Difference between revisions of "Team:UFSCar-Brasil/Description"

 
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         Description
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         Overview
 
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       <h2 class="ui teal header">What's our project?</h2>
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       <h2 class="ui teal header">What's our project about?</h2>
 
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<p> Neglected diseases have their importance in public health system, causing millions of deaths every year (MOREL, 2006). In Brazil dengue stands out; a disease which afflicted more than 1,350,406 people in 2015, according to data from the Brazilian Health Ministry epidemiological report. The microorganisms responsible for these diseases are usually transported by insects, thus, repellents represent a good strategy to fight these microorganisms (STEFANI et al., 2009; SORGE et al., 2007; FRADIN & DAY, 2002).</p>
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<p>Besides the problem previously reported, it is interesting to highlight the toxicity associated to high concentrations of N,N-diethyl-m-toluamide (DEET), which is the molecule currently used on market. (CHEN-HUSSEY et al., 2014; ROBBINS et al., 1986; OSIMITZ et al., 2010; MCGREADY et al., 2001).</p>
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<p>Repellents extracted from natural oils are an alternative to toxicity, but they have a shorter lifespan compared to DEET, necessitating more topical applications; between natural molecules with proved efficiency we highlight the D-limonene, that besides being safe to human skin it is also a GRAS (Generally recognized as safe) product (SUN, 2007; KARLBERG et al., 1991).</p>
        <h2 class="ui center aligned header">Project Description</h2>
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<p>With the objective to prolong the duration of the natural repellent based on D-limonene, the use of synthetic biology to promote its continuous production was proposed in this project. However, previous efforts to produce D-limonene in bacterial chassi were not efficient due the insolubility of limonene synthase, enzyme responsible to convert geranyl pyrophosphate to limonene.</p>
        <p>Diseases transmitted by insect vectors, such as malaria, dengue fever, yellow fever and leishmaniasis, affect millions of people and caused about 600.000 deaths around the world in 2012, especially in tropical regions(1). In Brazil, population
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<p>To make possible the production of D-limonene, the construction of a genetic circuit with three modules was proposed. The first module has a promoter responsible to different stresses (UspA), which induces the production of limonene synthase. The stresses create conditions that are likely to produce molecular chaperones, improving the efficiency in the process of protein folding (PURVIS et al., 2005; SHIMIZU et al., 2013). Combining this condition to the problem of storage of our product we chose to use osmotic stress, created by an ethylene glycol polymer (PEG). It showed to be efficient due to its flexible and hydrophilic features, which creates high osmotic pressures, and its chemical arrangement makes it unlikely to interact with other biological molecules present in the genetically modified microorganism (MONEY et al., 1989). The second module is composed by three proteic chaperones, natural from <i>Escherichia coli</i>, overexpressed to obtain a better protein folding. The last module consists in a killswitch. It is a biosafety mechanism responsible for programmed cell death of the bacteria used in this project, after the required time to produce limonene. The killswitch’s function is associated to the action of <i>znuABC</i> operon, and to protein Zur. With this it is possible to control bacterial lifespan, depending on the concentration of zinc present at the final product.</p>
          is affected significantly by those diseases. In the summer of 2014/2015 there were <b>1.350.406 cases of dengue in Brazil</b>, resulting in 614 deaths (57% higher than the same period in 2013/2014) (2). Brazilian health authorities affirmed
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          that the dengue fever epidemic had a raise of 229% (3). In our city, Sao Carlos, the epidemy was so severe that the public health agency has not been able to account for the total number of cases (WIki Political). Unofficial reports count more
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          than 20.000 victims and at least 5 dead people (4, 5).</p>
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        <p>Dengue fever is an urban disease, caused by 4 different types of viruses. Those viruses are transmitted by female mosquitoes <i>Aedes aegypti</i> bites (6,7). Most of times, symptoms can be confused with a bad cold. In several cases, however,
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          patients exhibit haemorrhagic fever, which can lead to death. There is no specific treatment or vaccine to prevent dengue fever (8). The only actions that may help avoiding the disease are controlling the environment where the insects reproduce
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          and using repellents. Every year, we see the same sad situation repeat itself, so far without prospects of change.</p>
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        <p>Considering this problem, our project consisted in developing an alternative repellent of that currently sold in the market, more or equally effective in preventing mosquitoes bites that transmits diseases. The main compound in current repellents
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          is <b>DEET (N, N-diethyl-m-toluamide)</b>, a toxic molecule which at certain concentrations could be lethal, and therefore must have strict control on its use (9). The main characteristic of our repellent is a longer duration when compared to
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          other products and the replacement of DEET by <b>D-limonene</b>, a less toxic compound.</p>
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        <p>Our proposal was building a bacteria carrying out the production of D-limonene via limonene synthase. To enable long term storage at room temperatures, the bacterial cells that make up the repellent would be plasmolyzed, with suspended metabolism,
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          in a dormant state. The maintenance of this state would be obtained by a solution of polyethylene glycol (PEG) that will raise the osmotic pressure. Once in contact with the skin, PEG solution would be diluted by sweat and inducing osmotic shock
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          in bacteria cells. Then the universal stress protein promoter (UspA) would be activated, inducing expression of limonene synthase. In addition to PEG, other compounds such as glycerol and metal ions in low concentrations would constitute our
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          insect repellent cream, in order to keep bacteria at the required metabolic condition and posterior enzyme activities. This way, the distribution of our repellent may become feasible, allowing its use.</p>
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        <p>The expression of limonene synthase was the aim of other iGEM team (TU_Munich, 2012). However, protein folding has been an unsolved problem. To overcome those hindered limonene synthase folding, we used constitutive promoters for expression of
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          chaperones from all Escherichia coli available classes (e.g. ClpB, DnaK and IbpA / IbpB). It would reinforce chaperones stocks naturally produced during osmotic shocks (heat-shock proteins) in bacteria. Besides improving limonene production,
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          our goals were to improve protein solubility, indirectly creating a toolkit for protein solubility enhancement for future iGEM teams, reducing the occurrence of inclusion bodies.</p>
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        <p>Finally, we proposed a system of programmed death - a killswitch - to ensure biosafety and biosecurity of the engineered bacteria. The mechanism involves two suicide genes: Killer Red and Barnase. The first consists in a protein that triggers
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          the generation of reative oxygen species, destroying both plasmid and chromosomal DNA. The second results in a protein with ribonuclease activity. The genes would be activated by a zinc sensitive promoter triggered by zur proteins that, when
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          associated with zinc, inhibit killswitch expression, allowing bacteria to grow. After some hours of repellent action, bacteria activities would decrease zinc concentration in medium, leading to gene activation and, thus, to death of the bacteria.
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<img class="ui centered image" src="https://static.igem.org/mediawiki/2015/0/0f/UFSCar-Brasil_circuit_overview_10.png">
  
