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Team:Brasil-SP
From 2014.igem.org
Template:Team:Brasil-SP/templateUP
Project Description
Our project consists of a biological molecular device (using Bacillus subtilis as chassis) for detection of Cystatin C, a biomarker of chronic kidney disease. The genetic circuit being assembled is based on the outstanding project of the Imperial College of London team of iGEM 2010 (special thanks to the ex-iGEMer Christopher Hirst, who helped us a lot sending some important BioBricks). Part of our mission is also to improve the characterization of the BioBricks developed on 2010 and to validate the molecular design as a generic detection system. This flexibility of detection is based on a protease cleavage of a membrane protein who triggers the genetic circuit. Since any cleavage site could be designed, virtually any protease could be used as a signal for the detection. In our case, the disease biomarker will inhibit the action of our chosen protease (Cathepsin S) and the detection will be made indirectly and negatively - i.e. by the Cathepsin lack of protease activity and absense of the system output. We are on the way to assemble all the parts and properly characterize each part of our construction on time for the Jamboree.
To address a real world situation, we are working on the same principle and aesthetics of the well known devices for biodetection like pregnancy or HIV tests: easy-to-use microfluidic devices. The plan is to design a microchip able to store spores of the developed strains of B. subtilis and safely expose blood samples to our biodetection system, successfully containing the biomaterial and enabling a proper discard of the chip. A priori, the device output monitoring would require a fluorescence detector tool, but we also propose a naked eye output observation as a concept for future prospects.
Since we are working on a solution for a problem directly related to ordinary people, having a public feedback about synthetic biology is very important to analyze the social impact of our work and it help us to evaluate the biosafety and bioethical issues beyond a simple risk analysis - a sociological characterization of the values of our project. Thus, as a policy and practices approach, we will try to report public opinion of Brazil on these issues using a questionnaire to evaluate our actual scenario and, in a certain way, our own project.
To address a real world situation, we are working on the same principle and aesthetics of the well known devices for biodetection like pregnancy or HIV tests: easy-to-use microfluidic devices. The plan is to design a microchip able to store spores of the developed strains of B. subtilis and safely expose blood samples to our biodetection system, successfully containing the biomaterial and enabling a proper discard of the chip. A priori, the device output monitoring would require a fluorescence detector tool, but we also propose a naked eye output observation as a concept for future prospects.
Since we are working on a solution for a problem directly related to ordinary people, having a public feedback about synthetic biology is very important to analyze the social impact of our work and it help us to evaluate the biosafety and bioethical issues beyond a simple risk analysis - a sociological characterization of the values of our project. Thus, as a policy and practices approach, we will try to report public opinion of Brazil on these issues using a questionnaire to evaluate our actual scenario and, in a certain way, our own project.
Wiki Pre Structure (Under Construction!)
This is the initial wiki pre structure that might be changed over its development. Everything here is merely provisional!
- Home
- The Issue
- Overview
- Avaliable Solutions
- Our Solution
Overview
A census conducted by the Brazilian Society of Nephrology, in 2012, says that the number of patients on dialysis is approximately 97,500 per year. This number generates a cost of 1.4 billion dollars annually to the Brazilian Federal Government, corresponding to 10% of public funds addressed to health in the country. The earlier the diagnosis, the bigger the chances of success of kidney disease treatment. However, the commonly used methods that only diagnose renal dysfunction in late stages and the silent nature of some diseases, such as Chronic Kidney Disease, hampers an early diagnosis and the development of an appropriate treatment.AvaliableSolutions
Kidney dysfunction is diagnosed through the evaluation of glomerular filtration rate in the kidney (GFR, measured in mL/min), in which the determination of serum creatinine concentration is the predominant method. Changes in the levels of creatinine are detectable only at later stages of renal dysfunction, when the kidney has already lost about 30% of its filtration efficiency. Moreover, the serum creatinine concentration is extremely sensitive to several variables such as diet, gender, ethnicity, age, muscle mass, and others; impairing significantly its correlation rate with the GFR. Moreover, some renal complications are asymptomatic, such as Chronic Kidney Disease (CKD), not allowing the diagnosis of the disease in its early stage. Therefore, there is a lack of tools with the precision and sensitivity needed to measure GFR in early stages of kidney disease. The urea nitrogen is also a biomarker used in the diagnosis of kidney disease, but like creatinine, it is only capable of detecting advanced stages.Our Solution
Several studies support Cystatin C as the best biomarker of renal dysfunction when compared to classical biomarkers (urea nitrogen and serum creatinine), because Cystatin C is very sensitive to changes in GFR. However, the available methods to evaluate the levels of Cystatin C are often very expensive and inefficient, such as the immunofluorescence method. Our solution to this problem is to develop a genetic circuit with the ability to detect different levels of Cystatin C in the blood. When the detectable levels of Cystatin C are higher than the normal, it will lead us to diagnose CKD and other renal dysfunctions in early stages. The genetic circuit is shown in the Figure below and the input information is based on Cystatin C inhibitory activity against cysteine proteases, in this case, cathepsin S. - The Project
- Overview
- Cystatin C
- Detection Module
- Diagnosis Module
- Response Module
- The Device
Overview
XXXXX.Cystatin C
