Designed by: Matthias Otto Group: iGEM18_Bielefeld-CeBiTec (2018-09-02)
Glutathione gamma-glutamylcysteinyltransferase 1
Short Summary
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, containing the superoxide dismutase A and catalase under the control of a strong RBS and the pBAD promoter on pSB1C3, or BBa_K2638112, containing the glutathione synthetase, the glutathione peroxidase and the glutathione reductase under the control of a strong RBS and the pBAD promoter on pSB1C3, does are not subject to increased growth rate but to a significantly higher viability when the cells are exposed to elevated concentrations of heavy metals, namely CuSO4.
Phytochelatin synthase
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 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: 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. This was carried out via this protocol. For the enzyme assay three samples contained 1 mM GSH and another three samples contained 5 mM GSH. The phytochelatin synthase was activated by adding 500 µM CdCl2. The last three samples contained 5 mM GSH and were activated by addition of 50 µM CuSO4. The blank sample contained no phytochelatin synthase but 500 µM CdCl2.
Figure 3: Chromatogram of mass spectrometry connected with a liquid chromatography of glutathione and the different phytochelatins.
In figure 3 the different m/z ratio of the substrate glutathione and the different phytochelatins (PC) can be seen. The difference of the m/z ratio between glutathione and PC2 is about 232. The difference is the same between PC2, PC3, PC4 and PC5. In this Figure it becomes clear that the phytochelatin synthase was catalytically active and produced different phytochelatins with n = 5.
Figure 4: Chromatogram of liquid chromatography of glutathione and the different phytochelatins.
Further Figure 4 shows the retention time of the substrate glutathione and the different phytochelatins. The chromatogram is from the assay sample with 1 mM glutathione. The highest peak is number 4 which is glutathione. This reveals that the substrate was still available in high amounts and not the whole amount of glutathione was converted to phytochelatins. Number 3 shows the second highest peak which is PC2 with a peak area of 479843. The peak area of peak number 1 is with 197103 more than half as high as peak number 3. The lowest peak area reveals peak number 2 with a peak area of 50905. It becomes obvious that the amount of phytochelatins decreases as the number of γ-Glu-Cys moieties in the phytochelatins increases. The whole overview of the samples can be seen in Table 1. This table also shows that the three samples which were activated by CuSO4 contained PC2, PC3 and PC4. This ensures that the phytochelatin synthase can not only be activated by CDCl2 but also by CuSO4. In comparison with the Cadmium activated samples it becomes obvious that the peak area of the CuSO4 treated samples is lower.
Table 1: Phytochelatin assay was conducted with 1 mM and 5 mM of GSH. Three samples with 5 mM GSH were activated by 50 µM CuSO4 instead of 500 µM CdCl2.
