Part:BBa_K385002:Experience

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Part:BBa K385002:Experience
Aberdeen iGEM 2010 Bio-brick Experience

This MS2 coat protein from phage MS2 was tested for its capacity to inhibit the translational expression of GFP translationally fused to Npeptide in the yeast expression construct GAL1p-[Npep-GFP]. This was possible as there were two MS2 coat protein binding stem loops upstream of the Npep-GFP mRNA sequence of GAL1p-[Npep-GFP]. The MS2 coat protein was expressed in a separate construct regulated by a MET17 promoter which was induced in the absence of methionine and repressed by its presence. The two constructs were transformed into yeast. Transformant cultures were then grown with galactose to induce the GAL promoter whilst varying concentrations of methionine was added to separate cultures. This allowed the translational inhibition of GFP expression by MS2 coat protein to be characterised using FACS analysis (see Aberdeen 2010 wiki for details ). This analysis showed that there is a linear relationship between GFP expression levels and methionine concentrations, (i.e Increasing methionine concentration resulted in increasing GFP expression as MS2 coat protein expression is repressed by the presence of methionine) which confirms the capacity for MS coat protein to bind to its corresponding stem loops, thereby inhibiting translation of downstream fusion proteins in the mRNA sequence.

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