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Detecthol
Click on the images below to understand how it works.
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.
The Chassis
Looking for a chassis that would resist the presence of ethanol, and specially of methanol, the simplest solution was to use yeast cells, largely employed in the production of biofuels, such as Saccharomyces cerevisae [6]. The best choice turned out to be a methylotrophic organism, that is, one that can use methanol as a carbon source [7]. Pichia pastoris is a species of methylotrophic yeast with its genome sequenced [11]that is commonly used in the production of recombinant proteins [8], mainly due to its populational characteristics, such as growth rate and cell density, which make cell suspensions paste-dense [9], and to its methanol-responsive promoters, that could be part of a genetic circuit that would respond to the presence of methanol. In addition, P.pastoris cultures are able to grow in media with up to 10% of ethanol [10], which makes it a perfect candidate four our chassis.
Imagem da Pichia pastoris, the methylotrophic yeast.
Molecular detection
Overview
In order to detect methanol in alcoholic drinks, we searched for P.pastoris promoters inducible by methanol, and preferably known to have well-established genetic recombination techniques.
***** tabela só com os 3 promotores
The three possible choices (see table above) were PAOX1, PFLD1 and PDAS. Since we did not find much information on PDAS, while PAOX1 and PFLD1 are well characterized, both of them having commercial plasmids for genomic integration [13][14], we chose the latter two as our molecular detectors. That, in a simple genetic construction, could work as a proof of concept. A priori, our output system would be monomeric RFP (mRFP1, Biobrick BBa_E1010), being used for fluorescence tests and for efficiently characterizing the relative promoter strength. We also expect to be able to see the production of RFP with the naked eye, once in E.coli it is shown to be possible.
PAOX1
PAOX1 is a strong promoter which can be controlled by simple changes in its carbon source [16], and it 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 [12].
The challenge in using PAOX1 is its regulation. This promoter is prone to a strong catabolic repression by hexoses and ethanol [17] — the main component of alcoholic beverages. Fortunately, ethanol is also involved in the degradation of peroxisomes, cellular compartments where P. pastoris realizes the metabolism of methanol; this aspect is interesting for our application, since it means methanol will not be degraded as fast as it would in the absence of ethanol. Therefore, the degradation of peroxisomes would enhance the activation of PAOX1, by allowing methanol to stay for longer in the cell.
In order to develop this biosensor, it is necessary to evaluate the rates at which the promoter PAOX1 is activated via methanol or inhibited via ethanol. In addition, we decided to study a modification on Pichia pastoris' Mxr1p transcription factor [18] that should alter its interaction with PAOX1 by turning ethanol into an activator of the promoter [19]. If the rate of activation by ethanol stays below the rate of activation by methanol, the latter should be identifiable when the drink is diluted. It would then be possible to create a color guide that would help one differ pure and contaminated beverages (For more information, see the Modeling section).
In addition to testing the PAOX1 that is native in Pichia pastoris, we have synthesized a modified version of this promoter with up to 33% more strength than the wild type promoter [20], according to the hypothesis that a stronger promoter should allow better visualization of the colorimetric output.
PFLD1
This second promoter has a high transcription rate when activated, just like PAOX1, but unlike the previous one it activated by methylamine or methanol [12] [21]. Also, it is not repressed by hexoses, like PAOX1. Since an extensive search in scientific literature did not uncover any data on the regulation of PFLD1 by ethanol, this promoter represents a great alternative to PAOX1 if it is confirmed that they do not share the same repression characteristic related to this dicarbonyl alcohol, eliminating an issue in our project.
Preservation mechanism
Overview
In order to allow portability and storing of the device, and to create a robust and resistant and fast-responding methanol detector, some lyophylization tests were realized, aiming to produce results similar to Saccharomyces cerevisae yeast granules. That was made possible by adapting some protocols [29], originally for different yeast species, to Pichia pastoris. Some tests were realized, essentially looking for answers to the questions below:
- How many cells would have to be lyophilized?
- What is the ideal dilution of the drink?
- How long would it take between the contact between yeast and product and the obtaining of results?
These values predicted via mathematical modelling and tested in laboratory, find the necessary conditions for the use of this modified yeast as a methanol biosensor. The purpose was to test the predictions in non-contaminated beverages, and later in artificially contaminated ones, both as a proof of concept and as a way to test the sensibilities of the sensor.
Freeze-dry
Otto preparando conteúdo.
The device
Descrição só do device mesmo. Ximena?
Também acho que deveria sair esse título e deixar só o link para o Application:
