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Team:USP-Brazil/Project
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Detecthol
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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 eles três
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:
