Team:Bielefeld-CeBiTec/Toxicity Results

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Toxicity Results

Abstract

In our experiments to improve the tolerance of Escherichia coli DH5α to heavy metals, we created a collection of several composite parts designed to combat oxidative stress. These parts mainly convey the ability to dismutate the superoxide anion and its secondary products like hydrogen peroxide into nontoxic forms. We were able to demonstrate that cells carrying our construct BBa_K2638118 or BBa_K2638112 does not lead to anare not subject to increased growth rate but to a significantly higher viability when the cells are exposed to elevated concentrations of heavy metals, namely cupric sulfate.

Phytochelatin synthase

The phytochelatin synthase produces phytochelatin which plays a major role in heavy metal detoxification processes in Arabidopsis thalina. The phytochelatin synthase BBa_K2638150

was cloned into pSB1C3 in Escherichia Coli (E.coli) DH5α. For an enzyme assay it was cloned downstream of T7 promoter and upstream of the intein tag in E. coli ER2566. After overexpression and purification the protein was analyzed via SDS-PAGE and MALDI-TOF. An enzyme assay ensured the catalytic activity of the BBa_K2638150.
The gene for the phytochelatin synthase (PCS1) has been ordered as gene synthesis from IDT. The gene synthesis was designed containing overlapping sequences to the iGEM standard backbone pSB1C3 to incorporate it directly via Gibson Assembly. The resulting BioBrick containing the phytochelatin synthetase is BBa_K2638150. After successful transformation in E.coli DH5 α different promoters were used to construct different composite parts. The Anderson promoter of BBa_J23111 with the ribosomal binding site (RBS) BBa_B0030 was cloned upstream of the phytochelatin synthase for BBa_K2638152 as well as the pTet promoter BBa_R0040 and the RBS BBa_J61101 for BBa_K2638151. For inducible expression pBad/araC promoter BBa_I0500 was cloned together with the RBS BBa_B0030 for BioBrick BBa_K2638153. For characterization we wanted to overexpress and purify the phytochelatin synthase. Therefore, BBa_K2638150 was cloned downstream of a T7 promoter and fused to an intein tag and chitin binding domain. This construct was transformed into E. coli ER2566 and the phytochelatin synthase was overexpressed by induction of the T7 promoter. After cultivation, purification was carried out with the NEB IMPACT system. Briefly, the phytochelatin synthase was bound to the column with its chitin binding domain. Afterwards, washing the column with cleavage buffer resulted in self-cleavage of the intein leading to a separation of the protein from the column. The protein concentration was determined by Roti-Nanoquant assay, showing a protein concentration of 20.21 mg/mL. To confirm successful expression and purification the protein was loaded onto a SDS-PAGE (Figure 1).

Figure 1:Figure 1: SDS-PAGE of the purified phytochelatin synthase (BBa_K2638150). Phytochelatin synthase was expressed and purified and loaded on a SDS-PAGE in different dilutions. Lanes 1 and 2 show a 1:6 dilution of the sample, lanes 3 and 4 show 1:12 dilutions, lanes 5 and 6 show 1:24 dilutions and lanes 7 and 8 show 1:48 dilutions. The holes show cut bands which were examined by MALDI-TOF. Lanes 1 and 2 show a 1:6 dilution of the sample. The lanes 3 and 4 show dilutions 1:12, lanes 5 and 6 show 1:24 dilutions and lanes 7 and 8 show dilutions 1:48.

The SDS-PAGE shows an intense band at around 50 kDa. This band gets less intense in samples with a higher dilution but is still strongly present in the 1:48 dilution. As the phytochelatin synthase has a molecular weight of 53.946 kDa, this band indicates successful expression and purification of the enzyme. To proof that the band is indeed the phytochelatin synthetase, matrix associated laser desorbtion ionization – time of flight analysis (MALDI-TOF mass spectrometry) was performed. Therefore, the bands were cut out as indicated and prepared as described for MALDI-TOF analysis (Figure 2).

