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.

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.

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.

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.

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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.

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.

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.

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]).

Bacillus subtilis is Gram-Positive bacteria, considered a model organism for, which has been studied in the last 50 years. Our intention here was to summarize the information we get so other teams can use our page as a basis to their work. Who is it? When we begin to outline our project, we first decided to use Escherichia coli as a chassis. However, looking at Bacillus subtilis more closely we concluded it would be a very better choice to what we aimed. More than a thousand years ago humans started working with B. subtilis. Japaneses discovered that fermented soybeans could be delicious at breakfast, and for centuries Bacillus subtilis has been used to produce natto1. Nowadays, Bacillus subtilis is also used as a biological factory, to produce enzymes and fine biochemicals. It’s also an interesting host for the heterologous protein production. This Gram +ve bacteria can produce and secrete large amounts of protein to the medium. In addition, the genre Bacillus is noticeable by his high capacity of adsorbing and uptaking exogenous DNA2. This is one of the main reasons why it has been extensively used in both applied and fundamental research in the past decades. As a model organism, there is available a lot of detailed data about it. Bacillus subtilis was the first Gram-positive bacteria to have its entire genome sequenced and annotated (strain 168)3. Following this project, all essential genes were indentified4. Currently, large-scale data such as transcriptome5, proteome6, secretome7 and metabolome8 are available. Furthermore, its use is promoted mainly due to the non-pathogenic nature of this organism. It has been awarded the GRAS (Generally Recognized as Safe) status by the US Food and Drug Administration - FDA. In the opposite, Bacillus subtilis is close enough to other clinically relevant Gram-positive pathogens to be used as an organism relevant to research new targets to antimicrobials and anti-infectives2. Well, Bacillus subtilis can easily uptake exogenous DNA, secrete a lot of proteins in the medium, has been used through centuries and is a safe chassis. Can it be better? Yes it can. We haven’t mentioned before what made us choice Bacillus subtilis as our favorite “pet”-chassis and why you should use it too: the endospores. Bacteria's life can be stressful and complex, needing to face a lot of adversities and we can use this in our favor. In this situation, like starvation, many bacteria trigger a cellular response involving many genes. In Bacillus subtilis this process entails the activity of more than 500 genes and take approximately ten hours.9 In a sweetable growth medium, cells double in length and then divide centrally to produce two identical daughter cells. The sporulation process starts with an asymmetric division. The biggest cell (called mother) engulfs the smallest (called prespore). When this process is completed, the membrane which surrounds the cytoplasm of the prespore (now called the forespore) gets a very amorphous appearance, probably due to the absence of peptidioglycan layer to define the cell shape. Next, a modified form of cell wall, known as cortex, is synthesized between the prespore membranes, providing it with an oblong shape. Simultaneously, the proteinaceous spore coat begins to be deposited on the outside surface of the prespore. The lysis of a mother cell releases the mature spore. The spore is a resistant structure, conceived to resist to hazards like heat, radiation and toxic chemicals, remaining dormant until be exposed to mild conditions when it get germinated. As a commercial product, it’s perfect. The storage and the cells switch on is equally easy, bringing to open-and-shut product.10 The primary question is: exposing our cells to a stressful medium will lead them all to form spores? Certainly no. Sporulation is the ultimate bacteria's decision. And when they finally decide to it, always there are some of them who betray the movement. Many of other strategies are tried before sporulation. Those include secretion of antibiotics and other chemical weapons to kill other microbes that are in the same niche; the activation of cellular motility by flagella to search for new nutrients source and the secretion of hydrolytic enzymes to remove extracellular polysacharides and proteins. Furthermore, before sporulation starts the cells check chromosome integrity and the state of chromosomal replication, so if the process begins the bacterium is sure that it will be finished.9 Once situation is extreme, cells must choice between three ways: sporulation, competence or cannibalism. Most cells make the commitment to sporulation. However, the genetic material realized by cell lysis