PCR amplification and Gibson assembly

Since terA, terB, terC and terD are adjacent within the terABCD operon, we were able to amplify the entire terABCD cluster by PCR using a single primer pair. We designed the primers to generate overhangs, creating homologous regions between the pET28a backbone and the cluster. These homologous regions were required for Gibson assembly. We then assembled the amplified fragments and backbone via Gibson assembly and transformed them into E.coli DH5α, a strain optimized for efficient cloning. We verified positive clones by colony PCR and sequencing. Finally, we transferred the confirmed constructs into E. coli BL21 (DE3) to enable high-level, IPTG-inducible expression of the operon.

Figure 2: Amplification PCR of terABCD cluster and the pET28 backbone. A 1. DNA ladder (5kb +), 2. amplified terABCD operon of 3381bp. B. 1. DNA ladder, 2. amplified control (backbone used for REE/curli project) of 5263bp, 3. amplified backbone for the tellurium project of 5301bp.

The backbone amplification from the pET28a plasmid was successful, matched with the previously amplified backbone for the REE/Curli project (Fig. 2B). The terABCD cluster was amplified according to the colony PCR protocol using Phanta high-fidelity polymerase instead of Taq polymerase (Fig. 2A). Since the terZABCDE operon was on the pTTS12 megaplasmid of 583 kb, a standard plasmid extraction kit was not fitted to isolate the plasmid (Kusumawardhani et al, 2020).

Figure 3: Colony PCR and transformation of pET28a-TerABCD into E. coli DH5α and BL21. A. 1. DNA ladder (5kb +), 2-4. positive DH5α colonies for pET28a-terABCD showing a band of 3381bp. B. Plate of BL21 cells after transformation with pET28a-terABCD. Colonies appear after selection on kanamycin-containing medium. C. 1. DNA ladder, 2-5. positive BL21 colonies for pET28a-terABCD showing a band of 3381bp.

The Gibson assembly was successful. Positive colonies were selected via kanamycin resistance encoded in the pET28a plasmid (Fig. 3B). The presence of the insert was confirmed by colony PCR (Fig. 3A,C), and the insert was sequenced after the transformation into DH5α to check for mutations. Only colonies without mutations were further transformed into BL21. The pET28a had two origins of replication, which enabled replication both in DH5α and BL21.

Plate assay and optimal conditions determination

In the first experiment, we aimed to perform a semi-quantitative determination of the optimal conditions for tellurite reduction into elemental tellurium. We tested the engineered E. coli BL21 pET28a-terABCD against P. putida S12 pTTS12. We used E. coli pET28a-mCitrine (harboring the mCitrine fluorescent protein under the PT7-lacO promoter instead of the terABCD gene cluster) as a negative control.

To determine the tellurite concentration range compatible with bacterial growth, we prepared plates supplemented with 0 ng/ml, 50 ng/ml, 100 ng/ml, 500 ng/ml, 1 µg/ml, 5 µg/ml, 10 µg/ml, 50 µg/ml, 100 µg/ml, and 200 µg/ml tellurite. Since P. putida and E. coli have different optimal incubation temperatures, we plated them separately. We supplemented plates for E. coli with 1 mM IPTG to induce terABCD expression.

Figure 4: E. coli BL21 pET28-mCitrine, P. putida S12 pTTS12, E. coli BL21 pET28-terABCD on tellurite supplemented plates after 48h of incubation. Plates supplemented with 1mM IPTG and with a concentration of 0 ng/ml, 50ng/ml, 100ng/ml, 500ng/ml, 1μg/ml, 5μg/ml, 10μg/ml, 50μg/ml, 100μg/ml or 200μg/ml tellurite. Colony darkening is the result of tellurite reduction into tellurium. A. corresponds to P. putida S12 pTTS12 growth. B. corresponds to E. coli pET28a-terABCD and E. coli pET28a-mCitrine growth.

