Team:USP-Brazil/Solution

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Problem

The Solution

Detection of Methanol in Alcoholic Drinks

Methanol, when present in alcoholic drinks, even when it is not intentionally added to lower production cost, is a metabolic product of the degradation of fruit organic residues [14]. Analytical chemistry methods for methanol detection in ethanol solutions have been developed since late nineteenth century. The most common is Chapin's colorimetric method, now an ISO rule [15][16] in ethanol-producing industries. Most methanol detection devices rely on highly specific equipment and techniques, such as infrared spectroscopy, liquid and gaseous chromatography [6][17]. There are also chemical methods [18], but no alternative suitable for the scale of the problem, once they all are too elaborate and expensive, whether because of the equipment, or because of the reagents. A problem as global as this needs a solution that is cheap, user-friendly and scalable.

The greatest advantage of a biochemical sensor could be, in addition to accessibility and easy use, its production: once the microorganism that has been modified to act as a detector is built, cell culture growth is itself responsible for producing the detector. Not only that simplifies the process, but it also reduces its cost: the only expenses after the development and construction of the biosensor would be with culture media and with preparing the product (e.g. lyophilization).

Bottlenecks

The difficulties faced in methanol detection consist in two categories: accessibility and efficiency. Methanol analysis today is still expensive, at a mean price of 58 USD [1], which is too high for noncommercial producers. Also, most populations exposed to methanol intoxication have no access to chromatography equipment, to the reagents necessary for analysis, nor to the technical knowledge involved. Also, time and efficiency play a major role: the fastest tools take up to a week to return their results [1], and usually provide information that is useless to the producer – once the main concern is whether the drink is safe. Essentially, there is no method developed for individual, small-scale producers: only for the companies that can afford cutting-edge tools.

