Difference between revisions of "Team:BioMarvel/Description"
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| − | <h1>Description</h1> | + | <h1>Description</h1> |
| − | <p>Heart failure (HF) is a major public health problem, with a prevalence of over 5.8 million in the USA, and over 40 million worldwide (Lancet 388:1545-1602, 2016). Heart failure is when the heart is unable to pump sufficiently to maintain blood flow to meet the body's needs. Common causes of heart failure include coronary artery disease including a previous myocardial infarction (heart attack), high blood pressure, atrial fibrillation, excess alcohol use, infection, and cardiomyopathy of an unknown cause. Heart failure is a common, costly, and potentially fatal condition. (Lancet 365:1877–89, 2005). Early diagnosis of heart failure is essential for the successful medical treatment of the disease. However, current diagnosis of heart failure patients takes considerable time from initial sampling to detection results due to sample transportation time and operating costs. Also, there is no convenient and affordable service system for continuous monitoring of patient status.</p> | + | <p> |
| + | <span style="color:blue">Heart failure (HF) is a major public health problem, with a prevalence of over 5.8 million in the USA, and over 40 million worldwide</span> | ||
| + | <span style="color:red">(Lancet 388:1545-1602, 2016).</span> | ||
| + | Heart failure is when the heart is unable to pump sufficiently to maintain blood flow to meet the body's needs. Common causes of heart failure include coronary artery disease including a previous myocardial infarction (heart attack), high blood pressure, atrial fibrillation, excess alcohol use, infection, and cardiomyopathy of an unknown cause. | ||
| + | <span style="color:red">Heart failure is a common, costly, and potentially fatal condition. (Lancet 365:1877–89, 2005). Early diagnosis of heart failure is essential for the successful medical treatment of the disease.</span> | ||
| + | <span style="color:blue">However,</span> | ||
| + | current diagnosis of heart failure patients takes considerable time from initial sampling to detection results due to sample transportation time and operating costs. Also, there is no convenient and affordable service system for continuous monitoring of patient status. | ||
| + | </p> | ||
| − | <p>Therefore, biosensors that is portable and permits on-site analysis of samples are very useful for effective diagnosis and monitoring of heart failure patients. Among them, electrochemical immunosensors have several advantages of simplicity, inexpensive cost, accuracy, and high sensitivity. In the immunosensors, biological recognition events between antigens and antibodies immobilized on a sensing platform generate. | + | <p>Therefore, biosensors that is portable and permits on-site analysis of samples are very useful for effective <span style="color:blue">diagnosis and</span> monitoring of <span style="color:blue">heart failure</span> patients. <span style="color:blue">Among them,</span> electrochemical immunosensors have several advantages of simplicity, inexpensive cost, accuracy, and high <span style="color:blue">sensitivity. In the immunosensors,</span> biological recognition events between antigens and antibodies immobilized on a sensing platform <span style="color:blue">generate.</span> |
| − | </p> | + | </p> |
| − | <p>It is necessary to produce effective, high sensitivity, and low-cost immunosensors. Effective immobilization of antibodies on a sensing platform is crucial in designing immunosensors. Self-assembled monolayers (SAMs) method are widely used to immobilize antibodies on gold surfaces of a sensing area via covalent binding in biosensors, but the chemical method is very complex and takes a long time. Therefore, it is necessary to develop a simple method of antibody immobilization on gold sensing surfaces. To overcome this problem, we will presents a synthetic biology method by producing a novel fusion protein constructed by genetically fusing gold binding polypeptides (GBP) to protein G (ProG) as a crosslinker for effective immobilization of antibodies on gold sensing surfaces. The resulting GBP-ProG fusion protein will directly be self-immobilized onto gold surfaces via the GBP portion, followed by the oriented binding of antibodies onto the ProG domain targeting the Fc region of antibodies. As a result, we expect that the GBP-ProG protein can be a valuable crosslinker for simple and oriented immobilization of antibodies onto gold sensing surfaces without any surface chemical modification.