Plate assays are not highly precise, but they can provide semi-qualitative information about the optimal conditions for tellurite reduction. P. putida does not grow efficiently on tellurite concentrations above 50 µg/ml (Fig. 4A). Colony darkening indicates that the bacteria take up tellurite and reduce it into elemental tellurium, since elemental tellurium is insoluble and forms black granules. In P. putida, this darkening becomes visible at concentrations as low as 1 µg/ml (Fig. 4A). For BL21, the strain carrying the operon does not appear to grow better than the strain without the operon; both cannot grow after 5 µg/ml (Fig. 4B). This observation raises three questions:

• Is the terABCD operon not correctly expressed?
• Is IPTG induction insufficient?
• Is the engineering design itself unsuccessful?

To improve terABCD operon expression, we increased the IPTG concentration to 5 mM and allowed the bacteria to grow in liquid Luria Bertani (LB) medium. We also calibrated the tested tellurite concentrations based on the optimal growth and reduction we observed in P. putida. The new test conditions included 0 µg/ml, 2.5 µg/ml, 5 µg/ml, 10 µg/ml, 25 µg/ml, and 50 µg/ml tellurite.

Figure 5: Tellurite reduction comparison between P. putida S12 pTTS12 and E. coli BL21 pET28a-terABCD. A. E. coli BL21 pET28a-terABCD and P. putida S12 pTTS12 both incubated for 24h in presence of 10 µg/ml tellurite, supplemented with 5 mM IPTG for BL21. B. E. coli BL21 pET28a-terABCD and P. putida S12 pTTS12 both incubated for 48h in presence of 25 µg/ml tellurite, supplemented with 5 mM IPTG for BL21.

We observed a clear qualitative difference between P. putida S12 pTTS12 and E. coli BL21 pET28a-terABCD. With 5 mM IPTG, the operon was well expressed, and BL21 effectively reduced tellurite into tellurium, as evidenced by the darkening of the LB solution (Fig. 5A,B).

An IPTG concentration of 1 mM did not allow visible tellurite reduction. At 5 mM IPTG, E. coli BL21 pET28a-terABCD clearly reduced tellurite at concentrations ranging from 1 to 50 µg/ml. The cultures also displayed high turbidity, indicating that E. coli BL21 pET28a-terABCD grew more than in the plate assay (Fig.4B).

Then, we quantified tellurite reduction and evaluated the effectiveness of our engineered strain. For all subsequent experiments, we applied the following conditions:

• Negative control: E. coli BL21 pET28a-mCitrine
• Positive control: P. putida S12 pTTS12
• Test sample: E. coli BL21 pET28a-terABCD

We incubated E. coli strains at 37 °C and P. putida strains at 30 °C.

Growth kinetics at low bacterial concentration

The aim of this experiment was to assess the growth capacity of our engineered E. coli BL21 pET28a-terABCD in LB medium supplemented with tellurite at concentrations of 0 µg/ml, 2.5 µg/ml, 5 µg/ml, 10 µg/ml, 25 µg/ml, and 50 µg/ml. This setup allowed us to visualize how tellurite impacts bacterial growth kinetics. We added 5 mM IPTG to the growth medium to ensure effective terABCD expression.

Figure 6: Measurement of the OD₆₀₀ to assess bacterial growth kinetics. OD₆₀₀ measurements were made after 5h, 24h, 48h and 72h with tellurite concentrations of 0μg/ml, 2.5μg/ml, 5μg/ml, 10μg/ml, 25μg/ml and 50μg/ml. A. Growth curves for P. putida S12 pTTS12. B. Growth curves for E. coli BL21 pET28a-mCitrine. C. Growth curves for E. coli BL21 pET28a-terABCD.

Tellurite inhibited bacterial growth in all strains at the tested concentrations. E. coli BL21(DE3) lacking the terABCD operon did not grow in tellurite-supplemented medium, whereas E. coli BL21 pET28a-terABCD grew at all concentrations, albeit more slowly at higher concentrations. In contrast, P. putida S12 pTTS12 grew more rapidly than engineered E. coli at 5 and 10 µg/mL but could not grow at higher concentrations (Fig. 6A).

