Part:BBa_K525517

Fusion protein of BisdA and BisdB behind constitutive promoter

Fusion protein of BisdA and BisdB behind constitutive promoter leading to enhanced bisphenol A degradation compared to polycistronic bisdAB gene.

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_K525517.

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	59.3 kDa
	Theoretical pI	4.99
	High absorbtion	450 nm (due to CYP)
Degradation of BPA	Completely degradation of 0.53 mM BPA	21 - 24 h
	Specific BPA degradation rate	1.29 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 664
  Illegal BamHI site found at 1402
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 65
  Illegal AgeI site found at 1655
  
• 1000
  COMPATIBLE WITH RFC[1000]

Bisphenol A degradation with E. coli

The bisphenol A degradation with the BioBricks BBa_K123000 and BBa_K123001 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 gene we were able to completely degrade 120 mg L^-1 BPA in about 30 - 33 h. By fusing BBa_K123000 and BBa_K123001 together (using Freiburg BioBrick assembly standard) we could improve the BPA degradation of E. coli even further, so 120 mg L^-1 BPA can be degraded in 21 - 24 h. This data is shown in the following figure:

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Figure 2: BPA degradation by E. coli KRX carrying genes for BisdA and BisdB (only bisdA (black), polycistronic bisdAB (red) and fusion protein between BisdA and BisdB (green)) 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 24 h and 36 h, respectively, with automatic sampling every three hours in 300 mL shaking flasks without baffles with silicon plugs. At least three biological replicates were analysed (three for bisdA alone, seven for bisdAB polycistronic and five for the fusion protein).

We also carried out these cultivations at different temperatures and BPA concentrations, but the chosen conditions (30 °C and 120 mg L^-1 BPA) seem to be the best. Higher BPA concentrations have an effect on the growth of E. coli and higher temperature leeds to a worse BPA degradation (probably due to misfolding of the enzymes). Lower temperature also leeds to less BPA degradation (probably due to slower growth, expression and reaction rate at lower temperatures). These data on the effect of the temperature on the BPA degradation is shown in fig. 3.

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Figure 3: BPA degradation by E. coli KRX carrying genes for BisdA and BisdB (polycistronic bisdAB (black) and fusion protein between BisdA and BisdB (striped)) behind the medium strong constitutive promoter BBa_J23110 with RBS BBa_B0034. Cultivations were carried out at different temperatures in LB + Amp + BPA medium (starting concentration 120 mg L^-1 BPA) for 24 h in 300 mL shaking flasks without baffles with silicon plugs. Samples were taken at the end of the cultivation. Three biological replicates were analysed.

As shown by [http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2008.03843.x/full Sasaki et al. (2008)], BisdB expressed in E. coli leeds to hardly no BPA degradation. In our experiments we could not detect the BPA degradation products 1,2-Bis(4-hydroxyphenyl)-2-propanol and 2,2-Bis(4-hydroxyphenyl)-1-propanol in cultivations with E. coli expressing BBa_K123000 or BBa_K123001 alone (neither via UV- nor MS-detection). The BPA degradation products 1,2-Bis(4-hydroxyphenyl)-2-propanol and 2,2-Bis(4-hydroxyphenyl)-1-propanol were identified via MS-MS (m/z: 243 / 225 / 211 / 135) and only occured in cultivations with E. coli expressing BisdA and BisdB together. [http://www.springerlink.com/content/q7864l02734wg32m/ Sasaki et al. (2005)] reported the same MS-MS results for 1,2-Bis(4-hydroxyphenyl)-2-propanol and 2,2-Bis(4-hydroxyphenyl)-1-propanol when degrading BPA with S. bisphenolicum AO1 as we observed in our BPA degradation experiments.

We could also identify the BPA degradation products when working with E. coli TOP10 and MACH1 (data not shown) but because we want to fuse BisdA and BisdB to S-layer proteins which we express in E. coli KRX the characterizations of BBa_K123000 and BBa_K123001 were carried out in this strain.

Specificity of bisphenol A degradation with E. coli

In order to access the specificity of the bisphenol A degradation by the bisdA | bisdB fusion protein we tested how well two similar bisphenols, bisphenol F (BPF) and bisphenol S (BPS), were employed. The structure of those bisphenols differs only in the chemical group linking the two phenols from that of bisphenol A (see Figure 4).

Figure 4: Chemical structure of BPA, BPF and BPS showing the different chemical groups linking the two phenols.

BPF and BPS are used in a broad range of applications that involve the use of polycarbonates or epoxy resins and thus can often be found were BPA is also present. Accordingly their presence is a potential disruptive factor that could lead to a false positive signal with our biosensor. This is especially true for BPS that in some cases is used as a substitute for BPA in baby bottles [http://www.nytimes.com/2011/04/18/business/global/18iht-rbog-plastic-18.html]. Studies concerning the environmental pollution with BPF ([http://www.sciencedirect.com/science/article/pii/S0043135401003670 Fromme et al. (2002)]) and the acute toxicity, mutagenicity and estrogenicity of BPF and BPS ([http://onlinelibrary.wiley.com/doi/10.1002/tox.10035/abstract Chen et al. (2001)] and [http://toxsci.oxfordjournals.org/content/84/2/249 Kitamura et al. (2005)]) indicate their potential harmfulness but further research is needed to fully access their impact on human health.

