Team:USP-Brazil/Parts

Parts Submitted to the Registry

<groupparts>iGEM013 USP-Brazil</groupparts>

BBa_K1078000
Mxr1 (methanol expression regulator 1) modified

Mxr1 (methanol expression regulator 1) is a key regulator, at transcriptional level, of methanol metabolism in the methylotrophic yeast Pichia pastoris. It is a transcriptional factor that binds upstream of the MUT (methanol utilizing) pathway and peroxisome biogenesis gene´s promoters using its zinc finger domain. Among the MUT genes is the AOX1 gene that is regulated by the pAOX1 promoter. The pAOX promoter is found three times in the registry of parts; Part:BBa_K431007, Part:BBa_K945000 and BBa_I764001; it is used for heterologous protein expression in Pichia.

The 14-3-3 proteins are ubiquitous, and have important roles in controlling a wide variety of cellular processes, like gene expression, metabolism, cell cycle and apoptosis. These 14-3-3 proteins are involved in the carbon source-dependent regulation of Mxr1, which is inactive in ethanol and glycerol, but is active in methanol.

Our submitted Mxr1 is mutated in order to NOT be repressed by ethanol, as the original Mxr1 is. This is done by substituted the Ser215 of the protein with Ala, inactivating the 14-3-3 protein interaction (phosphorylation) with Mxr1. It is also smaller than the original Mxr1 because it was already reported that the major activation domain of Mxr1 is located within the first 400 amino acids.

The modified Mxr1 is able to activate the pAOX promoter in ethanol, methanol or in a ethanol/methanol solution. This is useful for our biosensor design, which aims to detect levels of methanol above 2% in common alcoholic drinks (normally containing 10 to 60% ethanol). This will allow government to make a preliminary high-throughput screening of ethanol drinks tainted will methanol.

BBa_K1078001
Strong reporter device optimised for Pichia pastoris, activated by methanol

This device is composed of three parts. The first part is a modified promoter pAOX1; it was found in a library where it showed more than 60% stronger activation when compared with the wild-type, as reported by Hartner FS et al., in Promoter library designed for fine-tuned gene expression in Pichia pastoris. Nucleic Acids Res 2008, 36:e76. The second part is a kozak sequence for Pichia pastoris, and the third is a Red fluorescent protein condon optimized for expression in Pichia pastoris.

This device was created to express a reporter gene, in this case RFP, in the presence of ethanol, its expression is inactivated by ethanol. When combined with the modified Mxr1 (BBa_K1078000), it is not inactivated by ethanol. This is useful for our biosensor design, which aims to detect levels of methanol above 2% in common alcoholic drinks (normally containing 10 to 60% ethanol). This will allow government to make a preliminary high-throughput screening of ethanol drinks tainted will methanol.