Based on these observations, we selected 10 µg/mL tellurite as the optimal concentration for subsequent experiments, as it allowed growth of both P. putida S12 pTTS12 and E. coli BL21 pET28a-terABCD.

Focus on bacterial growth at optimal concentration: 10 μg/ml

We assessed whether this modified culture environment influenced growth in tellurite-supplemented medium. We incubated cultures for 48 h at 10 µg/mL, the highest concentration at which both P. putida S12 pTTS12 and E. coli BL21 pET28a-terABCD exhibited robust growth. We grew cultures in larger volumes (5 mL in 15 mL culture tubes) to provide increased aeration and space for bacterial proliferation.

Figure 7: Bacterial growth in 10μg/ml tellurite supplemented liquid LB. OD₆₀₀ absorbance measurements were made after 24h and 48h. Tested samples were incubated in LB supplemented with 10μg/ml tellurite.

Consistent with the previous experiment, P. putida S12 pTTS12 reached the highest OD₆₀₀ values, reflecting the greatest biomass after both 24h and 48h. E. coli BL21 pET28a-terABCD showed moderate growth, reached an OD₆₀₀ above 0.5, and resisted tellurite better than BL21 pET28a-mCitrine, as indicated by higher biomass after 1 and 2 days.

To determine whether this apparent resistance associated with the reduction of tellurite to elemental tellurium, we employed the diethyldithiocarbamate (DDTC) assay for tellurite quantification, adapting the method described by Turner et al. (Turner et al, 1992) for our experimental conditions (see contribution page).

Calibration curves using diethyldithiocarbamate

The goal of this experiment was to generate a calibration curve using known concentrations of tellurite, enabling the derivation of a calibration equation to calculate the amount of tellurite taken up by bacteria in later experiments.

To achieve this, we tested and adapted the protocol described by Turner and colleagues to establish a calibration curve for subsequent quantification experiments. According to Turner and colleagues, the maximum tellurite concentration that provides reliable absorbance measurements is 50 µg/ml (Turner et al, 1992). However, we extended the range up to 200 µg/ml to assess the response at higher concentrations. We measured OD₃₄₀ at tellurite concentrations of 0 µg/ml, 2.5 µg/ml, 5 µg/ml, 10 µg/ml, 25 µg/ml, and 50 µg/ml.

Figure 8: Calibration curve for tellurite concentrations using diethyldithiocarbamate. Quantification was made through absorbance measurement at OD₃₄₀. A. Calibration curve up to 200 μg/mL with a R squared of 0.9204. The fitted formula is OD₃₄₀ = 0.018 × Tellurite concentration [μg/ml]. B. Calibration curve up to 50 μg/mL with a R squared of 0.9918. The fitted formula is OD₃₄₀ = 0.024 × Tellurite concentration [μg/ml].

Diethyldithiocarbamate (DDTC) proved to be an efficient reagent for quantifying tellurite in solution. We obtained reliable and precise measurements up to 50 µg/ml, within which the relationship between absorbance and tellurite concentration remained linear, with a strong fit (R² = 0.9918) (Fig. 8B). We successfully quantified tellurite both in water and in LB medium. At concentrations above 50 µg/ml, the calibration curve deviated from linearity and began to plateau or decline, suggesting signal saturation or interference at higher concentrations. In this range, the fit was noticeably weaker (R² = 0.9204).

Luria Bertani mediated tellurite reduction

The objective of this experiment was to evaluate the stability of tellurite in LB over time. Turner and colleagues demonstrated in 1992 that tellurite remains stable in bacterial culture for at least 6 h at room temperature (Turner et al, 1992). To this end, calibration solutions were incubated at 37 °C in a shaking incubator under the same conditions used for E. coli BL21 pET28a-TerABCD for 48h.

