Difference between revisions of "Part:BBa K1456004:Experience"

 
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
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<li>Hypoxia response elements (HREs) are 234 bp long small DNA sequences present in most of the body cells which work as binding domains for hypoxia inducible factor-1α (HIF-1α), a common transcription factor found in our body cells released during hypoxic conditions. (Semenza et al. 1992)
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how you used this part and how it worked out.
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<li>We aimed to demonstrate its functionality by inserting it into pTRE-Luc vector.
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<li>We expect that HRE, as an enhancer, would activate the promoter existing on the downstream region of it, depending on the level of HIF1alfa in the media which is increased in hypoxic conditions.
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<li>The design of our vector possessing our part is shown below.
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==Luciferase Assay==
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<li>The vectors isolated from the colonies we identified correct are co-transfected into HEK293 and HepG2 cell lines. Transfected cells are incubated in hypoxic and normoxic conditions and the luminescence levels are measured by performing luciferase assay. To conduct hypoxic condition, we used 100 uM CoCl2. Here, the graphics we obtained after the measurement of luminescence is shown below.
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<center>[[File:Atoms hre 4.png|400px|]]</center> <br/>
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<li>According to the results, in hypoxic conditions comparing with normoxia, HRE been inserted into pTRE-luciferase vector shows 7 times more activity by producing more luciferase in HepG2 cell line. In HEK293 cell line, this activity is measured in hypoxia 1,5 times more than normoxia. This results prove that HRE sequence improves the production rate of desired protein in hypoxia comparing with normal oxygen levels.
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<center>[[File:Atoms hre 5.png|400px|]]</center> <br/>
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===Applications of BBa_K1456004===
 
===Applications of BBa_K1456004===

Revision as of 14:38, 21 October 2014

  • Hypoxia response elements (HREs) are 234 bp long small DNA sequences present in most of the body cells which work as binding domains for hypoxia inducible factor-1α (HIF-1α), a common transcription factor found in our body cells released during hypoxic conditions. (Semenza et al. 1992)
  • We aimed to demonstrate its functionality by inserting it into pTRE-Luc vector.
  • We expect that HRE, as an enhancer, would activate the promoter existing on the downstream region of it, depending on the level of HIF1alfa in the media which is increased in hypoxic conditions.
  • The design of our vector possessing our part is shown below.

    Luciferase Assay

  • The vectors isolated from the colonies we identified correct are co-transfected into HEK293 and HepG2 cell lines. Transfected cells are incubated in hypoxic and normoxic conditions and the luminescence levels are measured by performing luciferase assay. To conduct hypoxic condition, we used 100 uM CoCl2. Here, the graphics we obtained after the measurement of luminescence is shown below.
    Atoms hre 4.png

  • According to the results, in hypoxic conditions comparing with normoxia, HRE been inserted into pTRE-luciferase vector shows 7 times more activity by producing more luciferase in HepG2 cell line. In HEK293 cell line, this activity is measured in hypoxia 1,5 times more than normoxia. This results prove that HRE sequence improves the production rate of desired protein in hypoxia comparing with normal oxygen levels.
    Atoms hre 5.png


    Applications of BBa_K1456004

    User Reviews

    UNIQ7e1e71aaf3dd0b39-partinfo-00000000-QINU

    UNIQ7e1e71aaf3dd0b39-partinfo-00000001-QINU