E. coli KRX carrying genes for the bisdA | bisdB fusion protein behind the medium strong constitutive promoter BBa_J23110 with RBS BBa_B0034 was cultivated at 30 °C for 36 h with LB-Medium containing 120 mg L^-1 BPA, BPF respectively BPS. The BPF and BPS concentration where determined with a HPLC using the same method as with BPA. Figure 5 shows the degradation of the respective bisphenol after 24 h of cultivation in percent.

Figure 5: Degradation of BPA, BPF and BPS after 24 h cultivation with E.coli KRX carrying genes for the bisdA | bisdB fusion protein behind the medium strong constitutive promoter BBa_J23110 with RBS BBa_B0034. Cultivations were carried out at different temperatures in LB + Amp + bisphenol medium (starting concentration 120 mg L-1 BPA, BPF or BPS respectively) for 24 h in 300 mL shaking flasks without baffles with silicon plugs. Samples were taken at the end of the cultivation. Two biological replicates were analyzed. While BPA is fully degraded only a small fraction of BPF (~7%) and BPS (~3%) was degraded.

The results of the experiment indicate that the bisdA | bisdB fusion protein has a high specificity for the degradation of BPA. In addition it is possible that the decrease in BPF and BPS concentration is due to internalization of those bisphenols or a endogenous enzyme of E. coli KRX and not the bisdA | bisdB fusion protein was responsible. It can be assumed that false positive signals because of BPF or BPS present in a sample are unlikely.

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

(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. 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

(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. (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:

(5)

Fig. 6 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.

Fig. 6: Comparison between modelled (lines) and measured (dots) data for cultivations of E. coli KRX carrying BPA degrading BioBricks. The BioBricks BBa_K525512 (polycistronic bisdAB genes behind medium strong promoter, shown in black) and BBa_K525517 (fusion protein between BisdA and BisdB, expressed with medium strong promoter, shown in red) were cultivated at least five 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_K525512	BBa_K525517
X0	0.112 108 mL-1	0.138 108 mL-1
µmax	1.253 h-1	1.357 h-1
KS,1	2.646 AU-1	1.92 AU-1
KS,2	265.1 AU-1	103.1 AU-1
S1,0	1.688 AU	1.166 AU
qS,1	0.478 AU 10-8 cell-1	0.319 AU 10-8 cell-1
S2,0	16.091 AU	6.574 AU
qS,2	0.295 AU 10-8 cell-1	0.191 AU 10-8 cell-1
BPA0	0.53 mM	0.53 mM
qD	8.76 10-11 mM cell-1	1.29 10-10 mM cell-1

The specific BPA degradation rate per cell q_D is about 50 % higher when using the fusion protein compared to the polycistronic bisdAB gene. This results in an average 9 hours faster, complete BPA degradation by E. coli carrying BBa_K525517 compared to BBa_K525512 as observed during our cultivations. The fusion protein between BisdA and BisdB improves the BPA degradation by E. coli.

Interpretation of the results

Degrading BPA with E. coli using the BisdA | BisdB fusion protein, both domains (BisdA and BisdB) are active and correctly folded because otherwise there would be no BPA degradation measured and no BPA degradation products 1,2-Bis(4-hydroxyphenyl)-2-propanol and 2,2-Bis(4-hydroxyphenyl)-1-propanol detected which could definetely be identified via MS-MS. The about 50 % higher specific BPA degradation rate in E. coli expressing BisdA | BisdB fusion protein could be explained either by improved folding properties of the fusion protein or by the closer distance of BisdA and BisdB in the fusion protein leeding to a more efficient reaction.

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].

Sasaki M, Tsuchido T, Matsumura Y (2008) Molecular cloning and characterization of cytochrome P450 and ferredoxin genes involved in bisphenol A degradation in Sphingomonas bisphenolicum strain AO1, Journal of Applied Microbiology [http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2008.03843.x/full 105(4):1158-1169]

Sasaki M, Maki J, Oshiman K, Matsumura Y, Tsuchido T (2005) Biodegradation of bisphenol A by cells and cell lysate from Sphingomonas sp. strain AO1, Biodegradation [http://www.springerlink.com/content/q7864l02734wg32m/ 16(5):449-459]

Fromme H, Küchler T, Otto T, Pilz K, Müller J, Wenzel A (2002) Occurrence of phthalates and bisphenol A and F in the environment, Water Research [http://www.sciencedirect.com/science/article/pii/S0043135401003670 36(6):1429-1438]

Chen M, Ike M, Fujita M (2002)Acute toxicity, mutagenicity, and estrogenicity of bisphenol-A and other bisphenols, Enviromental Toxicology [http://onlinelibrary.wiley.com/doi/10.1002/tox.10035/abstract 17(1):80-86]

Kitamura S, Suzuki T, Sanoh S, Kohta R, Jinno N, Sugihara K, Yoshihara S, Fujimoto N, Watanabe H, Ohta S (2005) Comparative Study of the Endocrine-Disrupting Activity of Bisphenol A and 19 Related Compounds, Toxicol. Sci. [http://toxsci.oxfordjournals.org/content/84/2/249 84(2):249-259]