Figure 9: Effect of LB medium on tellurite in incubation conditions. Calibration samples were incubated at 37°C, 180 rpm.

We tested the tellurite stability at a 10 µg/ml concentration, as this level provides suitable conditions for both bacterial growth and tellurite reduction in E. coli pET28a-terABCD and P. putida S12 pTTS12. LB medium alone significantly reduced tellurite within 24 hours under incubation conditions (Fig. 9). The absorbance at 340 nm decreased by 41.62% after one day, and by 57.36% after two days, corresponding to a 29.96% decrease between day one and day two (Fig. 9). We also observed tellurite reduction in the test tubes, indicated by the formation of a black pellet.

We attribute this abiotic reduction to 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 address this issue, we tested incubating the calibration samples alongside the bacterial samples to distinguish between medium-driven and bacteria-driven tellurite reduction.

Calibration curves at 37°C and 30°C

Turner and colleagues tested tellurite stability at room temperature. Because we observed a strong influence of time and temperature on tellurite reduction in LB medium, we decided to examine whether tellurite remains stable during the first six hours of incubation.

Figure 10: Tellurite calibration curves over 6h under incubation conditions. Absorbance measurements at OD₃₄₀ with tellurite concentrations of 0, 5, 10, 25 and 50 μg/ml. Measurements were made after 0.5h, 1.5h, 3h and 6h in incubation conditions. Calibration samples were incubated at 30°C or 37°C. A. Calibration curves after 0.5h, 1.5h, 3h and 6h incubation at 30°C. B. Mean calibration curve of panel A as well as the calibration formula. The fitted formula at 30°C is OD₃₄₀ = 0.018 × Tellurite concentration [μg/ml]. C. Calibration curves after 0.5h, 1.5h, 3h and 6h incubation at 37°C. D. Mean calibration curve of panel C as well as the calibration formula. The fitted formula at 37°C is OD₃₄₀ = 0.019 × Tellurite concentration [μg/ml].

Tellurite remained stable in LB medium for up to 6 hours, with only minor variations between different incubation durations. We can conclude that DDTC allows reliable quantification of tellurite under bacterial incubation conditions within this timeframe. The calibration curves obtained for incubation at 30 °C and 37 °C are highly similar :

OD₃₄₀ = 0.018 × tellurite concentration [µg/ml] and OD₃₄₀ = 0.019 × tellurite concentration [µg/ml], respectively (Fig. 10C,D).

To ensure greater accuracy in our tellurite reduction quantification experiments, we applied the calibration curve at 37 °C for the E. coli BL21 strains and the calibration curve at 30 °C for P. putida S12.

Tellurite quantification starting with a low bacterial concentration

The first tellurite quantification using DDTC aimed to evaluate the ability of our strains to reduce soluble tellurite into elemental solid tellurium. We tested bacterial growth in presence of tellurite concentrations of 0 µg/ml, 5 µg/ml, 10 µg/ml, 25 µg/ml, and 50 µg/ml. The OD₃₄₀ was measured after 5 h, 24 h, 48 h, and 72 h of incubation in 2-ml 96-well plates at 30 °C or 37 °C. E. coli strains were supplemented with 5 mM IPTG. The OD₃₄₀ measurements were then used to quantify tellurite reduction based on the calibration curves (see Improvements of calibration curves for tellurite quantification experiments part).

Figure 11: Tellurite reduction quantification using DDTC. Amounts of reduced tellurite in percentage after 5h, 24h, 48h and 72h of incubation are shown. Tellurite reduction was determined through OD₃₄₀ measurement of the bacterial cultures using the formula provided by the calibration curve : Reduced tellurite (%) = 1 – (OD₃₄₀ / 0.025). A. Tellurite reduction for 5μg/ml. B. Tellurite reduction for 10μg/ml. C. Tellurite reduction for 25μg/ml. D. Tellurite reduction for 50μg/ml.