Cystatin C, an inhibitor of cysteine proteases, has 120 amino acid residues and it is produced by all nucleated cells. It is an excellent biomarker for renal dysfunction due to its constant rate in the blood and its independence of the aforementioned variables (diet, gender, ethnicity, age, muscle mass, and others). Several scientific studies showed that Cystatin C has inhibitory activity against Papain and Calpain II. Cystatin C, likewise other low molecular weight proteins, is freely filtered by the glomeruli and it is almost completely reabsorbed in the proximal tubules. The level of Cystatin C remain constant when its production is equivalent to the nonreabsorbed portion. In patients with renal dysfunction, the GFR is lower because a smaller amount of filtered blood is filtered; as a consequence, a smaller amount of Cystatin C is reabsorbed by the proximal tubules, resulting in lower levels of excreted Cystatin C. Logically, a decrease in GFR implies an increase of Cystatin C concentration in the blood. Thus, the Cystatin C concentration in the blood is totally dependent on the GFR.Detection Module
The genetic circuit that is being developed by the Brasil-SP team is a biodetection system designed for Cystatin C. We will use the Quorum Sensing bacterial recognition system, based on communication between bacteria. This system consists in the recognition and release of substances that diffuse through the environment; those substances are called autoinducers. The autoinducers are responsible for the activation of their own synthesis, allowing the bacterial cells to respond appropriately to cell density. Thus, it is possible to control the expression of specific genes that are only activated when a certain cell concentration is achieved; as a result the behavior of the group and the formation of communities can also be controlled. In our biosensor, the Quorum Sensing substance is called AIP and its receiver is called ComD. Both AIP and ComD are anchored in the cell membrane. The AIP is attached to a linker that is cleaved in the presence of the protease cathepsin S, which can be inhibited by Cystatin C activity. After being cleaved and released from the membrane, AIP binds to the receptor triggering the phosphorylation of ComE (intracellular signalling molecule), which binds to a specific promoter sensitive to ComE, initiating the expression of the downstream gene. (lasR) One of the crucial steps in this project is to establish a threshold between normal and abnormal Cystatin C level. This threshold will be established using the threshold system constituted by Pseudomonas aeruginosa QteE and LasR genes. In P. aeruginosa, the expression of QteE is controlled by a constitutive promoter while LasR is indirectly induced by AIP through the action of ComE. We will also use a promoter inducible by isopropyl β-D-1-thiogalactopyranoside (IPTG) to control QteE transcription and translation. The QteE protein destabilizes LasR protein so that LasR cannot induce the expression of downstream genes. While the concentration of QteE is equal or greater than LasR, almost all LasR proteins are destabilized. Thus, QteE creates a barrier to cell response. By controlling expression levels of QteE gene, we manipulate the barrier to standardize the AIP concentration, that it is closely related to the Cystatin C concentration. If the concentration of LasR is high enough to overcome the barrier imposed by QteE, the LasR promoter is induced and the reporter gene Green Fluorescent Protein (GFP) is transcribed. However, the LasR and QteE proteins are part of the Quorum Sensing system of gram-negative bacteria, while our genetic circuit will be built in Bacillus subtilis, a gram-positive bacteria. To guarantee the appropriate folding of LasR protein and the induction of the promoter by LasR, substances called HSLs (homoserine lactone) must be added to the system. HSLs are intermediate products of a pathway triggered by the LasI gene. As these intermediates are found in Bacillus subtilis, the activation of transcription by a constitutive promoter meets the demand for HSLs. On the other hand, the aiiA gene in gram-positive bacteria leads to HSLs degradation. In order to guarantee a good performance of our circuit, the aiiA gene will be knocked out.Diagnosis Module
If the concentration of Cystatin C is normal, the activity of Cathepsin S is not sufficiently inhibited. Thus, the linker will be cleaved, releasing AIP. The AIP activates ComD, which, in turn, phosphorylates ComE. ComE activates the LasR production, so that it achieves optimal concentration to activate the promoter, even if several LasR proteins interact with qteE. Therefore, the transcription of the reporter gene, GFP, is activated. If the concentration of Cystatin C is above normal, indicating a possible kidney disease, the activity of Cathepsin S is inhibited. In this way, there will not be cleavage of the linker nor the release of AIP. Thus the chain of events is not triggered, so there is no production of the reporter gene.Response Module
Blood sample from a person with low levels of circulating Cystatin C triggers the expression of the reporter gene GFP in the biodetector, resulting in fluorescent signals. On the other hand, blood sample with high levels of Cystatin C does not induce the production of GFP, resulting in absent of fluorescence. Our test is carried out with patients plasma (without blood cells) filtered through a microfluidics device. In this device, our modified Bacillus subtilis will stored as spores, which are activated by rich medium (Luria-Bertani [LB]) before being used in the device. In order to induce the spore formation, the bacteria are incubated in a minimal nutrient medium (Difco Sporulation Medium [DSM]). - Modeling (Question: how many pages we'll really need here!? Modelling guys must have the answer.)
- Results
- Outreach (The "Human Practices" part)
- Notebook
- Team
- Members ("Team" + "Team Official Profile" (linking it) + "Attributions")
- Collaborations (Maybe the page "Working Together" is enought for this subject, right!?)
- Sponsors & Support
Requirements
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Visit the Wiki How To page for a complete list of requirements, tips and other useful information.
Tips
We are currently working on providing teams with some easy to use design templates.
In the meantime you can also view other team wikis for inspiration! Here are some very good examples
For a full wiki list, you can visit iGEM 2013 web sites and iGEM 2012 web sites lists.
This wiki will be your team’s first interaction with the rest of the world, so here are a few tips to help you get started:
- State your accomplishments! Tell people what you have achieved from the start.
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