Contribution

Introduction

During our project, we needed a reliable and easy way to quantify tellurite reduction in bacterial cultures. Tellurite is both toxic and scarce, and only a few simple, standardized methods exist to measure its concentration in a biological setting. Although Sodium diethyldithiocarbamate trihydrate (DDTC)-based spectrophotometric methods were described in the literature (Turner et al, 1992), they were not written for day-to-day iGEM lab use.

As far as we can determine from reviewing past iGEM wikis, no previous iGEM project has primarily focused on tellurite. Starting from scratch without an "all-in-one" protocol was challenging; we would have appreciated a community-ready method at the outset. This motivated us to refine, optimize, and document an iGEM-adapted DDTC assay that teams can reuse immediately.

Why this is a contribution

By documenting this assay in detail and adapting it for common iGEM lab conditions, we present a ready-to-use method for rapid, low-cost, and reproducible quantification of tellurite. We also include a robust calibration workflow so teams can convert absorbance into tellurite concentration in their own matrices. This "all-in-one" guide helps future teams to explore tellurite interactions with biology, validate engineered pathways, or compare resistance across strains.

We also evaluated an alternative NaBH₄-based colorimetric method (absorbance near 500 nm), which has been reported for tellurite detection (Molina et al, 2010). While potentially more sensitive in some contexts, we considered it less suitable for iGEM labs because sodium borohydride (NaBH₄) reacts violently with water and releases flammable hydrogen, increasing hazard and handling complexity for student teams. For these reasons we prioritized the DDTC method for its safety and simplicity, while noting NaBH₄ as an option for well-equipped labs that require extra sensitivity.

Principle

DDTC reacts with tellurite (TeO₃²⁻) to form a yellow-colored complex, which can be quantified using spectrophotometry. In aqueous solution, DDTC dissociates to release diethyldithiocarbamate anions (DDTC⁻), which act as the active ligands binding to tellurite. The resulting DDTC-tellurite complex exhibits a strong absorbance at 340 nm, allowing precise determination of tellurite concentrations.

Overnight cultures (day before the assay):

• Inoculate strains in LB medium without tellurite or inducers
• Add appropriate antibiotics for plasmid maintenance

Experimental cultures (day of assay):

• Inoculate fresh LB medium supplemented with tellurite and any required inducers

Reagents

• DDTC stock solution (10 mM): Dissolve sodium diethyldithiocarbamate trihydrate in distilled/deionized water. Store at 4 °C, protected from oxidation. Prepare fresh weekly.
• Tris-HCl buffer (0.5M, pH 7.0): Store at 4 °C.
• Sodium tellurite (Na₂TeO₃): For preparing calibration standards.
• Distilled/deionized water

Calibration Samples

Prepare calibration standards with tellurite under the same culture medium (LB in our case), incubation, and shaking conditions.

Sampling

1. Withdraw 250 µL of culture at your time point.
2. Spin 1 min at 13,000 rpm to pellet cells.
3. Withdraw 200 µL of the supernatant (see Assay Procedure step 2).

Assay Procedure (final volume 1.0 mL)

1. Pipette 600 µL of 100 mM Tris-HCl buffer (pH 7.0) into a 1 cm cuvette.
2. Add 200 µL of the tellurite-containing sample (see Sampling).
3. Add 200 µL of 10 mM DDTC stock solution.
4. Mix gently and incubate at room temperature (20–25 °C) for 5 min.
5. Measure absorbance at 340 nm against a reagent blank (buffer + DDTC without tellurite).

1. Prepare tellurite standards (e.g., 0, 5, 10, 25, 50 µg/mL sodium tellurite) and measure absorbance at 340 nm under the same conditions as your samples.
2. Fit a linear model and force it through zero.
3. Report the slope (m). For example, in our calibration we obtained m = 0.024.
4. Calculate the tellurite concentration using the formula:

[TeO₃] = A₃₄₀ / m

Here is an example of a calibration curve with the corresponding regression line:

Important: The absorbance value cannot be used above 1.2 because the model values are no longer linear and difficult to read on the spectrometer. For accurate quantification of tellurite, it is preferable to dilute the sample. The most linear and therefore best range is between 0.1 and 1 absorbance.

LB Use

For our experiments, we used liquid LB medium to grow bacteria and as the background medium for tellurite assays. We observed that tellurite remains stable in LB for at least 6 hours. However, in longer incubations (24 h and 48 h), LB alone was able to reduce tellurite, even in the absence of bacteria.

This abiotic reduction could be explained by several factors, including incubation temperature (37 °C), reduced oxygen availability, and the presence of low-molecular-weight thiols (e.g., cysteine, glutathione) naturally present in yeast extract and tryptone (Ollivier et al, 2011).

To illustrate this effect, the figure below shows the measured tellurite concentration at t = 0 h, t = 24 h, and t = 48 h, starting from an initial concentration of 10 µg/mL and only exposed to LB.

Figure 3: Effect of Luria Bertani medium on tellurite in incubation conditions.
Calibration samples were incubated at 37°C, 180 rpm. At t=0, a tellurite concentration of 10 µg/mL shows an OD₃₄₀ of 0.197. At t=24h, an absorbance of 0.115, showing a 41.62% decrease. At t=48h, an absorbance of 0.084, a 57.36% decrease compared to t=0. Between t=24h and t=48h, the absorbance at 340 nm has decreased by 29.96%.