After 5 hours of incubation, the extent of tellurite reduction was approximately the same across all strains and concentrations, with ~5 µg/ml of reduced tellurite. At an initial concentration of 5 µg/ml, all strains, including E. coli without the operon, achieved between 90% and 100% tellurite reduction after 24 hours (Fig. 11A). A similar outcome was observed after 48 hours with a concentration of 10 µg/ml (Fig. 11B).

In presence of high tellurite concentrations, E. coli BL21 pET28a-terABCD and P. putida S12 pTTS12 reduced more tellurite after 24 hours than the control strain E. coli BL21 pET28a-mCitrine (38.2% and 48.9% vs. 25.9%, respectively). However, after 48 and 72 hours, our engineered strain outperformed both controls in presence of 25 µg/mL and 50 µg/mL of tellurite, reaching approximately 71% and 15% reduction, respectively (Fig. 11C,D). The comparatively lower reduction observed in P. putida S12 pTTS12 can likely be explained by its limited growth in presence of tellurite concentrations above 25 µg/mL (see Bacterial growth in tellurite supplemented media part, Fig. 6C).

At the highest concentration tested (50 µg/mL), E. coli BL21 pET28a-terABCD was the only strain still reducing measurable amounts of tellurite after 72 hours, though it achieved only about one-fifth of total reduction (Fig. 11D).

Interestingly, E. coli BL21 pET28a-mCitrine showed a higher tellurite reduction than expected. This effect is likely multifactorial, but our main explanation is that the LB medium itself can reduce tellurite when incubated at 37 °C for more than 6 hours. This hypothesis is supported by both the bacterial growth kinetics (see Bacterial growth in tellurite supplemented media part, Fig. 6B) and the experiments on tellurite stability in LB (see Improvements of calibration curves for tellurite quantification experiments part, Fig. 9). For example, we found that for a tellurite concentration of 10 µg/ml, over 41% of the tellurite was reduced after 24 hours and more than 57% after 48 hours when incubated at 37 °C with shaking (Fig. 9). This matches the apparent reduction observed in E. coli BL21 pET28a-mCitrine for 10 µg/ml tellurite after 24 hours, even though this strain grows poorly in tellurite-supplemented LB (Fig. 6).

To improve the precision of our quantification, we decided to incubate the calibration samples together with the bacterial samples in subsequent experiments. We also focused on the 10 µg/ml concentration, since it represents the highest concentration at which both P. putida S12 and E. coli BL21(DE3) carrying the terABCD operon display robust growth and efficient tellurite reduction.

Tellurite quantification at optimal concentration: 10 μg/ml

The goal of this experiment was to determine whether culture volume influences tellurite reduction. This test was performed in presence of 10 µg/ml tellurite, the highest concentration for which both P. putida S12 andE. coli BL21(DE3) carrying the terABCD operon exhibited strong growth and efficient tellurite reduction. This time, the bacteria were tested in a larger volume: 5 ml in 15-ml culture tubes instead of 1.2 ml in 2-ml wells. To reduce medium-driven tellurite reduction bias during quantification, calibration samples were incubated in a shaking incubator at 37 °C. Since the previous experiment showed that measurements after 5 hours were similar across all samples, OD₃₄₀ was measured only after 24 and 48 hours.

Figure 12: Quantification of tellurite reduction at concentration of 10μg/ml. OD₃₄₀ was measured after 24 h and 48 h. Absorbance values were converted into tellurite reduction percentages using the calibration curves. For P. putida S12 pTTS12, the formula applied was: Reduced tellurite (%) = 1 – (OD₃₄₀ / 0.018). The same formula was used for E. coli BL21 pET28a-mCitrine and E. coli BL21 pET28a-terABCD.