References

…

See the Detector…

@@ Line 8: / Line 8: @@
 			<h2> The Solution</h2>
-			<h3>The bootlenecks</h3>
+		<h3>Detection of Methanol in Alcoholic Drinks</h3>
+		<p>Methanol, when present in alcoholic drinks, even when it is not intentionally added to lower production cost, is a metabolic product of the degradation of fruit organic residues [14]. Analytical chemistry methods for methanol detection in ethanol solutions have been developed since late nineteenth century. The most common is Chapin's colorimetric method, now an ISO rule [15][16] in ethanol-producing industries. Most methanol detection devices rely on highly specific equipment and techniques, such as infrared spectroscopy, liquid and gaseous chromatography [6][17]. There are also chemical methods [18], but no alternative suitable for the scale of the problem, once they all are too elaborate and expensive, whether because of the equipment, or because of the reagents. A problem as global as this needs a solution that is cheap, user-friendly and scalable.</p>
-			<p>Através da compreensão do desafio escolhido, definimos seis principais gargalos que o problema apresenta para direcionar o design do nosso projeto:</p>
+		<p>The greatest advantage of a biochemical sensor could be, in addition to accessibility and easy use, its production: once the microorganism that has been modified to act as a detector is built, cell culture growth is itself responsible for producing the detector. Not only that simplifies the process, but it also reduces its cost: the only expenses after the development and construction of the biosensor would be with culture media and with preparing the product (e.g. lyophilization). </p>
-			<ol>
+		<h3>Bottlenecks</h3>
-				<li><b>Métodos de Detecção Caros</b><br />Se estamos falando de populações com condições econômicas desprivilegiadas (principal motivo por produzirem e consumirem bebidas não-registradas{noncommercial}), os métodos de detecção utilizados atualmente para fiscalização ainda representam um obstáculo grande para análise de Metanol. Apesar de custarem em média 58 USD [1], ainda é um preço exorbitante para os produtores não-comerciais [2].</li>
+		<p> The difficulties faced in methanol detection consist in two categories: accessibility and efficiency. Methanol analysis today is still expensive, at a mean price of 58 USD [1], which is too high for noncommercial producers. Also, most populations exposed to methanol intoxication have no access to chromatography equipment, to the reagents necessary for analysis, nor to the technical knowledge involved. Also, time and efficiency play a major role: the fastest tools take up to a week to return their results [1], and usually provide information that is useless to the producer – once the main concern is whether the drink is safe. Essentially, there is no method developed for individual, small-scale producers: only for the companies that can afford cutting-edge tools. </p>
-				<li><b>Acesso</b><br />O principal método utilizado por órgãos de fiscalização governamentais (quando de fato realizam a fiscalização) é a cromatografia gasosa acoplada a detectores de ionização por chama {flame-ionization detector} ou a espectrômetros de massa. Como a maioria das populações expostas ao risco de intoxicação por etanol é muito grande e marginalizada [2], é praticamente impossível esperar que essa metodologia possa ser acessível o suficiente para ser usada em larga escala.</li>
-			</ol>
-			<h3>The chassis</h3>
-			<p>The organism to be modified to work as a detector is the yeast <i>Pichia pastoris</i>, which is an interesting choice due to its methylotrophic metabolism [5]&#8212;in other words, it uses methanol as a carbon source. <i>P. pastoris</i> is commonly used in the production of recombinant proteins [6], mainly due to its populational characteristics, such as growth rate and cell density, which make cell suspensions paste-dense [7], and to its methanol-responsive promoter, PAOX1. This promoter could be part of a genetic circuit that would respond to the presence of methanol by regulating the transcription of a reporter gene, responsible for indicating the presence of methanol by colouring the suspension (Figure 1).</p>
-			<p class="figure">
-				<img src="https://static.igem.org/mediawiki/2013/1/15/USPBrasilCultivo.jpg" width="640" height="395" /><br />
-				<b>Figure 1:</b> Fluorescent proteins expressed in an <i>E. coli</i> suspension. Respectively, amilCP BBa_K592009 (blue), amilGFP BBa_K592010 (yellow) and RFP BBa_E1010 (red).
-			</p>
-			<h3>Molecular system</h3>
-			<p>Texto aqui</p>
-			<p class="table">
-				<b>Table 1:</b> Legenda aqui
-			</p>
-			<table>
-				<tr class="tr_first"><td style="width:10%">Gene name</td><td style="width:35%">Gene product</td><td style="width:35%">Regulation</td><td style="width:20%">Expression level</td></tr>
-				<tr><td>AOX1</td><td>Alcohol oxidase 1</td><td>Induced by methanol</td><td>Strong (naturally &sim;5% of mRNA and &sim;30% of total protein)</td></tr>
-				<tr><td>GAP</td><td>Glyceraldehyde 3-phosphate dehydrogenase</td><td>Constitutive</td><td>Strong (similar to P<sub>AOX1</sub>)</td></tr>
-				<tr><td>AOD</td><td>Alternative oxidase</td><td>Expression on glucose but not on methanol or upon glucose depletion if integrated in natural locus</td><td>&sim;40% of P<sub>GAP</sub></td></tr>
-				<tr><td>AOX2</td><td>Alcohol oxidase 2</td><td>Induced by methanol</td><td>&sim;5%&#8211;10% of P<sub>AOX1</sub></td></tr>
-				<tr><td>DAS</td><td>Dihydroxyacetone synthase</td><td>Induced by methanol</td><td>Strong (similar to P<sub>AOX1)</sub></td></tr>
-				<tr><td>ENO1</td><td>Enolase</td><td>Constitutuve</td><td>&sim;20%&#8211;70% of P<sub>GAP</sub></td></tr>
-				<tr><td>FLD1</td><td>Formaldehyde dehydrogenase</td><td>Induced by methanol and methylamine</td><td>Strong (similar to P<sub>AOX1</sub>)</td></tr>
-				<tr><td>GPM1</td><td>Phosphoglycerate mutase</td><td>Constitutive</td><td>&sim;10%&#8211;40% of P<sub>GAP</sub></td></tr>
-				<tr><td>HSP82</td><td>Cytoplasmic chaperone (Hsp90 family)</td><td>Constitutive</td><td>&sim;10%&#8211;40% of P<sub>GAP</sub></td></tr>
-				<tr><td>ICL1</td><td>Isocitrate lyase</td><td>Depression and ethanol induction</td><td>Not compared to P<sub>AOX1</sub> or P<sub>GAP</sub></td></tr>
-				<tr><td>ILV5</td><td>Acetohydroxy acid isomeroreductase</td><td>Constitutive</td><td>&sim;15% of P<sub>GAP</sub></td></tr>
-				<tr><td>KAR2</td><td>ER resident chaperone (also termed Bip)</td><td>Constitutive</td><td>&sim;10%&#8211;70% of P<sub>GAP</sub></td></tr>
-				<tr><td>KEX2</td><td>Endopeptidase involved in the processing of secreted proteins</td><td>Constitutive</td><td>&sim;10% of P<sub>GAP</sub></td></tr>
-				<tr><td>PET9</td><td>ADP/ATP carrier of the inner mitochondrial membrane</td><td>Constitutive</td><td>&sim;10%&#8211;1700% of P<sub>GAP</sub></td></tr>
-				<tr><td>PEX8</td><td>Peroxisomal matrix protein</td><td>Induced by methanol or oleate</td><td>Weak</td></tr>
-				<tr><td>PGK1</td><td>Phosphoglycerate kinase</td><td>Constitutive</td><td>&sim;10% of P<sub>GAP</sub></td></tr>
-				<tr><td>PHO89 or NSP</td><td>Sodium-coupled phosphate symporter</td><td>Induced by phosphate limitation</td><td>Strong (similar to P<sub>GAP</sub>)</td></tr>
-				<tr><td>SSA4</td><td>Heat shock protein</td><td>Constitutive</td><td>&sim;10%&#8211;25% of P<sub>GAP</sub></td></tr>
-				<tr><td>TEF1</td><td>translation elongation factor 1 alpha</td><td>Constitutive and strong growth association</td><td>Strong (similar to  P<sub>GAP</sub>)</td></tr>
-				<tr><td>THI11</td><td>Protein involved in thiamine biosynthesis</td><td>Completely repressed by thiamin</td><td>&sim;70% of P<sub>GAP</sub> on medium lacking thiamin</td></tr>
-				<tr><td>TPI1</td><td>Triose phosphate isomerase</td><td>Constitutive</td><td>&sim;10%&#8211;80% of P<sub>GAP</sub></td></tr>
-				<tr><td>YPT1</td><td>GTPase involved in secretion</td><td>Constitutive</td><td>Weak</td></tr>
-			</table>
-			<h4>pFLD1 Promoter</h4>
-			<p>PAOX1 is a strong promoter which can be controlled by simple changes in its carbon source [8], and is the most common choice for expression of heterologous proteins in <i>P. pastoris</i>, having a naturally elevated expression rate, of circa 5% of the RNA and 30% of total protein production [11]. Here, the chosen reporter was RFP (Red Fluorescent Protein).</p>
-			<p>The challenge in the building of this sensor was the regulation of PAOX1. This promoter is prone to a strong catabolic repression [12] by hexoses and ethanol&#8212;the main component of alcoholic beverages. Ethanol is also involved in the degradation of peroxisomes, cellular compartments where <i>P. pastoris</i> realizes the metabolism of methanol. This aspect is actually interesting to our application, since it means methanol will not be degraded as fast as it would, in the absence of ethanol. Therefore, methanol would stay for longer in the cell, being able to activate PAOX1.
-			<h4>pAOX1 Promoter</h4>