Team:ATOMS-Turkiye/Results1

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<p>We are currently testing the chemotactic response of our chassis to physiologically relevant malate concentration levels below 10<sup>-6</sup> 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.</p>
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<p>NF-kappaB (NF-kB) proteins comprise a family of structurally-related eukaryotic transcription factors that are involved in the control of a large number of normal cellular and organismal processes, such as immune and inflammatory responses, developmental processes, cellular growth, and apoptosis In some circumstances, NF-kB/IkB complex can be separated by external effects such as radiation, cellular stress, pathogens, inflammation etc. In this case, NF-kB can enter into nucleus and integrate with compatible kB-RE sites in order to initiate transcription.</p>
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<img src="https://static.igem.org/mediawiki/2014/5/5c/ATOMS_KB_results_1.png">
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<img src="https://static.igem.org/mediawiki/2014/thumb/9/98/ATOMS_KB_results_2.png/800px-ATOMS_KB_results_2.png">
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<p>KBRE partını yukarıdaki şekilde görüldüğü gibi CMVmini promotorun up stream kısmına klonladık.</p>
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<p>NF-kappaB (NF-kB) was synthesized to GenScript™ company and it came in pUC57 plasmid.we digested it with BamHI & PstI and fosfatını antartic ALP ile uzaklaştırdık.(CIP)after that we made phenol chloroform. Then  we digested our vector, pTRE-luciferase, with BamHI & PstI  and again we made phenol chloroform for it. Afterwards we ligated them.(pTRE-luciferase kB-RE)</p>
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<p>We transformated our plasmid(pTRE-luciferase kB-RE) to DH5α and made colony pcr with CMV forward and kB reverse primers. So we seperated our correct plasmid from the others which is not involved kb. Bu deney sonucunda 20-30 bp hizasında bant görmeyi hedefledik.</p>
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<img src="https://static.igem.org/mediawiki/2014/e/ef/ATOMS_KB_results_3.png">
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<p>  And we observed bands of KBRE at the correct possition of agarose gel.</p>
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<img src="https://static.igem.org/mediawiki/2014/2/21/ATOMS_KB_results_4.png">
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<li>pTRE Luciferase – KBRE  HEK 293 T hücre hattına transfekte edildi ve KBRE operatör dizisi TNF-α and H2O2 ile hücreler inkübe edilerek construct ın downstreamindeki reporter olan luciferasın üretilmesini hedefledik ve lüsiferaz assay uygulayarak üretilen lüsiferaz miktarını ölçtük. Bu deneyde stimulan olarak H2O2 kullandığımız zaman hücrelerimizin bir kısmı H2O2 ye bağlı olarak öldü ancak elde edilen sonucumuz anlamlıydı. Bununla beraber literatürde KBRE’in çalıştığını göstermek amacıyla uygulanan efficient bir stimulan olan TNFα[1]’yı kullandık. Elde ettiğimiz sonuçlar aşağıda gösterilmiştir.</li>
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<img src="https://static.igem.org/mediawiki/2014/d/d4/ATOMS_KB_results_5.png">
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<li>Yukarıda görüldüğü gibi KBre nin güçlü bir uyaranı olan TNFα yı ve etkili bir Reactive oxygen türü olan H2O2 yi hücrelere exposure yaptığımızda KBRE nin down streamindeki reporter olan luciferase ın aktivitesinde anlamlı bir artış görmekteyiz. Bu sonuçta response elementlerine novel olarak bir araya getirip igem library ye bir regülatör olarak sunduğumuz KBRE in başarılı bir şekilde çalıştığını göstermektedir.</li>
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<h3>Reference</h3>
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<li>1. Zhang Y., Yang X., Bian F., et al. TNF-α promotes early atherosclerosis by increasing transcytosis of LDL across endothelial cells: Crosstalk between NF-κB and PPAR-γ. Journal of Molecular and Cellular Cardiology 72 (2014) 85–94</li>
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Revision as of 00:43, 18 October 2014

