Measurement

Part 1: Protein Engineering Module: Characterization and measurement of lanthanide binding protein TFD

1.1 Summarize

In our protein engineering modification module, it is very important to quantify the ability of the TFD protein to adsorb rare earth ions. Having a sensitive assay for the detection of rare earth elements (REEs) is essential both for identifying potential sources of objects for REE recovery and for the quantitative monitoring of operations related to REE in the industry. Element analysis commonly used technology is inductively coupled plasma mass spectrometry (ICP-MS) or inductively coupled plasma optical emission spectroscopy (ICP-OES). However, their testing is expensive and not portable, and it is not easy to put into industrial-scale testing applications.

Fortunately, the unusual spectral properties of rare earth elements give us a new option for the convenient quantitative detection of rare earth binding by luminous methods. Before browsing our Measurement page, it is necessary to introduce the fundamentals of our measurement. Not only will this help readers understand the mechanics of our measurement design, but it will also help other iGEM teams migrate and apply our measurement methods in the future when they want to use them. Our protein-REE-binding measurements are based on two mechanisms: the unique spectral properties of rare earth elements, and the lanthanide ion luminescence mechanism sensitized by the antenna effect.

1.2 Measurement principle

1.2.1 Spectral properties of rare earth elements

Lanthanides have characteristic spectral properties, and they have super-sharp emission peaks in the visible light range (Figure 1) [1]. However, lanthanide luminescence fundamentally differs from fluorescence, which occurs when molecules transition from one electronic state to another. However, the 4f-4f transitions in trivalent lanthanide cations are “disallowed” by the Laporte selection rule. Because the 4f orbitals are relatively insensitive to the ligand field, these optical bands are line-like and unique characteristics of each metal. As a consequence of the parity rule (and sometimes due to the change in spin multiplicity), the extinction coefficients of lanthanide ions that are several orders of magnitude lower than those of commonly used fluorophores (<10 M−1 cm−1versus 10,000 to 100,000 M−1 cm−1). This makes direct excitation of metals very inefficient unless excitation is done using a high-power laser, which in turn can be destructive to biological samples. [2] [3]

Figure 1. Emission spectra of trivalent lanthanide ions [1]

1.2.2 Lanthanide ion luminescence mechanism for antenna sensitization

However, this problem can be overcome by using a strongly absorbing chromophore to sensitize Ln(III) emission in a process known as the antenna effect (Figure 2). Most of these chromophores absorb 300 to 400 nm light and have extinction coefficients that are greater than 10,000 M⁻¹ cm⁻¹, and the photosensitizer adjacent to the metal ion to absorb and transfer energy to the metal ion excited state (luminescence resonance energy transfer, LRET) (Figure 2). Several lanthanide(III) ions, such as Tb(III) and Eu(III), display intrinsic luminescence with relatively long luminescence lifetimes, which makes it easy for us to select them as representative rare earths for quantitative detection. The indole ring on tryptophan can act as a sensitizer for Tb(III) at a distance of 5-8 Å [4] [5]. In addition, emitting specific luminescence of Tb(III) or Eu(III) by introducing unnatural amino acids as sensitizers has also been reported [6].

Figure 2. Lanthanide ion luminescence mechanism for antenna sensitization

1.3 Measurement method

When the 280 nm wavelength of light is directly radiated against it, it does not emit fluorescence, or emit a faint green fluorescence. The high-frequency vibration of the surrounding medium such as the O-H bonds in the coordination water molecules causes non-radiation inactivation, causing energy loss, and has a certain quenching effect on its luminescence. Installing tryptophan as an antenna group near the rare earth binding site in the lumen of the TFD protein can solve this problem, as detailed in our Model page. When 280 nm wavelength exciting light is given, the indole ring on tryptophan absorbs the energy of light and transfers it to terbium ion. Through the energy transfer mechanism, terbium ions are excited to a high-energy state. In the excited state, terbium ion releases photons through a radiative transition to produce characteristic luminescence at 545 nm wavelength. [5]

The amount of rare earth ions adsorbed by TFD-S ( BBa_K5261001) was quantitatively detected by the microplate reader. The mode of time-resolved fluorescence (TRF) was selected, which allows all background signals to be suppressed, only long-lived terbium emission was collected in the pulse experiment, and fluorescence values in the wavelength range of 520-570 nm were detected. Under the same control, the higher the fluorescence intensity, the more terbium ions adsorbed by TFD protein in solution, indicating that the protein adsorbed rare earth ions stronger.