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  <h6 class="ui center aligned header"><b>Figure 1</b>: Genetic circuit of Bug Shoo.</h6>
  
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  <h2 class="ui center aligned header"> References</h2>
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<p>MOREL, C. M. Inovação em saúde e doenças negligenciadas. Cad. Saúde Pública, Rio de Janeiro. 2006.</p>
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<p>STEFANI G. P., PASTORINO A. C., CASTRO A. P. B. M., FOMIN A. B. F., JACOB C. M. A. Insect repellents: recommendations for use in children. Rev Paul Pediatr 2009</p>
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<p>SORGE F., IMBERT P., LAURENT C., MINODIER P., BANERJEE A., KHELFAOUI F. Children arthropod bites protective measures: insecticides and repellents. Arch Pediatr 2007</p>
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<p>FRADIN M. S., DAY J. F. Comparative efficacy of insect repellents against mosquito bites. N Engl J Med 2002</p>
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<p>CHEN-HUSSEY V., BEHRENS R., LOGAN J. G. Assessment of methods used to determine the safety of the topical insect repellent N,N-diethyl-m-toluamide (DEET). Parasit Vectors. 2014</p>
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<p>ROBBINS P. J., CHERNIAK M. G. Review of the biodistribution and toxicity of the insect repellent N, N-diethyl-m-toluamide (DEET). J Toxicol Environ Health. 1986</p>
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<p>OSIMITZ T. G., MURPHY J. V., FELL  L. A., PAGE B. Adverse events associated with the use of insect repellents containing N, N-diethyl-m-toluamide (DEET). Regul Toxicol Pharmacol. 2010</p>
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<p>MCGREADY R., HAMILTON K. A., SIMPSON J. A., CHO T., LUXEMBURGER C., EDWARDS R., LOOAREESUWAN S., WHITE N. J., NOSTEN F., LINDSAY S. W. Safety of the insect repellent N, N-diethyl-m-toluamide (DEET) in pregnancy. Am J Trop Med Hyg. 2001</p>
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<p>SUN J. D-Limonene: safety and clinical applications. Altern Med Rev. sep 2007</p.>
 +
<p>KARLBERG T., BOMAN A., MELINJ B. animal experiments on the allergenicity of d-limonene—the citrus solvent. AM. omip Hrg.. Vol 35. No. 4. pp. 419-426. 1991</p>
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<p>PURVIS J. E., YOMANO L. P., INGRAM L. O. Enhanced Treahalose Production Improves Growth of <i>Escherichia coli</i> under Osmotic Stress. Appl. Environ. Microbiol. Jul 2005</p>
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<p>SHIMIZU K. Regulation Systems of Bacteria such as <i>Escherichia coli</i> in Response to Nutrient Limitation and Environmental Stresses. Metabolites. Mar 2014</p>
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<p>MONEY P. N. Osmotic Pressure of Aqueous Polyethylene Glycols, Relationship between Molecular Weight and Vapor Pressure Deficit. Plant Physiol. 91, 766-769, 1989.</p>
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Latest revision as of 22:12, 18 September 2015

Overview

What's our project about?