This experiment allowed us to distinguish between LB-mediated and bacteria-mediated tellurite reduction. Reduction was observed in all samples, but in E. coli BL21 pET28a-mCitrine it appeared to be largely attributable to the autonomous reduction occurring in LB. After both 24 h and 48 h,E. coli BL21 pET28a-terABCD and P. putida S12 pTTS12 displayed higher tellurite reduction than E. coli BL21 pET28a-mCitrine, whose reduction closely matched that of the calibration.

After 24 h, LB alone reduced up to 39.47% of the soluble tellurite. E. coli BL21 pET28a-mCitrine also showed ~39% reduction, suggesting that this reduction was mainly due to the medium. In contrast, E. coli BL21 pET28a-terABCD and P. putida S12 pTTS12 reduced 74.73% and 75.97%, respectively, indicating that E. coli BL21 pET28a-terABCD achieved ~35.26% reduction independently of LB. After 48 h, E. coli BL21 pET28a-TerABCD reduced over 90% of the tellurite, with ~35% attributable to its own activity beyond the LB effect.

Although LB-mediated tellurite reduction strongly influenced the results, the presence of the terABCD cluster clearly enhanced the reduction capacity of E. coli. To minimize the confounding effect of medium-driven reduction, we next tested our strains starting from fully grown bacterial cultures and quantified tellurite reduction within the first 6 hours.

Tellurite quantification during the first 6h

Our previous experiments indicated that our engineered E. coli strain resists and reduces tellurite. To avoid unwanted medium-driven reduction and improve quantification accuracy, we tested tellurite reduction in fully grown bacterial cultures, previously grown overnight without IPTG or tellurite.

As shown in the "Improvements of calibration curves for tellurite quantification experiments" part (see Improvements of calibration curves for tellurite quantification experiments part, Fig. 10A,C), calibration curves remain stable for up to 6h of incubation at both 30 °C and 37 °C. We incubated calibration samples under the same conditions as the bacterial samples to maximize accuracy.

We assessed how rapidly our strains reduce tellurite by measuring absorbance at 340 nm after 0.5h, 1.5h, 3h, and 6h at tellurite concentrations of 5 µg/ml, 10 µg/ml, 25 µg/ml, and 50 µg/ml.

Figure 13: Tellurite reduction quantification within the first 6h. The absorbance at 340nm was changed into tellurite reduction percentage using calibration curves. Reduced tellurite (%) = 1 – (OD₃₄₀ / 0.018) formulas were used for P. putida S12 pTTS12. Reduced tellurite (%) = 1 – (OD₃₄₀ / 0.019) formulas were used for E. coli BL21 pET28a-mCitrine and E. coli BL21 pET28a-terABCD. A. is the tellurite reduction after 0.5h, 1.5h, 3h and 6h with a tellurite concentration of 5µg/ml . B.. is the tellurite reduction after 0.5h, 1.5h, 3h and 6h with a tellurite concentration of 10µg/ml. C. is the tellurite reduction after 0.5h, 1.5h, 3h and 6h with a tellurite concentration of 25µg/ml. D. is the tellurite reduction after 0.5h, 1.5h, 3h and 6h with a tellurite concentration of 50µg/ml. One tailed Student t-test was performed for significance evaluation.

E. coli BL21 pET28a-TerABCD displayed a significantly higher tellurite reduction capacity than BL21 without the terABCD cluster. After 6 h in a 10 µg/ml tellurite medium, E. coli BL21 pET28a-TerABCD reduced 80.80% of the tellurite, compared to 24.45% for E. coli BL21 pET28a-mCitrine (p = 9.71 × 10⁻³, Fig. 13B). At concentrations of 5 µg/ml, 10 µg/ml, and 25 µg/ml, E. coli pET28a-terABCD and P. putida S12 pTTS12 reduced comparable amounts of tellurite. Both strains reduced more than 90% of the soluble tellurite at 5 µg/ml and between 80–100% at 10 µg/ml. At 25 µg/ml, E. coli BL21 pET28a-TerABCD reduced 40.66%, while P. putida S12 pTTS12 reduced 51.18%. At 50 µg/ml, E. coli BL21 pET28a-terABCD reduced 27.40% and outperformed P. putida S12 pTTS12, which reduced 11.11%.