In addition, to detect the adsorption capacity of TFD protein to other rare earth ions, we replaced Tb³⁺ in the incubated protein with other lanthanide ions and measured the decay curve of the characteristic luminescence intensity of Tb³⁺ with time. The equilibrium dissociation constant KD of TFD protein binding to rare earth was characterized by an adsorption equilibrium curve. The value of KD can reflect the binding affinity, and the two are inversely proportional.

Figure 3. Luminescence determination of rare earth ions bound to the TFD protein

1.4 Measurement experiment

According to Shane J. Caldwell et al. [7] who titrated a pre-incubated TbCl₃ and EDTA mixture with metal-free TFD proteins, we learned that the equilibrium dissociation constant K_D(Tb³⁺/EDTA) = 1.6×10−18 M. Based on this, we examined the binding capacity of TFD-S protein to other rare earth ions.

1.5 Measurement protocols

Our protocols are documented in detail below.

1.6 Elimination of interfering factors

The interference factors of fluorescence value measured in this experiment mainly come from the spontaneous fluorescence of rare earth ion (trace), the spontaneous fluorescence of protein (trace), and the fluctuation of fluorescence value with time due to the dynamic factors in the binding equilibrium process after terbium ion binds to protein. The fluorescence intensity caused by the first two interfering factors is minimal. To eliminate the interference of time fluctuation factors, we set the blank control group as: 180 μL incubation mother solution and 20 μL buffer solution, that is, no replacement ions were added, so that the attenuation of fluorescence value only came from the binding and replacement process of TFD-REEs. The data of the experimental group were corrected by subtracting the control group's data at the corresponding time. At 0 h, the control group with terbium ion binding at all the protein adsorption sites had the strongest fluorescence intensity, so the other data were all negative.

1.7 Results

The adsorption process of TFD-REE can be approximated as a single-layer adsorption model, assuming that the adsorption process occurs at a specific position on the surface of the adsorbent, and each adsorption site can only be occupied by one molecule at most, resulting in a single-molecule adsorption layer. For details, see our Model page. Therefore, we used the langmuir adsorption isotherm equation q_e= \(\frac{K_LC_e} {1+a_LC_e}\) to perform curve fitting on the replacement results. (q_e is the concentration of lanthanide metal ions adsorbed by protein in an equilibrium state, where the fluorescence value A₅₄₅ is used, C_e is the concentration of lanthanide metal ions in the solution system, K_L and a_L are Langmuir isotherm constants).

We measured all non-radioactive rare earth ions except Tm(III), selected the relatively stable-binding ions La(III)/Nd(III)/Pr(III)/Sm(III) for analysis and mapping, and obtained the attenuation curve of the characteristic luminescence intensity of Tb³⁺ over time (Figure. 4, left), as well as the fitting curve of the replacement ion concentration with the equilibrium fluorescence intensity of Tb³⁺ (Figure 4, right).

Figure 4. The binding of other lanthanides, such as Pr(III), Nd(III), Sm (III), and Eu(III), was measured in displacement titrations using Tb(III)-bound TFD-S. The left figure showed the attenuation curve of the characteristic luminescence intensity of Tb³⁺ with time, and the right figure showed the fitting curve of the replacement ion concentration and the equilibrium fluorescence intensity.

When the fluorescence value of Tb(Ⅲ) decays by half, it can be considered that the number of protein adsorption sites occupied by the replacement ion X(Ⅲ) and Tb(Ⅲ) is equal, and they have the same binding ability to the protein, that is, \(\frac{K_D(Tb^{3+})} {K_D(X^{3+})}\)=\( \frac{c_1/2(Tb^{3+})}{c_1/2(X^{3+})} \), X represents the other rare earth element. Therefore, in the fitting curve of the replacement ion concentration and equilibrium fluorescence intensity, c_1/2(X³⁺) at half attenuation of A₅₄₅ was extracted, and \(\frac{K_D(Tb^{3+})}{K_D(X^{3+})}\) was obtained. The results are shown in the following table.

Table 1. Fitting results

Finally, we recorded all the characterization results of TFD-S in the page of (BBa_K5261001) as the characterization results of the New Basic Part.

1.8 Our advantages and possible help for future iGEM teams

The most common elemental analysis assays are ICP-MS or ICP-OES, but they are expensive and difficult to use for regular iGEM teams. The lanthanide ion luminescence detection based on antenna effect sensitization is simple and can be performed using only a microplate reader and a 96-well plate. In addition, our assay method is highly applicable and accurate, and can also be extended to other metal ions where fluorescence can be detected.

