Difference between revisions of "Part:BBa K385002:Experience"

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===Applications of BBa_K385002===
 
===Applications of BBa_K385002===
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The MS2 coat protein reading frame was fused in-frame to GFP to make a translational fusion. It was placed under control of the yeast GAL1 promoter (BBa_J63006), and transformed into yeast in the single copy shuttle vector pRS415.
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Part:BBa K385002:Experience
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Aberdeen iGEM 2010 Bio-brick Experience
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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
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(see also <a href="http://2010.igem.org/MS2_Coat-Protein_Effect_on_Expression_of_GFP_in_pRS415"> Aberdeen 2010 wiki for details </a>). 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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===User Reviews===
  
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The transformants were grown overnight in synthetic defined medium containing 2% w/v galactose, and observed usin a fluorescence microscope optimised for GFP visualisation (Figure 1).
 
  
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A control culture of the same transformant was grown using glucose as the carbon source;  these conditions do not activate the GAL promoter. The results (Figure 2) show no GFP fluorescence.
 
  
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Overall the results indicate that the MS2 coat protein can be successfully expressed as a protein fusion with other standard parts.
 
  
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This part was shown to be able to inhibit translation of fusion proteins if the MS2 coat protein stem loop binding sequence is upstream of the mRNA sequence that translates to the fusion protein.
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Latest revision as of 22:34, 4 November 2010

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K385002

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

MS2 experience.jpg



(see also 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.

User Reviews

UNIQf77a9bd3caa2c022-partinfo-00000002-QINU

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Slam

This part was shown to be able to inhibit translation of fusion proteins if the MS2 coat protein stem loop binding sequence is upstream of the mRNA sequence that translates to the fusion protein.

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UNIQf77a9bd3caa2c022-partinfo-00000004-QINU