Team:ATOMS-Turkiye/Results

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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.

    We cloned kB-RE and inserted it into the upstream region of CMV mini promoter as it’s shown above.

    NF-kappaB (NF-kB) was synthesized to GenScript™ company and it came in pUC57 plasmid. We digested it with BamHI & PstI and exposed it to Antarctic phosphatase.

    Afterwards, we purified our part via phenol chloroform method. We performed the same procedures onto the pTRE-luciferase vector. Eventually, we ligated them.

    We inserted our plasmid (pTRE-luciferase kB-RE) into DH5α strain and performed colony PCR by using CMV forward and kB reverse primers. At the end of this experiment, we expected a band seen in the 20-30 bp line.

    And we observed correct bands in the expected region.

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    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.

    Sentetik olarak sentezlenen ve pUC57 vektörü içerisinde gönderilen tPA genini EcorI ve BamHI enzimleri ile kesip pTRE vektörüne ligation yaptık ve tekrar DH5-α suşuna transforme ettik. Ligation doğruluğunu kontrol etmek amacıyla colony PCR yaptık.

    CLONING CONTROL-2

    Expected

    The received genes were ligated with the pTRE vector, and then transformated to E.coli strands DH5-α. To test the validity of the transformation, we again applied colony PCR through CMV forward and SV40 reverse primers. The expected base length was 1984 bp.

    Experimented

    Colony PCR was applied to the inserts acquired from transformation and ligation. As it can be seen in the figure above, the second colony contains the appropriate base length in respect to the ladder.

    WESTERN BLOTTİNG

    Expected

    The confirmed genes were transfected into HEK293 cells. The lysates acquired from the cells were run through Western Blot. tPA, that is known to have 63 kD, was expected to have the image on the left.

    http://www.emdmillipore.com/TR/en/product/Anti-tPA-%28Tissue-Plasminogen-Activator%29-Antibody%2C-clone-GMA-043,MM_NF-05-883

    Experimented

    The bands in the Western Blot were accurate. The transfection of the inserts were verified and assays of tPA were prepared from the lysates.

    After having proven the presence of tPA expression in the cells transfected with Western Blotting, the concentration of the amount of tPA in the cell and the amount of secreted tPA were measured using the ‘Human tPA Activity Kit’ to show that the expressed proteins are functionally active.

    The real parameter of the measurement in the Assay was the product of the reaction of the plasmin enzyme. Since tPA shifts the inactive plasminogen to active plasmine, the measured value also presents tPA activity. The yield gives absorbance at 405 nm.

    tPA ASSAY

    Experimented

    tPA assay sonucuna göre tPA enziminin hücremizde üretildiğini ve hücre dışına salgılandığını kanıtlayarak tPA’nın fonksiyonel olarak aktif olduğunu ve hücre içinde üretilerek hücre dışına salgılandığını kanıtladık.

    Through the result of the tPA Assay, we proved that the tPA is functionally active through showing that tPA was produced in the cell and was secreted out of the membrane.

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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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