Neglected diseases have their importance in public health system, causing millions of deaths every year (MOREL, 2006). In Brazil dengue stands out; a disease which afflicted more than 1,350,406 people in 2015, according to data from the Brazilian Health Ministry epidemiological report. The microorganisms responsible for these diseases are usually transported by insects, thus, repellents represent a good strategy to fight these microorganisms (STEFANI et al., 2009; SORGE et al., 2007; FRADIN & DAY, 2002).

Besides the problem previously reported, it is interesting to highlight the toxicity associated to high concentrations of N,N-diethyl-m-toluamide (DEET), which is the molecule currently used on market. (CHEN-HUSSEY et al., 2014; ROBBINS et al., 1986; OSIMITZ et al., 2010; MCGREADY et al., 2001).

Repellents extracted from natural oils are an alternative to toxicity, but they have a shorter lifespan compared to DEET, necessitating more topical applications; between natural molecules with proved efficiency we highlight the D-limonene, that besides being safe to human skin it is also a GRAS (Generally recognized as safe) product (SUN, 2007; KARLBERG et al., 1991).

With the objective to prolong the duration of the natural repellent based on D-limonene, the use of synthetic biology to promote its continuous production was proposed in this project. However, previous efforts to produce D-limonene in bacterial chassi were not efficient due the insolubility of limonene synthase, enzyme responsible to convert geranyl pyrophosphate to limonene.

To make possible the production of D-limonene, the construction of a genetic circuit with three modules was proposed. The first module has a promoter responsible to different stresses (UspA), which induces the production of limonene synthase. The stresses create conditions that are likely to produce molecular chaperones, improving the efficiency in the process of protein folding (PURVIS et al., 2005; SHIMIZU et al., 2013). Combining this condition to the problem of storage of our product we chose to use osmotic stress, created by an ethylene glycol polymer (PEG). It showed to be efficient due to its flexible and hydrophilic features, which creates high osmotic pressures, and its chemical arrangement makes it unlikely to interact with other biological molecules present in the genetically modified microorganism (MONEY et al., 1989). The second module is composed by three proteic chaperones, natural from Escherichia coli, overexpressed to obtain a better protein folding. The last module consists in a killswitch. It is a biosafety mechanism responsible for programmed cell death of the bacteria used in this project, after the required time to produce limonene. The killswitch’s function is associated to the action of znuABC operon, and to protein Zur. With this it is possible to control bacterial lifespan, depending on the concentration of zinc present at the final product.

Figure 1: Genetic circuit of Bug Shoo.

References

MOREL, C. M. Inovação em saúde e doenças negligenciadas. Cad. Saúde Pública, Rio de Janeiro. 2006.

STEFANI G. P., PASTORINO A. C., CASTRO A. P. B. M., FOMIN A. B. F., JACOB C. M. A. Insect repellents: recommendations for use in children. Rev Paul Pediatr 2009

SORGE F., IMBERT P., LAURENT C., MINODIER P., BANERJEE A., KHELFAOUI F. Children arthropod bites protective measures: insecticides and repellents. Arch Pediatr 2007

FRADIN M. S., DAY J. F. Comparative efficacy of insect repellents against mosquito bites. N Engl J Med 2002

CHEN-HUSSEY V., BEHRENS R., LOGAN J. G. Assessment of methods used to determine the safety of the topical insect repellent N,N-diethyl-m-toluamide (DEET). Parasit Vectors. 2014

ROBBINS P. J., CHERNIAK M. G. Review of the biodistribution and toxicity of the insect repellent N, N-diethyl-m-toluamide (DEET). J Toxicol Environ Health. 1986

OSIMITZ T. G., MURPHY J. V., FELL L. A., PAGE B. Adverse events associated with the use of insect repellents containing N, N-diethyl-m-toluamide (DEET). Regul Toxicol Pharmacol. 2010

MCGREADY R., HAMILTON K. A., SIMPSON J. A., CHO T., LUXEMBURGER C., EDWARDS R., LOOAREESUWAN S., WHITE N. J., NOSTEN F., LINDSAY S. W. Safety of the insect repellent N, N-diethyl-m-toluamide (DEET) in pregnancy. Am J Trop Med Hyg. 2001

SUN J. D-Limonene: safety and clinical applications. Altern Med Rev. sep 2007

KARLBERG T., BOMAN A., MELINJ B. animal experiments on the allergenicity of d-limonene—the citrus solvent. AM. omip Hrg.. Vol 35. No. 4. pp. 419-426. 1991

PURVIS J. E., YOMANO L. P., INGRAM L. O. Enhanced Treahalose Production Improves Growth of Escherichia coli under Osmotic Stress. Appl. Environ. Microbiol. Jul 2005

SHIMIZU K. Regulation Systems of Bacteria such as Escherichia coli in Response to Nutrient Limitation and Environmental Stresses. Metabolites. Mar 2014

MONEY P. N. Osmotic Pressure of Aqueous Polyethylene Glycols, Relationship between Molecular Weight and Vapor Pressure Deficit. Plant Physiol. 91, 766-769, 1989.

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