Part:BBa_K525562

Fusion protein of NADP+ Oxidoreductase and BisdA and BisdB with middle strong promoter,RBS

Fusion protein of ferredoxin-NADP+ oxidoreductase, BisdA and BisdB; RFC 25 (Freiburg BioBrick assembly standard) for characterization of intra- and extracellular BPA degradation.

Usage and Biology

Expressing this BioBrick in E. coli enables the bacterium to degrade the endocrine disruptor bisphenol A (BPA).

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_K525562.

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	87.1 kDa
	Theoretical pI	5.26
	High absorbtion	450 nm (due to CYP)
Degradation of BPA	Completely degradation of 0.53 mM BPA	> 36 h
	Maximal specific BPA degradation rate	1.32 10-10 mM cell-1

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1411
  Illegal BamHI site found at 2149
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 65
  Illegal AgeI site found at 2402
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 746
  

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 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^-1 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:

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 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_K525499, BBa_K123000 and BBa_K123001) 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

(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

(2)

with the substrate concentration S, the Monod constant K_S and the maximal specific growth rate µ_max ([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

(3)

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

(4)

The specific BPA degradation rate per cell q_D 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:

(5)

with the maximal specific BPA degradation rate per cell q_D,max and the constant K_D.

Fig. 2 shows a comparison between modelled and measured data for cultivations with BBa_K525562 in E. coli KRX. In Tab. 2 the parameters for the model are given, obtained by curve fitting the model to the data.

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 BBa_K525562. The BioBrick BBa_K525562 (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.

Tab. 2: Parameters of the model.

Parameter	BBa_K525562
X0	0.115 108 mL-1
µmax	1.730 h-1
KS,1	13.87 AU-1
S1,0	3.003 AU
qS,1	0.240 AU 10-8 cell-1
BPA0	0.45 mM
qD,max	1.32 10-10 mM cell-1
KD	0.121 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 micrOTOF_Q
• 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>