Team:USP-Brazil/Project

Molecular detection

Overview

The idea of our team this year was to build a biosensor (Chart 1) 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.

>>>>>> ISSO TUDO FOI FEITO? Se não, tem que tirar daqui, né.

(faz um chart) Chart 1: Biosensors

The idea of building sensors based in living organisms is not a recent concept. For more than 20 years [23], the advances in genetic engineering have made it possible for one to insert exogenous genetic systems in organisms as a step on creating biosensors for very specific purposes, such as detecting osmotic concentration or phenolic composites [24-27]. One interesting and successful example is from last year's iGEM competition: a strain of bacteria that is sensible to volatile composites from decomposing meat, essentially a spoiled meat detector (https://2012.igem.org/Team:Groningen).

Based on these technologies, our plan is to create a biosensor using the yeast Pichia pastoris, and taking advantage of its metabolic pathways, of its robustness and availability for genetic manipulation [5], and then to use this biosensor to perform a quick and cheap detection of methanol in alcoholic beverages.

P_AOX1

A montagem do circuito P_AOX1 + RFP é aqui? Estou me guiando pelo ProjetoOlimpiada-PessoaJr.

P_FLD

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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

o que é pra pôr aqui?

The device

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