Difference between revisions of "Part:BBa K1725000"
 
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<partinfo>BBa_K1725000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1725000 SequenceAndFeatures</partinfo>
  
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This promoter was characterised by driving expression of GFP with two different Ribosome Binding Sites, as found in the BioBricks E5501 and I13500.
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This promoter was characterised by using it to drive expression of GFP with two different Ribosome Binding Sites, <bbpart>BBa_B0032</bbpart> in E5501 and <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)
  
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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.
 
  
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https://static.igem.org/mediawiki/2015/d/df/Glasgow_2015_Repressors_Promoter_Graph_2.png
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https://static.igem.org/mediawiki/parts/e/ee/Glasgow_2015_Promoter_Strength_Graph.png
  
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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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<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>
  
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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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https://static.igem.org/mediawiki/parts/7/78/Glasgow_2015_Fold_Repression_Graph.png
  
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https://static.igem.org/mediawiki/2015/9/95/Glasgow_2015_Repression_Fold_Graph.png
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<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>
  
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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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<h2> <b> IIT Delhi 2017 - Characterization of Promoter Strength </b> </h2>
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<p> We see in Fig. 1 that the decreasing order of strengths for the five repressible promoters is pLac, pcI, pPhlF, pTet and pSrpR. This is also evident in Fig. 2 when all the 5 promoters settle to a steady state. Fig. 3 depicts the protein production rates for the promoters.  pLac has the largest production rate reaffirming its highest strength. However, despite dominating completely in terms of protein production rate, we see in Figs. 2 and 4 that in the initial transient phase, pLac has lower relative strength compared to pTet and pcI. This is due to the highest dilution rate for pLac in this regime, which can be seen in Figs. 5 and 6. Similar arguments can be made about pcI. </p>
  
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[[File:Photobleaching_Fig_1.png|900px|thumb|left|Fig 1. Relative Strength for the five repressible promoters, including the novel promoters pPhlF and pSrpR, used in the construction and analysis of 5n1. The decreasing order of relative strength is pLac > pcI > pPhlF > pTet > pSrpR. Rlative strengths have been averaged over 4 trials.]]
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[[File:Photobleaching_Fig_2.png|900px|thumb|left|Fig 2. Relative Strength over time for the five repressible promoters, including the novel promoters pPhlF and pSrpR, used in the construction and analysis of 5n1. The plots have been averaged over 4 trials.]]
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[[File:Photobleaching_Fig_3.png|900px|thumb|left|Fig 3. Protein production rate over time for the five repressible promoters, including the novel promoters pPhlF and pSrpR, used in the construction and analysis of 5n1. The plots have been averaged over 4 trials.]]
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[[File:Photobleaching_Fig_4.png|900px|thumb|left|Fig 4. Relative Strength versus OD over time for the five repressible promoters, including the novel promoters pPhlF and pSrpR, used in the construction and analysis of 5n1. The plots have been averaged over 4 trials.]]
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[[File:Photobleaching_Fig_5.png|900px|thumb|left|Fig 5. Dilution rate over time for the five repressible promoters, including the novel promoters pPhlF and pSrpR, used in the construction and analysis of 5n1. The plots have been averaged over 4 trials.]]
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[[File:Photobleaching_Fig_6.png|900px|thumb|left|Fig 6. OD over time for the five repressible promoters, including the novel promoters pPhlF and pSrpR, used in the construction and analysis of 5n1. The plots have been averaged over 4 trials.]]
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<p> As shown in Fig. 2, pLac and pcI have three distinct regions of behavior in terms of relative strength over time. In the initial transient phase, dilution rate is higher than the protein production rate, thus we see that relative strength per OD decreases over time. The middle corresponds to overcompensation phase, where protein production rate surpasses the dilution rate. Finally, the relative strength settles a steady state value, entering an exact compensation between protein production and dilution rates. Contrary to pLac and pcI, the other promoters don’t exhibit the overcompensation phase.  </p>
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
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Latest revision as of 03:09, 28 October 2017

PhlF repressible promoter

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
    COMPATIBLE WITH RFC[1000]

This promoter was characterised by using it to drive expression of GFP with two different Ribosome Binding Sites, BBa_B0032 in E5501 and 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.

IIT Delhi 2017 - Characterization of Promoter Strength

We see in Fig. 1 that the decreasing order of strengths for the five repressible promoters is pLac, pcI, pPhlF, pTet and pSrpR. This is also evident in Fig. 2 when all the 5 promoters settle to a steady state. Fig. 3 depicts the protein production rates for the promoters. pLac has the largest production rate reaffirming its highest strength. However, despite dominating completely in terms of protein production rate, we see in Figs. 2 and 4 that in the initial transient phase, pLac has lower relative strength compared to pTet and pcI. This is due to the highest dilution rate for pLac in this regime, which can be seen in Figs. 5 and 6. Similar arguments can be made about pcI.

Fig 1. Relative Strength for the five repressible promoters, including the novel promoters pPhlF and pSrpR, used in the construction and analysis of 5n1. The decreasing order of relative strength is pLac > pcI > pPhlF > pTet > pSrpR. Rlative strengths have been averaged over 4 trials.


Fig 2. Relative Strength over time for the five repressible promoters, including the novel promoters pPhlF and pSrpR, used in the construction and analysis of 5n1. The plots have been averaged over 4 trials.


Fig 3. Protein production rate over time for the five repressible promoters, including the novel promoters pPhlF and pSrpR, used in the construction and analysis of 5n1. The plots have been averaged over 4 trials.


Fig 4. Relative Strength versus OD over time for the five repressible promoters, including the novel promoters pPhlF and pSrpR, used in the construction and analysis of 5n1. The plots have been averaged over 4 trials.


Fig 5. Dilution rate over time for the five repressible promoters, including the novel promoters pPhlF and pSrpR, used in the construction and analysis of 5n1. The plots have been averaged over 4 trials.


Fig 6. OD over time for the five repressible promoters, including the novel promoters pPhlF and pSrpR, used in the construction and analysis of 5n1. The plots have been averaged over 4 trials.


As shown in Fig. 2, pLac and pcI have three distinct regions of behavior in terms of relative strength over time. In the initial transient phase, dilution rate is higher than the protein production rate, thus we see that relative strength per OD decreases over time. The middle corresponds to overcompensation phase, where protein production rate surpasses the dilution rate. Finally, the relative strength settles a steady state value, entering an exact compensation between protein production and dilution rates. Contrary to pLac and pcI, the other promoters don’t exhibit the overcompensation phase.