Part:BBa_K779302

eYFP-4xFF1 MammoBlock

This part is an engineered variant of the original green fluorescent protein from Aequorea victoria that fluoresces yellow. Additionally, this protein has four binding sites for the FF1 micro RNA (miRNA). In the presence of FF1 miRNA, translation of this protein is markedly attenuated.

Through the Gateway method of cloning, we created the expression vector Hef1A:eYFP-4xFF1 (BBa_K779603). We gathered data on eYFP-4xFF1 driven by the mammalian constitutive promoter Hef1A:

The circuit that MIT iGEM 2012 tested:

In this circuit, HEK293 cells transfected with pEXPR_1-2_Hef1A-eYFP-4xFF1 and Tag-BFP, express yellow and blue fluorescent proteins. However, when co-transfected with U6 TetO: FF1 plasmid, FF1 miRNAs block the expression of yellow fluorescent proteins via binding to FF1 sites on pEXPR_1-2_Hef1A-eYFP-4xFF1; HEK293 cells therefore only appear blue (due to Tag-BFP). As the ratio of mirFF1 increases from 0.25X to 8X, there is a corresponding decrease in the yellow fluorescent signal, indicating gene knockdown. The histograms show the population of cells shifting from yellow towards the non-fluorescent region.

Experimental Data:

100,000 HEK293 Cells were transfected with varying molar ratios of U6-tetO:mirFF1 to Hef1a:eYFP 4xFF1, and 1:1 molar ratio of Hef1a:eYFP 4xFF1 and Hef1a:TagBFP (a transfection marker), standardized to a total of 500ng plasmid DNA using 1.65 uL of lipofectamine 2000. As the ratio of mirFF1 increases from 0.25X to 8X, there is a corresponding decrease in the yellow fluorescent signal, indicating gene knockdown. The histograms show the population of cells shifting from yellow towards the non-fluorescent region, by 10². This region was determined by analyzing a no-transfection control.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 850
  Illegal PstI site found at 211
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 850
  Illegal PstI site found at 211
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 850
  Illegal PstI site found at 211
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 850
  Illegal PstI site found at 211
  
• 1000
  COMPATIBLE WITH RFC[1000]