BioBricks

style="text-indent:20px"> General design of our project has been architected; thus it is the time to learn more about the individual parts of the big picture.

Hypoxia Respond Element (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 intend to add several HREs to the upstream region of weak constitutive promoter, PminiCMV, in order to design a novel hypoxia inducible promoter. We expect that in normal conditions with enough oxygen presence, PminiCMV promoter will express very small amount of mRNA which will be degraded soon conceivably. However, in hypoxic conditions, more transcription factors will hopefully bind to our designed HRE sites to enhance the transcription rate of PminiCMV promoter.
• According to the studies in the literature, HRE sequence has provided significant high transcription rate in hypoxic cells when it was attached on the upstream region of Simian virus 40 (SV40) promoter. (Tang et al. 2002) In this study, plasmids which contain HRE inducible promoters were presented in cell lines and the amount of these plasmids were increased gradually to test whether the detection level and transcription rate of hypoxia would raise. As a result, the production rate of reporter gene attached to HRE promoters increased 410 times more. This result encouraged us to use these sequences as novel, efficient and powerful hypoxic promoters in human cells.

Reactive oxygen species(ROS) are effecting NFKB proteins and then this effection inducing Kappa ß response element(our ROS inducible promoter called KBRE) to product our antioxidant therapy proteins

Oxygen Dependent Degradation Domain (ODD)

• The second mechanism includes a few understructure elements called TetR-VP16 complex and two different plasmids, pTet-off and pTRE vectors. In pTet-off plasmid, PCMV constitutive promoter with medium strength codes TetR-VP16 protein complex which can bind its respond element present in second plasmid, pTRE. Tetracyclin respond element (TetRE) is a protein binding domain which allows the binding of TetR component of the protein complex. Whereas, VP16 component works as a transcription activator for the weak constitutive promoter (PminiCMV) existing in pTRE vector. TetR-VP16 protein complex can activate this weak promoter in pTRE vector.
• ODD is a peptide about 200 amino acid long which includes a hydroxylation site of prolyl hydroxylase. In normal oxygen levels, hydroxylation from this site causes peptide to be targeted by ubiquitine-proteosome degradation system to be eliminated swiftly. However, in hypoxic conditions, hydroxyl subgroups cannot be added to ODD domain which results as the survival and accumulation of peptide and the components cohesive to it. (Huang et al. 1998)
• We aim to insert an additional peptide called oxygen dependent degradation domain (ODD domain) between TetR and VP16 components. With this mechanism, TetR-ODD-VP16 complex can only initiate the transcription of only weak promoter in hypoxic conditions because of the degradation of the complex by proteasome in oxygen presence. ith this, protein complex is expected to behave like a hypoxia inducer.

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Tissue Plasminogen Activator (tPA)

• It has already been told that this protein is the most potent and efficient thrombolytic agent popular in clinical practice. It does this by activating plasminogen, a common anticoagulative factor zymogen found in normal bloodstream. By the conversion of plasminogen into plasmin, an enzyme that catalyzes the reaction of the cleavage of fibrin, the main clot component, thrombolysis begins to degrade the clot. (IUBMB Enzyme Nomeclature 1992; MetaCyc 2014) This activity has been shown synthetically in E.coli and experimentally in vitro. (Pennica et al. 1983; Van de Werf et al. 1984)

Superoxide Dismutase-1 (SOD-1)

• SOD is a powerful and essential antioxidant enzyme which converts and scavenges free radicals and reactive oxygen species (ROS) into hydrogen peroxide (H2O2), slightly less harmful compound in order to convey it to the second step reaction which detoxifies H2O2 into water. (Batelli et al. 1972; Baudry et al. 1993; Gonzalez et al. 1995; Doctrow et al. 1996)
• In our body cells, there are three different subtypes of SOD enzyme in different cellular levels. SOD-1 exists in cytoplasm, SOD-2 in mitochondria and SOD-3 is in extracellular matrix. We have chosen SOD-1 to take advantage of easy interference and activity capacity due to its location. Additionally, there is evidence in the literature for the synthetic production of SOD-1 enzyme by transfecting human cells. (Shuvaev et al. 2013)

