Difference between revisions of "Part:BBa K779400"
 
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<partinfo>BBa_K779400 short</partinfo>
 
<partinfo>BBa_K779400 short</partinfo>
  
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The U6-TetO-FF1 part encodes conditional expression of the short hairpin RNA sequence FF1. The mammalian RNA Pol III U6 promoter incorporates tetracycline-responsive elements that allow for tightly regulated RNA expression under specific conditions. In the presence of the Tet repressor (TetR), two Tet operator (TetO) sites become bound by the TetR and prevent RNA Pol III from binding, subsequently inhibiting transcription of the downstream sequence. In the absence of TetR, RNA transcription proceeds normally. The short hairpin FF1 RNA sequence can be used as an RNA interference mechanism through its binding to a complementary region present in the transcript of another gene, thus inhibiting translation of that sequence and silencing expression of that gene.  
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The U6-TetO-FF1 part encodes conditional expression of the short RNA sequence FF1. The mammalian RNA Pol III U6 promoter incorporates tetracycline-responsive elements that allow for tightly regulated RNA expression under specific conditions. In the presence of the Tet repressor (TetR), two Tet operator (TetO) sites become bound by the TetR and prevent RNA Pol III from binding, subsequently inhibiting transcription of the downstream sequence. In the absence of TetR, RNA transcription proceeds normally. The short FF1 RNA sequence can be used as an RNA interference mechanism through its binding to a complementary region present in the transcript of another gene, thus inhibiting translation of that sequence and silencing expression of that gene.  
  
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The design of the U6-TetO promoter is based on data from  "Comparison of RNAi efficiency mediated by tetracycline-responsive H1 and U6 promoter variants in mammalian cell lines" Henriksen et. al. (Nucleic Acids Research, 2007).
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https://static.igem.org/mediawiki/2012/a/a1/FF1-KD-Gated-Normalized.png
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<i><font size="1.5"> 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 10<sup>2</sup>. This region was determined by analyzing a no-transfection control.</font></i>
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https://static.igem.org/mediawiki/2012/a/a7/MIT2012_actuation_linhs.png
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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.
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This experiment allowed us to characterize the ability of U6 promoter to drive the expression of short strands of RNA (like FF1 miRNA): by varying molar ratios of miRNA to pEXPR_1-2_Hef1A-eYFP-4xFF1 plasmid, we can calibrate the strength of the U6 promoter. Furthermore, we can harness such ability of U6 promoter to drive the expression of RNA oligonucleotides involved in processing or actuation. Another advantage to this system is that it can serve as an actuation output: the miRNA expressed can inhibit the expression of a gene of interest or relieve the inhibition of its expression. </p>
 
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===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 08:07, 12 October 2012

U6-TetO-FF1 MammoBlock

The U6-TetO-FF1 part encodes conditional expression of the short RNA sequence FF1. The mammalian RNA Pol III U6 promoter incorporates tetracycline-responsive elements that allow for tightly regulated RNA expression under specific conditions. In the presence of the Tet repressor (TetR), two Tet operator (TetO) sites become bound by the TetR and prevent RNA Pol III from binding, subsequently inhibiting transcription of the downstream sequence. In the absence of TetR, RNA transcription proceeds normally. The short FF1 RNA sequence can be used as an RNA interference mechanism through its binding to a complementary region present in the transcript of another gene, thus inhibiting translation of that sequence and silencing expression of that gene.

The design of the U6-TetO promoter is based on data from "Comparison of RNAi efficiency mediated by tetracycline-responsive H1 and U6 promoter variants in mammalian cell lines" Henriksen et. al. (Nucleic Acids Research, 2007).

FF1-KD-Gated-Normalized.png
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.
MIT2012_actuation_linhs.png
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.

This experiment allowed us to characterize the ability of U6 promoter to drive the expression of short strands of RNA (like FF1 miRNA): by varying molar ratios of miRNA to pEXPR_1-2_Hef1A-eYFP-4xFF1 plasmid, we can calibrate the strength of the U6 promoter. Furthermore, we can harness such ability of U6 promoter to drive the expression of RNA oligonucleotides involved in processing or actuation. Another advantage to this system is that it can serve as an actuation output: the miRNA expressed can inhibit the expression of a gene of interest or relieve the inhibition of its expression.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 248