Moreover, our detection mechanism based on rare earth luminescence can also be applied to the development of protein-rare earth sensors. Using a small amount of TFD protein as a biosensor, it can be used to rapidly detect the content of terbium ions in environmental samples from different sources, such as different mine wastewater. Other antenna groups can also be introduced into the TFD protein to develop lanthanide-based optical probes for biological systems, which can be applied to the quantitative detection of other specific and more valuable lanthanide metals. Of course, this also requires the further development of future portable detection equipment. Anyway, this will greatly expand the potential applications of our project in the field of biosensors.

Finally, our design ideas and measurement methods can also provide a reference for the futher iGEM teams to carry out the rare earth protein-REEs combination research. By using the antenna effect to sensitize the mechanism of rare earth element-specific luminescence, the detection content in the field of rare earth is greatly enriched.

Part 2: Yeast Surface Display Module: Detection of lanthanide ion adsorption capacity of engineered yeast

2.1 Overview

In our yeast surface display module, after displaying the lanthanide binding protein TFD-S on the cell surface, we need to continue to measure the lanthanide ion adsorption and recovery capacity of the engineered yeast. Here, however, we cannot continue to use the lanthanide ion luminescence mechanism sensitized by the antenna effect, because it would be subject to multiple interference from the cellular system. We need to continue to find a convenient and inexpensive method for quantitative detection of rare earths. Here, we used the Arsenazo III-based spectrophotometry assay.

2.2 Measurement Principle

Arsenazo III has been shown to detect concentrations of REEs as low as 0.01 μg/mL [8]. It can form 1: 1 complexes with metals through coordination bonds and only one arseno group is involved in metal binding [9]. Rare earth metal ions such as trivalent lanthanum, europium, or praseodymium can be adsorbed under acidic conditions (generally citrate/phosphate buffer system, pH 2.8) [10], and the ion concentration can be quantitatively determined by spectrophotometry at A₆₅₀.

Figure 5. Arsenazo III molecular formula

2.3 Measurement method

To measure the rare earth adsorption capacity of engineered yeast, terbium ion was selected as the representative object, which is of great value in the high-tech field. Cells with induced TFD-S protein expression (see the Protocols page for more details) at a biomass of 50 mL OD₆₀₀ = 25 were incubated with 100 μM TbCl₃ by oscillating for 1 day, then the supernatant was collected by centrifugation. With the Arsenazo III-based spectrophotometry assay, A₆₅₀ was determined to detect the remaining Tb(III) concentration in the supernatant, so as to measure the adsorption capacity of lanthanide metal ions by yeast cells.

Figure 6. Arsenazo III-based assay measured by microplate reader or spectrophotometer

2.4 Measurement protocols

Arsenazo III-based spectrophotometry assay can be used as a general method for quantitative determination of rare earth concentration. To facilitate future iGEM teams, we have written the detailed protocols below.

2.5 Results

After the adsorption process, the TbCl₃ content in the solution decreased significantly, which was manifested in the obvious color depth difference of the Arsenazo III – REE complex formed before and after adsorption (Figure 7 A), which was also reflected by the value of A₆₅₀.

However, due to the extremely low concentration of Tb(III) ion in the adsorbed solution and the mismatch with the detection limit of the Arsenazo III assay, we cannot quantitatively detect the accurate concentration of rare earth ion by drawing a standard curve.

However, we could estimate it by the "two-point interval method" according to the value of A₆₅₀ For the solution after adsorption, three parallel data points were measured in a microplate reader, and the A₆₅₀ obtained was 0.0393 (Figure 7 A), which was between that of 2 μM and 10 μM TbCl₃ standard solution (Figure 7 B), so it could be preliminarily judged that c(Tb³⁺) in the solution after the adsorption was also in this range. Since we initially added c(Tb³⁺) = 100 μM (before adsorption), it could be considered that our surface display strain had a strong adsorption capacity for rare earth ions.

Figure 7. A. The absorbance at 650 nm of the solution before and after biosorption was determined by Arsenazo III assay, and an obvious color depth difference was observed. B. A₆₅₀ value of the standard solution of TbCl₃ from 0 to 150 μM.

2.6 Conclusion

Due to the late successful construction of our surface display engineered yeast strains, we did not further perform precise quantitative characterization later due to time constraints. However, we have been able to prove that our engineered yeast is already equipped with rare earth adsorption capacity, which will hopefully be further optimized in the future to become a rare earth element bio-adsorbent suitable for industrial applications. In addition, we believe that the Arsenazo III-based spectrophotometry assay is a convenient method for the quantitative detection of rare earth elements, and our experience can be used as a reference for future iGEM teams.

References

[1] J.-C.G. Bünzli. Handbook on the Physics and Chemistry of Rare Earths, vol. 50, Elsevier, 2016.

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