Team:ATOMS-Turkiye/Results1

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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 pHRE-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

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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.

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    Therapy

    4. tPA

    Tissue plasminogen activator (abbreviated tPA or PLAT) is a protein involved in the breakdown of blood clots. It is a serine protease (EC 3.4.21.68) found on endothelial cells and (hücreden plazmaya salgılanır), the cells that line the blood vessels. As an enzyme, it catalyzes the conversion of plasminogen to plasmin, the major enzyme responsible for clot breakdown. Because it works on the clotting system, tPA is used in clinical medicine to treat embolic or thrombotic stroke. Use is contraindicated in hemorrhagic stroke and head trauma.

    The question is, how can tPA enzyme activity be measure? We sought the answer to the question in the examination of the yield of a tPA catalyzed reaction. We searched the sector, in which we found the the Human tPA Activity Kit of the company ASSAYPRO that we then ordered and used. In the reaction which tPA catalyzed, we aimed to show that tPA was active through the transformation of plasminogen to plasmine.

    To acquire the tPA gene, the tPA forward and tPA revers primers were synthesized from the cDNA’s we were in possession of. Then, by also using these primers, we acquired the tPA genes through the PCR of the cDNA. The head and neck cancer cell line was used as the source for cDNA.

    GENE SYNTHESIS FROM cDNA OF 64A CELL LINE

    Expected

    The base length of tPA is 1762 bp’dir. The electrophoresis of the PCR was expected to show a base length of around 1700 bp. The image below shows the expected result of the electrophoresis.

    Experimented

    Through the primers that we ordered and the cDNA, we acquired tPA inserts. The insert was, as expected, portraying that the base length was around 1700 bp.

    Elde ettiğimiz insertleri ve pTRE vektörünü EcoRI ve BamHI enzimleri ile kestik. Ardından sahip olduğumuz ligation Protocolüne göre insert ve vektörü birleştirmeye çalıştık. Ligation neticesinde elde ettiğimiz plasmitleri E. coli’nin DH5-α suşuna transforme ettik. Transformasyon sonrasında ligationın doğruluğunu kontrol etmek amacıyla colony PCR yaptık.

    CLONING CONTROL-1

    Expected

    The acquired inserts were ligated to the pTRE vector and the ligate was transformated to the E.coli strands DH5-α. To confirm the results of the transformation, colony PCR was conducted using CMV forward and tPA revers primers. The picture above shows the expected base length of the insert, which was 1894 bp.

    Experimented

    The specified primers were put into colony PCR. As it can be seen in the results above, a right insert was not achieved. This process was repeated several times but no result was achieved. Seeing that ligation did not provide a solution to the problem, synthetically produced inserts were ordered.

    Expected

    The base length of tPA is 1762 bp’dir. The electrophoresis of the PCR was expected to show a base length of around 1700 bp. The image below shows the expected result of the electrophoresis.

    Experimented

    Through the primers that we ordered and the cDNA, we acquired tPA inserts. The insert was, as expected, portraying that the base length was around 1700 bp.

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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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    Expressional Check of Therapeutical Genes


  • His tagged Aprotinin (BPTI), SOD1, GPX1 and PLAT (tPA) was overexpressed in HEK 293T cells. 36 hours post transfection cells were collected and subjected to western blotting. Aprotinin, SOD1 and tPA were expressed. However, expression of GPX1 could not be observed. Previously, we realized that GPx-1 is a member of seleno-cystein proteins. To code seleno-cystein GPx has a internal stop codon. Expression in the absense of seleno-cystein GPX-1 expression stops at the internal stop codon thus, the his-tag at the and of GPx-1 can not be expressed. Therefore,we couldn’t see any band in westernblotting. Next , we planned to add sodiumselenite to induce production of seleno-cystein.

  • This is comassive blue flourorange view of blotted membrane.

  • The expression level of beta actin is the same among samples.

  • This is comassive blue flourorange view of blotted membrane.

  • We over expressed SOD-1 and tPA with different doses of SOD-1 and tPA expression vector. SOD-1 well responded to the increasing doses. tPA gave the same response to all different doses. To induce expression of GPx-1 the cells treated with different doses of sodiumselenite and GPx-1 proportionally response to the increasing doses of sodiumselenite with single dose of expression vector.

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    Geri İleri

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