10 eV
  • Collision RF: 150 Vpp
  • Transfer time: 70 µs
  • Pre puls storage: 7 µs

MS-MS

  • Isolated mass: 243.1 +/- 0.1
  • Collision energy: 30 eV


References

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].

<biblio>

  1. Sasaki pmid=18492046
  2. Sasaki05a pmid=16332782

</biblio>

[edit]
Categories
//chassis/prokaryote/ecoli
//function/degradation/bisphenol
Parameters
n/aNADP+ Oxidoreduktase and polycistronic BisdA and BisdB