Figure 2:MALDI-TOF results of the phytochelatin synthase BBa_K2638150. Purified phytochelatin synthase were analyzed by SDS-PAGE and characteristic bands were cut out and analyzed by MALDI-TOF MS.

Figure 2 shows the results of the MALDI-TOF measurements. Comparison with the Mascot database indicates that the examined sample is the phytochelatin synthase BBa_K2638150. In order to determine that the BioBrick BBa_K2638150 works as expected, an enzyme assay for the phythochelatin synthase (Chen et al.,1997) was conducted. The assay is based on the conversion of glutathione to phytochelatin Therefore, the enzymatic in vitro assay was performed and afterwards the sample was measured with a liquid chromatography which was connected with a mass spectrometer.

Evaluation

Heavy metal exposure poses many risks and dangers to living organisms and the environment. Certain heavy metal ions such as copper can interact with enzymes and lower their activity as well as their specificity. Reactive oxygen species (ROS) arise from processes such as Fenton chemistry and Haber-Weiss reactions. Therefore, a sophisticated approach to lower the toxic effects of heavy metals on the cell is desired. We evaluated several approaches of applying anti-oxidants against the generation of ROS. In our project, we set a focus on the accumulation of copper ions. Furthermore, using cupric salts is cheaper than gold ions and easier to solve than ferric salts. Its toxicity is lower than that of silver ions. Hence there is a broader spectrum in which anti-toxic measures can be explored. Therefore, we tested our approaches on anti-oxidant measures in different concentrations of cupric salts. Subject to our research were the five following composite parts: BBa_K2638109, BBa_K2638112, BBa_K2638114, BBa_K2638110 and BBa_K2638118.

Figure 1: Plotting the OD₆₀₀ of E. coli DH5alpha carrying an empty pSB1C3 plasmid versus time with varying copper concentrations. Experiments were performed in a total volume of 1 mL in a 24-well plate at 37 °C and 350 rpm (n=3).

Escherichia coli DH5alpha carrying an empty pSB1C3 vector was grown in LB medium containing different concentrations of cupric salt over a period of 13 hours. With no additional cupric salt added the cells reached an optical density of 0.848 and entered the stationary phase after 6.5 hours. Small additions of cupric salt to the medium appeared to have a benevolent effect on the growth: concentrations of 1 mM and 2 mM CuSO₄ resulted in a higher optical density of around 0.950. Growth started to deteriorate at a concentration of 4 mM CuSO₄. The maximal growth rate µmax dropped to 7.54E-3 min^-1 in comparison to the growth in absence of cupric salt (12.06E-3 min^-1) meaning it is only 62.52 % of the original µmax. Growth decreased even further with rising copper ion concentrations to 0.28E-3 min^-1 and 0.36E-3 min^-1. For the examination of antitoxic measures, three concentrations of cupric salts were chosen: 0 mM, 4 mM and 8 mM. 0 mM was used as a control. 4 mM CuSO₄ was chosen since growth could still be observed, even though growth was not as good as at lower concentrations of cupric salt. At 8 mM CuSO₄, no significant growth was observed. Therefore, it was interesting to see whether more growth occurs or whether a higher viability can be achieved by applying our approaches for antitoxic measures.

Figure 2: Plotting the OD₆₀₀ of E. coli DH5alpha each carrying different plasmid versus cultivation time in a volume of 1 mL LB medium containing 0 mM CuSO₄. Experiments were performed in a 24-well plate at 37 °C and 350 rpm (n=3).

The growth of strains carrying different constructs in absence of cupric salt was examined. No significant difference between the highest OD₆₀₀ values of the cells could be observed. However, cells carrying plasmids under the control of the pBAD promoter showed a different growth pattern since they can rely on another carbon source once glucose is metabolized. This leads to two consecutive exponential phases resulting in similar OD₆₀₀ values compared to those only relying on glucose as a carbon source. Other than that, no significant changes in growth behavior were observed.

