Team:ATOMS-Turkiye/Results1

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<div class="technology">1. HRE</div>
<div class="technology">1. HRE</div>
<div class="thelanguage">
<div class="thelanguage">
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<p>The assembled PA2652 construct (<a href="http://partsregistry.org/Part:BBa_K515102"><b>BBa_K515102</b></a>) and non-codon optimised mcpS (in pRK415 backone vector), have been inserted and tested for functionality in <i>E. coli</i> DH5α obtained from New England Biolabs. We carried out several tests in an attempt to characterise the rewired chemotaxis towards L(-)malic acid. We separated testing of chemotaxis towards malate into behavioural, qualitative &amp; quantitative analyses. </p>
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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)</li>
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<ul><li>We aimed to demonstrate its functionality by inserting it into pTRE-Luc vector.</li>
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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. </li>
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</ul>
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<li>The design of our vector possessing our part is shown below.</li>
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<img src="https://static.igem.org/mediawiki/2014/d/d5/ATOMS-Hre_results_1.jpg">
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<li>We started our experiments by cloning our parts. The forward primer is designed to produce HRE sequence inserted before the CMV promoter sequence presenting on pTRE vector. Reverse primer is synthesized to produce the cloning region of the vector. HRE-CMV sequence is cloned from pTRE vector via performing PCR. </li>
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<li>The PCR product is purified with phenol chloroform method and, afterwards, is cut with XhoI and BamHI restriction enzymes. The product is ligated with pTRE-Luc vector been cut with same enzymes and been treated with Antarctic phosphatase. The ligation product is inserted in DH5alfa strain and we performed colony PCR from the plate. </li>
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<table class="blueborder">
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<tr>
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<td>
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<div>
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<p><b>Expected</b></p>
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<img src="https://static.igem.org/mediawiki/2014/8/89/ATOMS-Hre_2.png" />
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<p>To understand which colony our gene is inserted among the colonies that we transformated HRE-pTRE-luciferase vector, we expected the picture above when we perform PCR when we use pTRE-Luc forward and MCS reverse primers.
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</p>
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</div>
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</td>
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<td>
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<div>
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<p><b>Experimented</b></p>
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<img src="https://static.igem.org/mediawiki/2014/d/da/ATOMS_hre_3.png" />
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<p>From the samples we perform colony PCR by using pTRE-Luc forward and MCS reverse primers, we obtained a band in 428 bp line. This image proves that our HRE sequnce is inserted into the vector, successfully.
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</p>
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</div>
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</td>
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</tr>
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</table>
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Revision as of 23:40, 17 October 2014

Sensing

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1. HRE
  • 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.
  • We started our experiments by cloning our parts. The forward primer is designed to produce HRE sequence inserted before the CMV promoter sequence presenting on pTRE vector. Reverse primer is synthesized to produce the cloning region of the vector. HRE-CMV sequence is cloned from pTRE vector via performing PCR.
  • The PCR product is purified with phenol chloroform method and, afterwards, is cut with XhoI and BamHI restriction enzymes. The product is ligated with pTRE-Luc vector been cut with same enzymes and been treated with Antarctic phosphatase. The ligation product is inserted in DH5alfa strain and we performed colony PCR from the plate.

    • Expected

    To understand which colony our gene is inserted among the colonies that we transformated HRE-pTRE-luciferase vector, we expected the picture above when we perform PCR when we use pTRE-Luc forward and MCS reverse primers.

    Experimented

    From the samples we perform colony PCR by using pTRE-Luc forward and MCS reverse primers, we obtained a band in 428 bp line. This image proves that our HRE sequnce is inserted into the vector, successfully.

    [Collapse]

    2. KB

    We are currently testing the chemotactic response of our chassis to physiologically relevant malate concentration levels below 10-6 M. To obtain these results we have reassessed the data collection method based on flow cytometry, because this should speed up the process of data collection and accuracy. To distinguish our bacteria from the background particles we have cotransformed our chassis with a low copy plasmid pTAC1 containing GFP, so our cells can be uniquely visualised using flow cytometer even if their size is similar to the size of backround particles. Hopefully this new strategy will produce results for presentation in Boston.

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    3. ODD

    As the amount of IAA needed for enhancing plant growth depends on whether our bacteria are producing the compound outside or inside the plants, we attempted to replicate these findings.

    [Collapse]

    Sensing

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    4. PLAT

    [1] National Institute of Health. (no date) Model Organisms for Biomedical Research. [Online]. Available from: http://www.nih.gov/science/models/arabidopsis/index.html [Accessed 21st September 2011].

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    5. Aprotinin

    Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat en ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. Aenean fermentum, elit eget tincidunt condimentum, eros ipsum rutrum orci, sagittis tempus lacus enim ac dui. Donec non enim in turpis pulvinar facilisis. Ut felis. Praesent dapibus, neque id cu

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    6. SOD

    Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat en ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. Aenean fermentum, elit eget tincidunt condimentum, eros ipsum rutrum orci, sagittis tempus lacus enim ac dui. Donec non enim in turpis pulvinar facilisis. Ut felis. Praesent dapibus, neque id cu

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    7. GPX

    Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat en ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. Aenean fermentum, elit eget tincidunt condimentum, eros ipsum rutrum orci, sagittis tempus lacus enim ac dui. Donec non enim in turpis pulvinar facilisis. Ut felis. Praesent dapibus, neque id cu

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    Geri İleri

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