Team:USP-Brazil/Project

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Detecthol

Molecular detection

Overview

The idea of our team this year was to build a biosensor that would respond to methanol by producing a red-coloured protein. 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 the Mxr1p transcription factor, that [Lin-cereghino et al 2006] should alter its interaction with Paox1 by turning ethanol into an activator of the promoter. 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.

As an alternative, we decided to study another promoter present in Pichia pastoris, namely PFLD1. This second promoter has a high transcription rate when activated, just like Paox1, but instead being activated by methylamine and methanol. Also, it is not repressed by hexoses, like the first one. Since an extensive search in scientific literature did not uncover any data on the regulation of PFLD1 by ethanol, this promoter represents a strong alternative to Paox1, and not only would its characterization be useful for our project, but also it could be very interesting for the expression of heterologous proteins.

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P_AOX1

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 P_AOX1 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 P_AOX1, 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 P_AOX1 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 P_AOX1 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.

P_FLD1

This second promoter has a high transcription rate when activated, just like P_AOX1, but unlike the previous one it activated by methylamine or methanol [12] [21]. Also, it is not repressed by hexoses, like P_AOX1. Since an extensive search in scientific literature did not uncover any data on the regulation of P_FLD1 by ethanol, this promoter represents a great alternative to P_AOX1 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

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References

Incluir referências do Projeto-Olimpiada: 5, 23 a 27, 29, e a Lin-cereghino acima.