</p> | + | <p>It is necessary to produce effective, high sensitivity, <span style="color:blue">and low-cost</span> immunosensors. Effective immobilization of antibodies on a sensing platform is crucial in designing immunosensors. Self-assembled monolayers (SAMs) method are widely used to immobilize antibodies on gold surfaces of a sensing area via covalent binding in biosensors, but <span style="color:blue">the chemical method</span> is very complex and takes a long time. Therefore, it is necessary to develop a simple method of antibody immobilization on gold sensing surfaces. To overcome this problem, we will presents a synthetic biology method by producing a novel fusion protein constructed by genetically fusing gold binding polypeptides (GBP) to protein G (ProG) as a crosslinker for effective immobilization of antibodies on gold <span style="color:blue">sensing</span> surfaces. The resulting GBP-ProG fusion protein <span style="color:blue">will</span> directly be self-immobilized onto <span style="color:blue">gold surfaces</span> via the GBP portion, followed by the oriented binding of antibodies onto the ProG domain targeting the Fc region of antibodies. As a result, we expect that the GBP-ProG protein can be a valuable crosslinker for simple and oriented immobilization of antibodies onto gold sensing surfaces without any surface chemical modification.</p> |
| − | <p> We created a vector capable of producing the fusion protein which includes GBP and ProG. PCR was conducted using plasmid pTGE (Park et al., Anal.Chem., 78:7197, 2006) and protein G gene of Streptocococcus G418 as a template. Eventually, 6His-GBP and Protein G was obtained the combined form of gene products. The DNA obtained by PCR was separated about 700 bps of DNA with electrophoresis. These were cut with two restrict enzymes, NdeI and BamHI, then cloned to plasmid pET-22b (+) cut with the same restriction enzyme to create recombinant plasmid pET-6HisGBP-ProG. </p> | + | <p> We created a vector capable of producing the fusion protein which includes GBP and ProG. PCR was conducted using plasmid pTGE (Park et al., Anal.Chem., 78:7197, 2006) and protein G gene of <span style="color:blue; font-style:italic;">Streptocococcus</span> G418 as a template. Eventually, 6His-GBP and Protein G was obtained the combined form of gene products. The DNA obtained by PCR was separated about 700 bps of DNA with electrophoresis. These were cut with two restrict enzymes, NdeI and BamHI, then cloned to plasmid pET-22b (+) cut with the same restriction enzyme to create recombinant plasmid pET-6HisGBP-ProG. </p> |
| − | <p>Then parts EcoR1 and PstI in the GBP-ProG sequence will be removed through DNA synthesis. Then PCR process for GBP-ProG sequence will be performed as a manufactured primer. These will be cut with two restricted enzymes, EcoRI and PstI, then clone to plasmid pSB1C3 cut with the same restriction enzyme to create recombinant plasmid pSB1C3-GBP-ProG. The vector produced above will be transformed to DH5α E. coli strain and then screened from LB plates with chloramphenicol. After the induction expression, the entire culture medium centrifugation and discard the upper layer. The remaining cells will be suspended in the solution and the cells will be crushed at 4 ℃ using an ultrasonic thresher. The crushed solution centrifugation at 4 ℃ for 30 minutes at 13,000 rpm to obtain the supernatant. Supernatant (total protein) will be refined using the Talon resin column and then quantified using the Bradford protein quantification method and dilute to 1mg/ml with a fixed buffer solution (PBS, pH 7.4). </p> | + | <p>Then parts EcoR1 and PstI in the GBP-ProG sequence will be removed through DNA synthesis. Then PCR process for GBP-ProG sequence will be performed as a manufactured primer. These will be cut with two restricted enzymes, EcoRI and PstI, then clone to plasmid pSB1C3 cut with the same restriction enzyme to create recombinant plasmid pSB1C3-GBP-ProG. The vector produced above will be <span style="color:blue">transformed</span> to DH5α E. coli strain and then screened from LB plates with chloramphenicol. After the induction expression, the entire culture medium centrifugation and discard the upper layer. The remaining cells will be suspended in the solution and the cells will be crushed at 4 ℃ using an ultrasonic thresher. The crushed solution centrifugation at 4 ℃ for 30 minutes at 13,000 rpm to obtain the supernatant. Supernatant (total protein) will be refined using the Talon resin column and then quantified using the Bradford protein quantification method and dilute to 1mg/ml with a fixed buffer solution (PBS, pH 7.4). </p> |
| − | <p> | + | <p> |
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Revision as of 05:29, 28 June 2018
Description
Heart failure (HF) is a major public health problem, with a prevalence of over 5.8 million in the USA, and over 40 million worldwide (Lancet 388:1545-1602, 2016). Heart failure is when the heart is unable to pump sufficiently to maintain blood flow to meet the body's needs. Common causes of heart failure include coronary artery disease including a previous myocardial infarction (heart attack), high blood pressure, atrial fibrillation, excess alcohol use, infection, and cardiomyopathy of an unknown cause. Heart failure is a common, costly, and potentially fatal condition. (Lancet 365:1877–89, 2005). Early diagnosis of heart failure is essential for the successful medical treatment of the disease. However, current diagnosis of heart failure patients takes considerable time from initial sampling to detection results due to sample transportation time and operating costs. Also, there is no convenient and affordable service system for continuous monitoring of patient status.
