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Team:USP-Brazil/Solution
From 2013.igem.org
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<table> | <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 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 ∼5% of mRNA and ∼ 30% of total protein)</td></tr> | + | <tr><td>AOX1</td><td>Alcohol oxidase 1</td><td>Induced by methanol</td><td>Strong (naturally ∼5% of mRNA and ∼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>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>∼40% of P<sub>GAP</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>∼40% of P<sub>GAP</sub></td></tr> | ||
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<tr><td>KAR2</td><td>ER resident chaperone (also termed Bip)</td><td>Constitutive</td><td>∼10%–70% of P<sub>GAP</sub></td></tr> | <tr><td>KAR2</td><td>ER resident chaperone (also termed Bip)</td><td>Constitutive</td><td>∼10%–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>∼10% 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>∼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>∼10%–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>∼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>∼10%–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>∼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>∼10%–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> | </table> | ||
Revision as of 14:47, 25 September 2013
Template:Https://2013.igem.org/Team:USP-Brazil/templateUP
The Solution
The bootlenecks
Através da compreensão do desafio escolhido, definimos seis principais gargalos que o problema apresenta para direcionar o design do nosso projeto:
- Métodos de Detecção Caros
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]. - Acesso
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.
The chassis
The organism to be modified to work as a detector is the yeast Pichia pastoris, which is an interesting choice due to its methylotrophic metabolism [5]—in other words, it uses methanol as a carbon source. P. pastoris 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).
Figure 1: Fluorescent proteins expressed in an E. coli suspension. Respectively, amilCP BBa_K592009 (blue), amilGFP BBa_K592010 (yellow) and RFP BBa_E1010 (red).
Molecular system
Texto aqui
Table 1: Legenda aqui
| Gene name | Gene product | Regulation | Expression level |
| AOX1 | Alcohol oxidase 1 | Induced by methanol | Strong (naturally ∼5% of mRNA and ∼30% of total protein) |
| GAP | Glyceraldehyde 3-phosphate dehydrogenase | Constitutive | Strong (similar to PAOX1) |
| AOD | Alternative oxidase | Expression on glucose but not on methanol or upon glucose depletion if integrated in natural locus | ∼40% of PGAP |
| AOX2 | Alcohol oxidase 2 | Induced by methanol | ∼5%–10% of PAOX1 |
| DAS | Dihydroxyacetone synthase | Induced by methanol | Strong (similar to PAOX1) |
| ENO1 | Enolase | Constitutuve | ∼20%–70% of PGAP |
| FLD1 | Formaldehyde dehydrogenase | Induced by methanol and methylamine | Strong (similar to PAOX1) |
| GPM1 | Phosphoglycerate mutase | Constitutive | ∼10%–40% of PGAP |
| HSP82 | Cytoplasmic chaperone (Hsp90 family) | Constitutive | ∼10%–40% of PGAP |
| ICL1 | Isocitrate lyase | Depression and ethanol induction | Not compared to PAOX1 or PGAP |
| ILV5 | Acetohydroxy acid isomeroreductase | Constitutive | ∼15% of PGAP |
| KAR2 | ER resident chaperone (also termed Bip) | Constitutive | ∼10%–70% of PGAP |
| KEX2 | Endopeptidase involved in the processing of secreted proteins | Constitutive | ∼10% of PGAP |
| PET9 | ADP/ATP carrier of the inner mitochondrial membrane | Constitutive | ∼10%–1700% of PGAP |
| PEX8 | Peroxisomal matrix protein | Induced by methanol or oleate | Weak |
| PGK1 | Phosphoglycerate kinase | Constitutive | ∼10% of PGAP |
| PHO89 or NSP | Sodium-coupled phosphate symporter | Induced by phosphate limitation | Strong (similar to PGAP) |
| SSA4 | Heat shock protein | Constitutive | ∼10%–25% of PGAP |
| TEF1 | translation elongation factor 1 alpha | Constitutive and strong growth association | Strong (similar to PGAP) |
| THI11 | Protein involved in thiamine biosynthesis | Completely repressed by thiamin | ∼70% of PGAP on medium lacking thiamin |
| TPI1 | Triose phosphate isomerase | Constitutive | ∼10%–80% of PGAP |
| YPT1 | GTPase involved in secretion | Constitutive | Weak |
pFLD1 Promoter
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 P. pastoris, 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).
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—the main component of alcoholic beverages. Ethanol is also involved in the degradation of peroxisomes, cellular compartments where P. pastoris 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.
pAOX1 Promoter
References
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