Team:USP-Brazil/Problem

The Challenge

The commercialization of “non-standard”, or simply adulterated beverage, despite being a neglected topic in Brazil, is an increasing risk in leisure events [15]. An estimate by the World Health Organization (WHO) suggests that about 30% of the alcoholic drinks sold in the world are not registered, reaching two-thirds of all the consumption in India, and shocking 90% in East Africa [15]. It is a global issue that has raised the concern of international organizations as ICAP and Amphora Project. Methanol is one of the most common contaminants found in alcoholic drinks, able to provoke serious health disturbances, from metabolic acidosis to neurological problems. In large-fscale, a cheap and efficient methanol detector would undoubtedly help save millions of lives.

Detection of chemical compounds has always been one of the goals of Chemistry. In spite of all the progress allowed by technological developments in analytical chemistry, like gas chromatography [1] and ionization flame detection [2], such a task remains largely unreachable for those without access to highly specific equipment. That is the case of methanol contamination in “non-standard” (adulterated) beverage, where one has to opt between heavy machinery or purely chemical methods—that is, those without the specific equipment - that are usually very elaborate and expensive [3]. It is also possible to use biochemical sensors [4], but those also lack accessibility, due to the usage of enzymes, as well as other expensive reagents, such as ubiquinone or luminol.

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).

The Solution

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).

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.

The Detector

• Como imaginamos o produto final em termos de relação com o usuário, e seu impacto pra resolver o problema. Será elaborado um protocolo de tratamento a ser seguido pelo usuário, que consistirá em tratamento químico com hipoclorito e posterior retorno do produto pra tratamento adicional.

Qual a diferença dessa seção com a Safe Drink Detector?

References

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