isn’t wasted by the colony. Some cells opt for a different state of competence, triggered by ComK exceeding a certain threshold level. If a cell chose this way, it can uptake exogenous DNA resulting from lysis and use it to repair his own DNA or sometimes new genetic information. In the case of Bacillus subtilis approximately 10% of cells choose this path and after approximately 20 h they switch back to the vegetative state. In On the other hand, it has been showed that some bacteria further along to sporulation produce and secrete antibacterial factors that block sibling cells from sporulating. This makes them to lysis. Such cannibalistic cells feed on the nutrients released by the lysed cells. So, in the end, they prevent their own progression toward sporulation by satiating their feeding needs.9 But it's even more important to understand how spore's germination occur. In our device, we provide nutrients to the bacteria. Those nutrients act as germinants. The spores can recognize them receptor proteins encoded by the gerA family of operons, which includes gerA, gerB and gerK. We can induce germination providing alanine (Ala) or a mixture of asparagine, glucose, fructose and potassium ions (AGFK) for example. The first one activate the alanine receptor GerA and the second the asparagine receptor GerB. The interaction between nutrient germinants and receptors triggers the replace of spore core's huge depot of dipicolinic acid and cations by water. This effect triggers the hydrolysis of the spore's peptidoglycan cortex by one of two redundant enzymes. The completion of cortex hydrolysis and subsequent germ cell wall expansion allows spore core hydration. This results in resumption of spore metabolism and macromolecular synthesis.11 Understanding this process we can chose when and how our product will start working. What we did Here you can see all our microbiology protocols. Feel free to use all of them. We acknowledge to LMU-Munich team 2012 for… (hyperlink para: https://2012.igem.org/Team:LMU-Munich ). We based our protocols on their work. In this project, it was used the 168 strain (TROCAR PELA LINHAGEM QUE USAREMOS) First step was to grow our chassis. Bacillus subtilis grows quickly at Luria-Bertani medium (agar or broth) and it was used to propagate our cultures. FOTO PLACA DE PETRI COM BACILLUS FORRADA APÓS 24 HORAS Next, sporulation was induced through extensive culture (18 h) in DSM medium (incluir Fórmula). Then, it was used a proper Spore Count Protocol, resulting in the graphic below: GRÁFICO A SER CONSTRUÍDO MOSTRANDO ESPORULAÇÃO TABELA COM OS DADOS DO GRÁFICO, SE NECESSÁRIO FOTO DOS ESPOROS OBSERVADOS AO MICROSCÓPIO We could achieve a high sporulation level (XX%). This is very important from industrial outlook. However, it’s important to know spores viability. To induce germination, it was used Luria-Bertani broth. As a rich medium, it induces the receptors before mentioned. The following graphic shows the efficiency of our method: GRÁFICO A SER CONTRSUÍDO MOSTRANDO ATIVAÇÃO FOTO DO BACILLUS APÓS TESTE DE GRAM EM DIFERENTES ZOOMS Our work showed the efficiency of both sporulation and germination. This is very important from industrial view. Once our circuit is finished, it will be simple and cheap to grow Bacillus subtilis cells and to sporulate them. Understanding both processes in theory and practice, we can achieve a high efficiency approaches, which could bring our device to market. 1. SCHALLMEY, M. et al Developments in the use of Bacillus species for industrial production. Canadian Journal of Microbiology, 2004, 50(1): 1-17. 2. ZWEERS, J. C. et al Towards the development of Bacillus subtilis as a cell factory for membrane proteins and protein complexes. Microbial Cell Factories, 2008, 7:10. The electronic version of this article is the complete one and can be found online at: http://www.microbialcellfactories.com/content/7/1/10 . Acess: 09/13/2014 3. KUNST, F. et al The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature, 1997, 390:249-256. 4. KOBAYASHI, K. et al Essential Bacillus subtilis genes. Proc Natl Acad Sci U S A, 2003, 100:4678-4683. 5. SIERRO, N. et al DBTBS: a database of transcriptional regulation in Bacillus subtilis containing upstream intergenic conservation information. Nucleic Acids Res, 2007. 6. WOLFF, S. et al Towards the entire proteome of the model bacterium Bacillus subtilis by gel-based and gel-free approaches. J Chromatogr B Analyt Technol Biomed Life Sci, 2007, 849:129-140. 7. TJALSMA, H. Proteomics of protein secretion by Bacillus subtilis: separating the "secrets" of the secretome. Microbiol Mol Biol Rev, 2004, 68:207-233. 8. FISCHER, E., SAUER, U. Large-scale in vivo flux analysis shows rigidity and suboptimal performance of Bacillus subtilis metabolism. Nat Genet, 2005, 37:636-640. 9. SCHULTZ, D. et al Deciding fate in adverse times: Sporulation and competence in Bacillus subtilis. PNAS, December 15, 2009. 106. 10. ERRINGTON, J. Bacillus subtilis Sporulation: Regulation of Gene Expression and Control of Morphogenesist. Microbiological Reviews, Mar. 1993, 57: 1-33 11. SETLOW, P. Spore germination. Current Opinion in Microbiology, December 2003, 6: 550-556. .