For the statistical analysis, we specifically tested whether E. coli BL21 pET28a-TerABCD exhibited higher tellurite reduction than E. coli BL21 pET28a. Although a one-tailed test provides more power with a low number of replicates, it is inherently more biased. In our case, based on prior results, we had strong evidence suggesting that the engineered strain displayed enhanced tellurite uptake compared to E. coli BL21 pET28a-mCitrine.

At concentrations of 5 μg/ml, 10 μg/ml, and 25 μg/ml, E. coli BL21 pET28a-terABCD and P. putida S12 pTTS12 showed comparable reduction rates. Both strains reduced more than 90% of the soluble tellurite at 5 μg/ml and between 80–100% at 10 μg/ml. However, at 50 μg/ml, E. coli BL21 pET28a-terABCD outperformed P. putida S12 pTTS12, reducing 27.40% versus 11.11% after 6 h, respectively.

In this experiment, LB alone did not reduce tellurite, confirming that the terABCD cluster significantly contributes to both resistance and reduction into elemental tellurium. To ensure maximum accuracy, we calculated tellurite reduction percentages using calibration curves at both 37 °C and 30 °C (see Improvements of calibration curves for tellurite quantification experiments part, Fig 10.B,D). The strong expression of terABCD allowed our engineered E. coli to match the reduction efficiency of P. putida S12 pTTS12 at moderate concentrations and surpass it at the higher concentration (50 µg/ml).

Our engineered E. coli BL21 pET28a-TerABCD grows in a medium supplemented with soluble tellurite by efficiently reducing it to elemental tellurium. At tellurite concentrations between 5 µg/ml and 10 µg/ml, it grows slightly slower than the naturally tellurite-resistant P. putida S12 pTTS12. At higher concentrations (25 µg/ml and 50 µg/ml), E. coli BL21 pET28a-terABCD grows within three days, whereas P. putida S12 pTTS12 cannot.

As REEvolutionate is the first iGEM team to focus on tellurite, we first had to develop and refine a protocol for tellurite reduction quantification. For this purpose, we employed a diethyldithiocarbamate (DDTC)-based spectrophotometric method to measure tellurite concentration in bacterial samples. During this project, we demonstrated that DDTC-mediated quantification is consistent and reliable, although it has some limitations: measurements become less precise at concentrations above 50 µg/ml, and tellurite is prone to abiotic reduction in LB medium after more than 24 hours of incubation.

Within 6 hours, both E. coli BL21 pET28a-terABCD andP. putida S12 pTTS12 can nearly completely reduce tellurite at 5 µg/ml and 10 µg/ml. At 50 µg/ml, our engineered E. coli strain even outperforms P. putida S12 pTTS12.

While these results are promising, there is still room for improvement in both the experimental design and the model. First, the terABCD cluster used in E. coli is unmodified from the pTTS12 terZABCD operon in P. putida, and these genes are not optimized for expression in E. coli. Optimizing the terABCD cluster for E. coli could enhance both tellurite resistance and reduction capacity. Additionally, reintroducing pET28a-terABCD into a P. putida strain lacking the pTTS12 megaplasmid could allow for operon upregulation, further improving tellurite reduction and resistance. In this case, the T7 promoter should be replaced with a promoter suitable for P. putida (e.g., Plac or Ptac)

​​Another improvement would be to limit abiotic tellurite reduction when incubating LB at 37 °C in a shaking incubator. Molina et al. investigated tellurite behavior in both LB and the minimal medium M9 (Molina et al, 2010). The advantage of using a minimal medium is the absence of amino acids (particularly cysteine) that may play a significant role in LB-mediated tellurite reduction (Fuentes et al, 2007). In addition, for experiments lasting several hours to a few days, measuring absorbance at 350 nm appears to provide more consistent results