Difference between revisions of "Part:BBa K1456002"

Latest revision as of 19:33, 20 October 2014

Glutathione Peroxidase-1 (GPX1)

GPx is a known antioxidant enzyme abundant in human body cells which drives the H2O2 scavenge into water. (Mills 1957; Sunde 1997, IUBMB Enzyme Nomenclature 1989)
Glutathione (GSH) is a peptide consisting of three amino acid residues, glutamate, cysteine and glycine, respectively. It is a natural cellular component for preventing damage to organelles caused by reactive oxygen species, free radicals or peroxides.
In cell, GSH appears in different components or biochemical structures. GSH is a cofactor for GPx enzyme that the thiol group of cysteine in GSH is able to donate a reducing equivalent (H++ e−) to other unstable molecules, such as reactive oxygen species. In donating an electron, GSH itself becomes reactive, but readily reacts with another reactive GSH to form glutathione disulfide. (GSSG)
GSH can be regenerated from GSSG by the enzyme glutathione reductase (GSR) which uses NADPH cofactor. The scheme of reduction cascade can be seen in figure X:
In literature, GPx-1 can be overexpressed in transfected cells and produced synthetically. This recombinant enzyme is shown to work against hydrogen peroxide, lipid peroxide and drugs interfering with redox cycle. (Taylor et al. 1993; Kelner et al. 1995)

The bands acquired through the Western Blotting were fitting the expected results. The cell lines, that now had their transfections verified, were purfied for the preparation of GPx assays.
The amounts of protein acquired were as the charts above and the these data were taken into consideration in the preparation of the assays.
A functional assay of GPx was done through NADPH to measure GPx activity. The same amount of genes were transferred to all samples but varying amount of Na2SeO3 was prepared in the samples, with 2 samples containing different Na2SeO3 concentrations and one control group. The GPx gene was not transfected to the control group. From these 3 groups, the assay of the control group gave the lowest yield as expected; the sample of 500 nM Na2SeO3 produced the mid-result. The sample of 1000 nM Na2SeO3 yielded the highest activity results in the assay. These results prove that the GPx production is directly proportional to the amount of added Na2Se2O3 and that the GPx enzyme was produced functionally. Sequence and Features
Assembly Compatibility:
- 10
  COMPATIBLE WITH RFC[10]
- 12
  INCOMPATIBLE WITH RFC[12]
  Illegal NheI site found at 31
  Illegal NotI site found at 5
- 21
  COMPATIBLE WITH RFC[21]
- 23
  COMPATIBLE WITH RFC[23]
- 25
  INCOMPATIBLE WITH RFC[25]
  Illegal NgoMIV site found at 89
- 1000
  INCOMPATIBLE WITH RFC[1000]
  Illegal BsaI site found at 471
  Illegal SapI site found at 346

@@ Line 8: / Line 8: @@
   <li>GSH can be regenerated from GSSG by the enzyme glutathione reductase (GSR) which uses NADPH cofactor. The scheme of reduction cascade can be seen in figure X:</li>
   <li>In literature, GPx-1 can be overexpressed in transfected cells and produced synthetically. <b>This recombinant enzyme is shown to work against hydrogen peroxide, lipid peroxide and drugs interfering with redox cycle.</b> (Taylor et al. 1993; Kelner et al. 1995)</li>
 <center>https://static.igem.org/mediawiki/2014/thumb/4/40/ATOMS-Biobricks-XO-SOD-GPx-Aprotinin.jpg/800px-ATOMS-Biobricks-XO-SOD-GPx-Aprotinin.jpg</center>
+<li>The bands acquired through the Western Blotting were fitting the expected results. The cell lines, that now had their transfections verified, were purfied for the preparation of GPx assays.
+<center>https://static.igem.org/mediawiki/2014/0/0e/ATOMS_gpx_results_16.jpg</center>
+<li>The amounts of protein acquired were as the charts above and the these data were taken into consideration in the preparation of the assays.
+<center>https://static.igem.org/mediawiki/2014/d/df/ATOMS_gpx_results_17.jpg</center>
+<li>A functional assay of GPx was done through NADPH to measure GPx activity. The same amount of genes were transferred to all samples but varying amount of Na2SeO3 was prepared in the samples, with 2 samples containing different Na2SeO3 concentrations and one control group. The GPx gene was not transfected to the control group. From these 3 groups, the assay of the control group gave the lowest yield as expected; the sample of 500 nM Na2SeO3 produced the mid-result. The sample of 1000 nM Na2SeO3 yielded the highest activity results in the assay. These results prove that the GPx production is directly proportional to the amount of added Na2Se2O3 and that the GPx enzyme was produced functionally.
 <!-- Add more about the biology of this part here