Difference between revisions of "Part:BBa K1725001"
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This reporter was used to characterise the promoter K1725000.
 
This reporter was used to characterise the promoter K1725000.
  
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GFP fluorescence of K1725001, K1725002, K1725021, K1725022, K1725082, and E5504 with plasmid backbone pSB3K3 was measured to compare the relative strengths of promoters K1725000 and K1725020 to a promoter already well documented in the registry, R0040. The figure below shows the fluorescence scan image and a graph of approximate molecules of GFP per cell. These results indicated that K1725000 is a significantly stronger promoter than R0040 or K1725020.
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GFP fluorescence of K1725001 (K1725000.I13500), K1725002 (K1725000.E5501), K1725021 (SrpR repressible promoter.I13500), K1725022 (SrpR repressible promoter.E5501), K1725082 (TetR repressible promoter.I13500), and E5504 (TetR repressible promoter.E5501) with plasmid backbone pSB3K3 was measured to compare the relative strengths of promoters K1725000 and K1725020 (SrpR repressible promoter) to a promoter already well documented in the registry, R0040 (TetR repressible promoter). Figure 1 the fluorescence scan image and a graph of approximate molecules of GFP per cell. These results indicated that K1725000 is a significantly stronger promoter than R0040 or K1725020.
  
 
https://static.igem.org/mediawiki/2015/d/df/Glasgow_2015_Repressors_Promoter_Graph_2.png
 
https://static.igem.org/mediawiki/2015/d/df/Glasgow_2015_Repressors_Promoter_Graph_2.png
  
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<b>All constructs with pSB3K3 plasmid backbone, in DH5α cells. Replicates of constructs and controls from three colonies, under the same conditions. Mean and standard deviation of replicates were calculated to give value and error bars.</b>
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<b>Figure 1. All constructs with pSB3K3 plasmid backbone, in DH5α cells. Replicates of constructs and controls from three colonies, under the same conditions. Mean and standard deviation of replicates were calculated to give value and error bars.</b>
  
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This reporter was also used to characterise repression of K1725000 by K1725040.
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K1725000 driven expression is repressed by K1725040 (<i>phlF</i> encoding PhlF repressor) as shown in Figure 2. K1725042 is K1725040 driven by the <i>lacI</i> regulated promoter K1725080. Our control was K1725083 (the Tet repressor C0040 also driven by K1725080) and K1725082 (the TetR repressible promoter R0040 driving expression of I13500).
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K1725000 driven expression is repressed by K1725040 (<i>phlF</i> encoding PhlF repressor) as shown in the figure below. K1725042 is K1725040 driven by the <i>lacI</i> regulated promoter K1725080. Our control was K1725083 (the Tet repressor C0040 also driven by K1725080) and K1725082 (the TetR repressible promoter R0040 driving expression of I13500).
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https://static.igem.org/mediawiki/2015/9/95/Glasgow_2015_Repression_Fold_Graph.png
 
https://static.igem.org/mediawiki/2015/9/95/Glasgow_2015_Repression_Fold_Graph.png
  
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<b>Represser constructs with pSB1C3 backbone; promoter driving GFP constructs with pSB3K3 backbone. Repressor protein expression induced with 100μM IPTG. Replicates of constructs and controls of three dilutions from one colony, under the same conditions. Mean and standard deviation of replicates were calculated to give value and error bars.</b>
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<b>Figure 2. Repressor constructs in pSB1C3 backbone; promoter driving GFP constructs in pSB3K3 backbone. Cells were grown overnight in 100μM IPTG, to induce expression of the repressor proteins. Three replicates of the sample were diluted and tested under the same conditions for each sample. Mean and standard deviation of replicates were calculated to give value and error bars.</b>
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Revision as of 19:10, 18 September 2015

PhlF repressible promoter + strong RBS + GFP

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 718

This reporter was used to characterise the promoter K1725000.

GFP fluorescence of K1725001 (K1725000.I13500), K1725002 (K1725000.E5501), K1725021 (SrpR repressible promoter.I13500), K1725022 (SrpR repressible promoter.E5501), K1725082 (TetR repressible promoter.I13500), and E5504 (TetR repressible promoter.E5501) with plasmid backbone pSB3K3 was measured to compare the relative strengths of promoters K1725000 and K1725020 (SrpR repressible promoter) to a promoter already well documented in the registry, R0040 (TetR repressible promoter). Figure 1 the fluorescence scan image and a graph of approximate molecules of GFP per cell. These results indicated that K1725000 is a significantly stronger promoter than R0040 or K1725020.

Glasgow_2015_Repressors_Promoter_Graph_2.png

Figure 1. All constructs with pSB3K3 plasmid backbone, in DH5α cells. Replicates of constructs and controls from three colonies, under the same conditions. Mean and standard deviation of replicates were calculated to give value and error bars.

K1725000 driven expression is repressed by K1725040 (phlF encoding PhlF repressor) as shown in Figure 2. K1725042 is K1725040 driven by the lacI regulated promoter K1725080. Our control was K1725083 (the Tet repressor C0040 also driven by K1725080) and K1725082 (the TetR repressible promoter R0040 driving expression of I13500).


Glasgow_2015_Repression_Fold_Graph.png

Figure 2. Repressor constructs in pSB1C3 backbone; promoter driving GFP constructs in pSB3K3 backbone. Cells were grown overnight in 100μM IPTG, to induce expression of the repressor proteins. Three replicates of the sample were diluted and tested under the same conditions for each sample. Mean and standard deviation of replicates were calculated to give value and error bars.