Figure 3: Plotting the OD₆₀₀ of E. coli DH5alpha each carrying different plasmids versus time in a volume of 1 mL LB medium containing 4 mM CuSO₄. Experiments were performed in a 24-well plate at 37 °C and 350 rpm (n=3).

No mutant carrying any of the BioBricks exhibits an advantage regarding the growth rate or the final OD₆₀₀ value. While BBa_K2638110 and BBa_K2638114 exhibit the same growth pattern as the mutant carrying an empty pSB1C3 vector, BBa_K2638112 and BBa_K2638109 showed significant lower growth rates and did not reach an OD₆₀₀ value as high as the other mutants. The construct BBa_K2638118 achieved an OD₆₀₀ value of 0.79.

Figure 4: Plotting the OD of E. coli DH5alpha each carrying different plasmid versus time in a volume of 1 mL LB medium containing 8 mM CuSO₄. Experiments were performed in a 24-well plate at 37 °C and 350 rpm (n=3).

Every mutant showed visible growth only during the first two hours at best. Afterwards, there was no significant increase in the OD₆₀₀ value. The highest OD₆₀₀ values were achieved by the construct BBa_K2638112, the lowest OD₆₀₀ values by the construct BBa_K2638109.
However, since no growth improving effect using our initial approaches against toxicity could be observed, we decided to conduct an experiment based on the viability of the cells at the measured points of time at the beginning of the experiment and after three, six and 9 hours of cultivation.
We grew E. coli containing the constructs BBa_K2638112, BBa_K2638110, BBa_K2638110, BBa_K2638118 and pSB1C3 in 10 ml LB medium supplemented with 30 ng/ml chloramphenicol for 9 hours and either 0 mM, 4 mM or 8 mM CuSO₄. Every three hours, a sample was taken and 50 µl were plated in the following dilutions: 10^-4, 10^-5 and 10^-6. Additionally, whenever a sample was taken, the optical density at 600 nm was measured.
In order to validate our results, we correlated the optical density and the determined CFU (Colony Forming Units).

Figure 5: Plotting of the optical density at 600 nm of cells grown in LB medium containing 30 ng/ml chloramphenicol and no cupric salt at 37 °C against the amount of colony forming units (CFU) in ml^-1, as well as linear fits (n=3).

The OD₆₀₀ clearly correlates with the amount of colony forming units (CFU) when no further cupric sulfate was added to the medium. The Pearson correlation coefficient R of the different constructs can be found in the table below, indicating a significant positive correlation between the two values. The adjoined coefficients of determination R² implicate that at least 90 % of all changes in the optical density result in a change in the amount of colony forming units. This value increases even further regarding the different implemented constructs to at least 97.3 %.

Table 1:R and R² values of the tested constructs.

	BBa_K2638112	pSB1C3	BBa_K2638114	BBa_K2638118	BBa_K2638110
R value	0.996	0.952	0.993	0.987	0.994
R2 value	0.992	0.907	0.985	0.973	0.989

The same measurements were performed for cells at cupric sulfate concentrations of 4 mM and 8 mM. The linear fits for the constructs grown in both supplemented media are shown in figures 6 and 7.

Figure 6: Plotting of the optical density at 600 nm of cells grown in LB medium containing 30 ng/ml chloramphenicol and 4 mM CuSO₄ at 37 °C against the amount of colony forming units (CFU) in ml^-1, as well as linear fits (n=3).

A positive correlation was indicated by determining the Pearson correlation coefficient and the coefficient for determination for the different constructs. However, according to its coefficient of determination, in the case of BBa_K2638110 only 83.7 % of all changes of the optical density could be referred to the changes in the amount of colony forming units,. according to its coefficient of determination. In general, a positive correlation between the optical density and the CFU could be clearly determined.

Table 2:R and R² values of the tested constructs.

