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− | This experiment was conducted in order to examine the influence of the constructs on the growth behavior of <i>E. coli</i> in general. The growth of strains carrying the constructs mentioned in figure | + | This experiment was conducted in order to examine the influence of the constructs on the growth behavior of <i>E. coli</i> in general. The growth of strains carrying the constructs mentioned in figure 7 in absence of cupric salt was examined. No significant difference between the highest OD<sub>600</sub> values of the cells could be observed. However, cells carrying plasmids under the control of the pBAD promoter (BBa_K2638112) showed a different growth pattern since they can rely on another carbon source other than those regularly appearing in LB medium. This leads to two consecutive exponential phases resulting in similar OD<sub>600</sub> values compared to those only relying solely on the carbon sources of LB medium. Other than that, no significant changes in growth behaviour were observed. |
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− | This experiment was conducted to evaluate the general effect of the applied constructs on growth in presence of CuSO<sub>4</sub>. No mutant carrying any of the BioBricks shown in figure | + | This experiment was conducted to evaluate the general effect of the applied constructs on growth in presence of CuSO<sub>4</sub>. No mutant carrying any of the BioBricks shown in figure 8 exhibits an advantage regarding the growth rate or the final OD<sub>600</sub> value compared to the strain carrying the empty plasmid. While BBa_K2638110 and BBa_K2638114 exhibit the same growth pattern as the strain carrying an empty pSB1C3 vector implying that these BioBricks do not exhibit any effect on the resistance against elevated concentrations of copper ions. BBa_K2638112 and BBa_K2638109 however showed significant lower growth rates and did not reach an OD<sub>600</sub> value as high as the other strains. Therefore, they appear to posses a negative effect on the resistance against copper ions. The construct BBa_K2638118 does not differ significantly from the strain with the empty vector and does not reach an OD<sub>600</sub> value as high. A negative effect on the copper resistance can therefore not be excluded. |
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− | To evaluate the effect of the constructs in concentrations of CuSO<sub>4</sub> lethal to <i>E. coli</i>, we observed the growth of the strains mentioned in figure | + | To evaluate the effect of the constructs in concentrations of CuSO<sub>4</sub> lethal to <i>E. coli</i>, we observed the growth of the strains mentioned in figure 9 in LB medium containing 8 mM CuSO<sub>4</sub>. The results can be viewed in figure 9. Every strain showed visible growth only during the first two hours at best. Afterwards, there was no significant increase in the OD<sub>600</sub> value. The highest OD<sub>600</sub> values were achieved by the construct BBa_K2638112, carrying the genes <i>gor</i>, <i>btuE</i> and <i>gshB</i>, the lowest OD<sub>600</sub> values by the construct BBa_K2638109, carrying the gene <i>crs5</i>.</br> |
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<h2>Viability</h2> | <h2>Viability</h2> | ||
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− | The same measurements were performed for cells at CuSO<sub>4</sub> concentrations of 4 mM and 8 mM. The linear fits for the constructs grown in both media are shown in figures | + | The same measurements were performed for cells at CuSO<sub>4</sub> concentrations of 4 mM and 8 mM. The linear fits for the constructs grown in both media are shown in figures 10 and 11. |
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− | In contrast to those cells grown in the absence of CuSO<sub>4</sub> and in the presence of 4 mM CuSO<sub>4</sub>, no positive correlation between the optical density and the CFU could be observed. Instead, the cells grown in LB medium with 8 mM CuSO<sub>4</sub> were proven to show a negative correlation, clearly demonstrating the lethal effect on cells. This is clearly visible in figure | + | In contrast to those cells grown in the absence of CuSO<sub>4</sub> and in the presence of 4 mM CuSO<sub>4</sub>, no positive correlation between the optical density and the CFU could be observed. Instead, the cells grown in LB medium with 8 mM CuSO<sub>4</sub> were proven to show a negative correlation, clearly demonstrating the lethal effect on cells. This is clearly visible in figure 12 (see below). |
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Revision as of 02:08, 18 October 2018
Toxicity Results
Short Summary
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).
Growth experiments
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. Furthermore, heavy metals promoted reactive oxygen species (ROS) formation from processes such as Fenton chemistry and Haber-Weiss reactions. In our modeling regarding the accumulation of copper ions by the importer OprC, we came to the conclusion that the cells would have to incubate too long in the mining drainage, ultimately leading to cell death in a short period of time. Therefore, a sophisticated approach to increase the tolerance against heavy metals is desired which is necessary for cases where biological systems are deployed in heavy metal rich environments. Since we worked in our project with the heavy metals copper, silver, gold and iron, we evaluated several approaches of applying anti-oxidants against the generation of ROS.
We decided to set a focus on the accumulation of copper ions due to its low costs and easy solubility. Additionally, its toxicity is lower than that of silver and gold. Hence there is a broader spectrum in which anti-toxic measures against the generation of ROS 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 (pSB1C3+pTet+CRS5), BBa_K2638112 (pSB1C3+pBAD+gshB+gor+btuE), BBa_K2638114 (pSB1C3+pTet+soxR+oxyR), BBa_K2638110 (pSB1C3+gshB+pcs1) and BBa_K2638118 (pSB1C3+sodA+katG).
Viability
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 |
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 |
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 |