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Cloning
  • Here is our results page, you can analyze our constructed vectors for coding proteins and the agarose gel electrophoresis result of inserts ligated with interest vectors. TetR-VP16 double plasmid system is used in our project as an empowering system of expressing.
  • For Aprotinin, GPx, SOD and tPA;
  • For ODD;
  • For HRE and KB regulator genes;
  • This system includes few understructured elements called TetR-VP16 complex and two different plasmids, pTet-off and pTRE vectors. In the pTet-off plasmid, PCMV constitutive promoter codes for TetR-VP16 protein complex in medium strength which can bind to its responding element present in the second plasmid, pTRE. Tetracycline respond element (TetRE) is a protein binding domain which enables the binding of TetR component of the protein complex. Whereas, VP16 component works as a transcription activator for the weak constitutive promoter (PminiCMV) which exists in the pTRE vector. TetR-VP16 protein complex can activate this weak promoter in pTRE vector.

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    Sensing

    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.

    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.
  • 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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    2. KB

    NF-kappaB (NF-kB) proteins comprise a family of structurally-related eukaryotic transcription factors that are involved in the control of a large number of normal cellular and organismal processes, such as immune and inflammatory responses, developmental processes, cellular growth, and apoptosis In some circumstances, NF-kB/IkB complex can be separated by external effects such as radiation, cellular stress, pathogens, inflammation etc. In this case, NF-kB can enter into nucleus and integrate with compatible kB-RE sites in order to initiate transcription.

    KBRE partını yukarıdaki şekilde görüldüğü gibi CMVmini promotorun up stream kısmına klonladık.

    NF-kappaB (NF-kB) was synthesized to GenScript™ company and it came in pUC57 plasmid.we digested it with BamHI & PstI and fosfatını antartic ALP ile uzaklaştırdık.(CIP)after that we made phenol chloroform. Then we digested our vector, pTRE-luciferase, with BamHI & PstI and again we made phenol chloroform for it. Afterwards we ligated them.(pTRE-luciferase kB-RE)

    We transformated our plasmid(pTRE-luciferase kB-RE) to DH5α and made colony pcr with CMV forward and kB reverse primers. So we seperated our correct plasmid from the others which is not involved kb. Bu deney sonucunda 20-30 bp hizasında bant görmeyi hedefledik.

    And we observed bands of KBRE at the correct possition of agarose gel.

  • pTRE Luciferase – KBRE HEK 293 T hücre hattına transfekte edildi ve KBRE operatör dizisi TNF-α and H2O2 ile hücreler inkübe edilerek construct ın downstreamindeki reporter olan luciferasın üretilmesini hedefledik ve lüsiferaz assay uygulayarak üretilen lüsiferaz miktarını ölçtük. Bu deneyde stimulan olarak H2O2 kullandığımız zaman hücrelerimizin bir kısmı H2O2 ye bağlı olarak öldü ancak elde edilen sonucumuz anlamlıydı. Bununla beraber literatürde KBRE’in çalıştığını göstermek amacıyla uygulanan efficient bir stimulan olan TNFα[1]’yı kullandık. Elde ettiğimiz sonuçlar aşağıda gösterilmiştir.
  • Yukarıda görüldüğü gibi KBre nin güçlü bir uyaranı olan TNFα yı ve etkili bir Reactive oxygen türü olan H2O2 yi hücrelere exposure yaptığımızda KBRE nin down streamindeki reporter olan luciferase ın aktivitesinde anlamlı bir artış görmekteyiz. Bu sonuçta response elementlerine novel olarak bir araya getirip igem library ye bir regülatör olarak sunduğumuz KBRE in başarılı bir şekilde çalıştığını göstermektedir.

    • Reference

  • 1. Zhang Y., Yang X., Bian F., et al. TNF-α promotes early atherosclerosis by increasing transcytosis of LDL across endothelial cells: Crosstalk between NF-κB and PPAR-γ. Journal of Molecular and Cellular Cardiology 72 (2014) 85–94

    • [Collapse]

    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]

    Therapy

    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

    [Collapse]

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    Geri İleri

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