	BBa_K2638112	pSB1C3	BBa_K2638114	BBa_K2638118	BBa_K2638110
R value	0.953	0.998	0.968	0.972	0.915
R2 value	0.908	0.997	0.937	0.945	0.837

In contrast to those cells grown in the absence of cupric sulfate and in the presence of 4 mM cupric sulfate, no positive correlation between the optical density and the CFU could be observed. Instead, the cells grown in LB medium with 8 mM cupric sulfate were proven to show a negative correlation. This is clearly visible in figure 7 (see below).

Figure 7: Plotting of the optical density at 600 nm of cells grown in LB medium containing 30 ng/ml chloramphenicol and 8 mM CuSO₄ at 37 °C against the amount of colony forming units (CFU) in ml^-1, as well as linear fits (n=3).

The negative correlation was revealed by Pearson correlation coefficient R and coefficient of determination R².

Table 3:R and R² values of the tested constructs.

	BBa_K2638112	pSB1C3	BBa_K2638114	BBa_K2638118	BBa_K2638110
R value	-0.576	-0.955	-0.817	-0.952	-0.924
R2 value	0.332	0.913	0.668	0.906	0.853

R2 was less than half as big as the R2 values of the other constructs and of all constructs at different concentrations of cupric sulfate. The negative correlation was likely due to cells dying at higher copper concentrations but not being degraded immediately so that the OD600 value suggests a higher cell density than viability. Therefore, the correlation of every construct shifts to the negative area. Furthermore, the highest R and R2 values have been determined for the mutant carrying the empty pSB1C3 vector.

Figure 8: Bar chart of the CFUs of the tested constructs in LB medium supplemented with either 0 mM or 4 mM cupric sulfate at 37 °C (n=3).

No significant difference in growth or viability in comparison to the empty pSB1C3 vector was observed for any tested construct at a concentration of either 0 mM or 4 mM cupric sulfate. According to the previously recorded growth trends, the growth rate decreased visibly with a rising concentration of copper ions. However, since no effect significantly improving the amount of colony forming units compared to the wildtype could be determined for these concentrations, the CFU at 8 mM cupric sulfate was evaluated.

Figure 9: Bar chart of the CFUs of the tested constructs in LB medium supplemented with 8 mM cupric sulfate at 37 °C (n=3), as well as the assigned significance levels to explain which constructs differ significantly from each other.

The number of colony forming units at the beginning of the measurement, after 0 hours, already shows indicates a great difference. Furthermore, the CFU in the course of time reveals differences for certain constructs in comparison to the wildtype. BBa_K2638112 and BBa_K2638118 appear to be more viable after 3, 6 and 9 hours of cultivation in 8 mM cupric sulfate containing medium. However, since no growth appeared to occur, the evaluation of this data concentrates on the preservation of the existing biomass rather than to improve its growth capabilities. After all, the cells are meant to be incubated and not cultivated in our remediation process of filtering water.
To examine the collected data regarding the influence of time and tested constructs, we conducted a two-factor ANOVA (analysis of variance) with repeated measurements for both factors. The aim is to discard the null hypothesis stating that there is no significant difference between the temporal developments of the tested constructs. Therefore, we conducted Mauchly’s Test of Sphericity to determine the samples sphericity. The test suggested highly with high significance to accept the assumption of sphericity with a significance level of .000 meaning that a correction of values by Greenhouse-Geisser was needed. The test of Within-Subject Effects resulted in the conclusion that the applied construct as well as the combination of the temporal development and the construct has a significant influence on the amount of colony forming units. In case of the construct, the influence was determined to be even highly significant. The constructs, the temporal development and the combination of those factors were examined, and each assigned to a significance level.
In conclusion, we were able to develop two composite parts which increase a cell’s tolerance for elevated concentrations of copper. This ameliorated tolerance could be verified for concentrations up to 8 mM of CuSO4 for a duration of up to 9 hours. This is a great first approach to realize our project and enable our cells to survive longer in the mining drainage. As our composite can also be used by iGEM teams in the future, we deem these results to be very useful to the community. Further applications could also be in the remediation of contaminated soils and seas, as well as industrial applications and processes in a heavy metal rich environment.