The phytochelatin synthase produces phytochelatin which plays a major role in heavy metal detoxification processes in Arabidopsis thaliana. 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 an intein tag and chitin binding domain 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 DH5α 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).
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).