Coding

Part:BBa_K1456003:Experience

Designed by: safa tapan   Group: iGEM14_ATOMS-Turkiye   (2014-07-21)

Western Blotting

Atoms sod 3.2.png

  • Our western Blotting results show a band at 18kDa meaning that our HEK 293 T cell line has been successful.
  • Functional assay of SOD was performed to test whether our enzyme was functioning as expected.
    Atoms sod 4.png

  • Dosage levels were adjusted whilst SOD- Ptre vector was transfected to HEK293 cell. 1. 125ng/24-well gene transfected to 105 cells present in well. 2. 250ng/24-well gene transfected to 105 cells present in well. 3. 500ng/24-well gene transfected to 105 cells present in well. The results show that the SOD1 gene is effected by the adjustement of dosage levels which in return proves that as the amount of transfected genes increase the gene expression also increases

    >SOD Assay

  • Superoxide Dismutase 1 changes the oxygen radicals into 02 and H2O2. Using this feature of SOD we placed the oxygen radicals into the same solution as tetrazolium dye which uses the radicals as substrates and enabled them to compete. Hence this allowed us to measure the functional activity of SOD produced by our transfected cells using the spectrophotometer at 560 nm.
    Atoms sod 2.png

    Atoms sod 6.png

  • Transfected Tet off - pTRE and Tet off - pTRE-SOD1 vectors were subjected to SOD assay and measured using the spectrophotometer at 560 nm. Cells which have the Tet off – pTRE SOD1 gene present can degrade the oxygen radicals 21 times faster. SOD assay was performed for the proteins obtained from the transfected SOD1 visible colour change could be seen.
    Atoms sod 7.png



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