Difference between revisions of "Part:BBa K1725001"
 
Line 9: Line 9:
 
<partinfo>BBa_K1725001 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1725001 SequenceAndFeatures</partinfo>
  
−
This reporter was used to characterise the promoter K1725000.
+
This reporter was used to characterise the promoter <bbpart>BBa_K1725000</bbpart> which was used to drive expression of GFP with two different Ribosome Binding Sites, <bbpart>BBa_B0032</bbpart> in E5501 and, for this part, <bbpart>BBa_B0034</bbpart> in I13500. PphlF is a stronger promoter than pL-tet (<bbpart>BBa_R0040</bbpart> or PsrpR (<bbpart>BBa_K1725020</bbpart>). (figure 1) PhlF (<bbpart>BBa_K1725040</bbpart>) gave 83-fold repression of GFP expression from PphlF, whereas the control, TetR (<bbpart>BBa_C0040</bbpart>), gave only 33-fold repression of pL-tet. (figure 2)
  
−
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/parts/e/ee/Glasgow_2015_Promoter_Strength_Graph.png
  
−
<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>
+
<b>Figure 1 Relative Promoter Strength. BioBricks: K1725082 (pL-tet driving GFP expression with B0034), E5504 (pL-tet driving GFP expression with B0032), K1725001 (PphlF driving GFP expression with B0034), and K1725002 (PphlF driving GFP expression with B0032). Mean and standard deviation of replicates were calculated to give value and error bars, and normalised against a negative control.</b>
  
−
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).
 
  
 +
https://static.igem.org/mediawiki/parts/7/78/Glasgow_2015_Fold_Repression_Graph.png
  
−
https://static.igem.org/mediawiki/2015/9/95/Glasgow_2015_Repression_Fold_Graph.png
+
<b>Figure 2 Fold Repression. BioBricks: K1725082 (pL-tet driving GFP expression), K1725031 (pL-lac driving TetR expression), K1725001 (PphlF driving GFP expression), and K1725042 (pL-lac driving PhlF expression). Repressor protein expression induced with 100μM IPTG. Mean and standard deviation of replicates were calculated to give value and error bars, and normalised against a negative control.</b>
  
−
<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>
 
  
  

Latest revision as of 19:26, 20 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 BBa_K1725000 which was used to drive expression of GFP with two different Ribosome Binding Sites, BBa_B0032 in E5501 and, for this part, BBa_B0034 in I13500. PphlF is a stronger promoter than pL-tet (BBa_R0040 or PsrpR (BBa_K1725020). (figure 1) PhlF (BBa_K1725040) gave 83-fold repression of GFP expression from PphlF, whereas the control, TetR (BBa_C0040), gave only 33-fold repression of pL-tet. (figure 2)


Glasgow_2015_Promoter_Strength_Graph.png

Figure 1 Relative Promoter Strength. BioBricks: K1725082 (pL-tet driving GFP expression with B0034), E5504 (pL-tet driving GFP expression with B0032), K1725001 (PphlF driving GFP expression with B0034), and K1725002 (PphlF driving GFP expression with B0032). Mean and standard deviation of replicates were calculated to give value and error bars, and normalised against a negative control.


Glasgow_2015_Fold_Repression_Graph.png

Figure 2 Fold Repression. BioBricks: K1725082 (pL-tet driving GFP expression), K1725031 (pL-lac driving TetR expression), K1725001 (PphlF driving GFP expression), and K1725042 (pL-lac driving PhlF expression). Repressor protein expression induced with 100μM IPTG. Mean and standard deviation of replicates were calculated to give value and error bars, and normalised against a negative control.