Therefore, biosensors that is portable and permits on-site analysis of samples are very useful for effective diagnosis and monitoring of heart failure patients. Among them, electrochemical immunosensors have several advantages of simplicity, inexpensive cost, accuracy, and high sensitivity. In the immunosensors, biological recognition events between antigens and antibodies immobilized on a sensing platform generate.
It is necessary to produce effective, high sensitivity, and low-cost immunosensors. Effective immobilization of antibodies on a sensing platform is crucial in designing immunosensors. Self-assembled monolayers (SAMs) method are widely used to immobilize antibodies on gold surfaces of a sensing area via covalent binding in biosensors, but the chemical method is very complex and takes a long time. Therefore, it is necessary to develop a simple method of antibody immobilization on gold sensing surfaces. To overcome this problem, we will presents a synthetic biology method by producing a novel fusion protein constructed by genetically fusing gold binding polypeptides (GBP) to protein G (ProG) as a crosslinker for effective immobilization of antibodies on gold sensing surfaces. The resulting GBP-ProG fusion protein will directly be self-immobilized onto gold surfaces via the GBP portion, followed by the oriented binding of antibodies onto the ProG domain targeting the Fc region of antibodies. As a result, we expect that the GBP-ProG protein can be a valuable crosslinker for simple and oriented immobilization of antibodies onto gold sensing surfaces without any surface chemical modification.
We created a vector capable of producing the fusion protein which includes GBP and ProG. PCR was conducted using plasmid pTGE (Park et al., Anal.Chem., 78:7197, 2006) and protein G gene of Streptocococcus G418 as a template. Eventually, 6His-GBP and Protein G was obtained the combined form of gene products. The DNA obtained by PCR was separated about 700 bps of DNA with electrophoresis. These were cut with two restrict enzymes, NdeI and BamHI, then cloned to plasmid pET-22b (+) cut with the same restriction enzyme to create recombinant plasmid pET-6HisGBP-ProG.
Then parts EcoR1 and PstI in the GBP-ProG sequence will be removed through DNA synthesis. Then PCR process for GBP-ProG sequence will be performed as a manufactured primer. These will be cut with two restricted enzymes, EcoRI and PstI, then clone to plasmid pSB1C3 cut with the same restriction enzyme to create recombinant plasmid pSB1C3-GBP-ProG. The vector produced above will be transformed to DH5α E. coli strain and then screened from LB plates with chloramphenicol. After the induction expression, the entire culture medium centrifugation and discard the upper layer. The remaining cells will be suspended in the solution and the cells will be crushed at 4 ℃ using an ultrasonic thresher. The crushed solution centrifugation at 4 ℃ for 30 minutes at 13,000 rpm to obtain the supernatant. Supernatant (total protein) will be refined using the Talon resin column and then quantified using the Bradford protein quantification method and dilute to 1mg/ml with a fixed buffer solution (PBS, pH 7.4).
Reference
- McMurray JJ, Pfeffer MA (2005). "Heart failure". Lancet. 365 (9474): 1877–89.
- GBD 2015 Disease and Injury Incidence and Prevalence, Collaborators (2016). Global, regional, and national incidence, prevalence, and years lived with disability for 310 diseases and injuries, 1990–2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet. 388 (10053): 1545–1602.
- 3. Dickstein K, Cohen-Solal A, Filippatos G, et al. (2008). ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure 2008: the Task Force for the Diagnosis and Treatment of Acute and Chronic Heart Failure 2008 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association of the ESC (HFA) and endorsed by the European Society of Intensive Care Medicine (ESICM). Eur. Heart J. 29 (19): 2388–442
- 4. Park TJ, Ko S et al. (2006). Protein Nanopatterns and Biosensors Using Gold Binding Polypeptide as a Fusion Partner. Anal.Chem. 78:7197-205
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