Glutathione peroxidase-1 (GPx-1)

• GPx is a known antioxidant enzyme abundant in human body cells which drives the H2O2 scavenge into water. (Mills 1957; Sunde 1997, IUBMB Enzyme Nomenclature 1989)
• Glutathione (GSH) is a peptide consisting of three amino acid residues, glutamate, cysteine and glycine, respectively. It is a natural cellular component for preventing damage to organelles caused by reactive oxygen species, free radicals or peroxides.
• In cell, GSH appears in different components or biochemical structures. GSH is a cofactor for GPx enzyme that the thiol group of cysteine in GSH is able to donate a reducing equivalent (H++ e−) to other unstable molecules, such as reactive oxygen species. In donating an electron, GSH itself becomes reactive, but readily reacts with another reactive GSH to form glutathione disulfide. (GSSG)
• GSH can be regenerated from GSSG by the enzyme glutathione reductase (GSR) which uses NADPH cofactor. The scheme of reduction cascade can be seen in figure X:
• In literature, GPx-1 can be overexpressed in transfected cells and produced synthetically. This recombinant enzyme is shown to work against hydrogen peroxide, lipid peroxide and drugs interfering with redox cycle. (Taylor et al. 1993; Kelner et al. 1995)

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Aprotinin

• Also known as pancreatic trypsin inhibitor or serine protease inhibitor, aprotinin is a potent protease inhibitor. According to our researches, aprotinin prevents the formation of xanthine oxidase (XO) from xanthine dehydrogenase (XDH). XO catalyzes the end step of xanthine degradation pathway resulting with ROS formation. In fact, about 80% of ROS during oxidative stress is produced by this enzyme. Furthermore, harmful free radicals such as peroxynitrite can be built up owing to the products of XO. Such free radicals have been found to react with DNA, proteins, and cells, causing cellular damage or even toxicity.
• Another reaction catalyzed by xanthine oxidase is the decomposition of S-Nitrosothiols (RSNO), a reactive nitrogen species, to nitric oxide (NO), which reacts with a superoxide anion to form peroxynitrite under aerobic conditions, additionally.This enzyme catalyzes the oxidation of hypoxanthine to xanthine and can further catalyze the oxidation of xanthine to uric acid. Both XO and XDH enzymes play an important role in the catabolism of purines.
• Inhibition of xanthine oxidase has been proposed as a mechanism for improving cardiovascular health. (Dawson et al. 2006) A study found that patients with chronic obstructive pulmonary disease (COPD) had a decrease in oxidative stress, including glutathione oxidation and lipid peroxidation, when xanthine oxidase was inhibited using allopurinol. (Heunks et al. 1999) Oxidative stress can be caused by hydroxyl free radicals and hydrogen peroxide, both of which are byproducts of XO activity. (Higgins et al. 2009)

Kappa-B Respond Element (kB-RE)

• After removing the clot, we need an additional sensor system to compensate the hypoxia response elements transcription rates for antioxidant enzymes and proteins. Thus, we targeted to sense the ROS presence to conduct an efficient and swift solution.
• Nuclear factor kappa-B (NF-kB) is a transcription factor complex of hypoxia inducible factor-1 alfa (HIF-1α) protein, which consists of two subunits, p50 and p65. This peptide is detained in cytoplasm by a complementary peptide called IkB. (Kabe et al. 2005) This detention prevents the activation of related promoter sequence existing in the cell nucleus which is named kappa-B response element (kB-RE).
• 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. (Gilmore 2006)
• It is later described that one of these stimulants is the presence of ROS. (Bonello et al. 2007) We want to use this advantage to design a novel ROS inducible promoter in human cells in iGEM to be able to sense oxidative stress.

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Composite Parts

• As a full designed system, we aim to combine our parts to gain maximum efficiency and robustness. We can categorize our parts into two classes: sensor genes and activist proteins.
• HRE, ODD and kB-RE parts are sensor genes to detect hypoxia and ROS presence, respectively. As we predict that the yield of our activist proteins must continue constitutively to conduct a successful treatment, we want to combine our all activist proteins with our sensor genes.
Click Here to read about the summary of our parts and respective part numbers.

References