Difference between revisions of "Team:HKUST-Rice/Expression/ParaBAD"
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Cambridge 2011 reported a threshold of all-or-none between 0.001mM and 0.01mM of arabinose and Groningen 2011 reported an input dynamic range from 0.05% to 1% of arabinose. Our result did not agree with either of theirs.</p> | Cambridge 2011 reported a threshold of all-or-none between 0.001mM and 0.01mM of arabinose and Groningen 2011 reported an input dynamic range from 0.05% to 1% of arabinose. Our result did not agree with either of theirs.</p> | ||
| − | <p>Our result shows that the sensing range for <i>P<sub>araBAD</sub></i> is graded and roughly spans from 10-4 mM (or 0.0001mM) to 10-2 mM (or 0.01mM), and the production of GFP saturates beyond these concentrations.</p> | + | <p>Our result shows that the sensing range for <i>P<sub>araBAD</sub></i> is graded and roughly spans from 10<sup>-4</sup> mM (or 0.0001mM) to 10<sup>-2</sup> mM (or 0.01mM), and the production of GFP saturates beyond these concentrations.</p> |
<p> | <p> | ||
<p>In comparison to result by Cambridge 2011, the claimed call all-or-none response was not observed and the fluorescence intensity on a population level can be tuned incrementally.</p> | <p>In comparison to result by Cambridge 2011, the claimed call all-or-none response was not observed and the fluorescence intensity on a population level can be tuned incrementally.</p> | ||
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<img style="width:90%" src="https://static.igem.org/mediawiki/2015/f/f6/HKUST_Rice_ParaBAD_Fig_2.png" alt="Transfer functions for BBa_I2031 on plasmid pSB3K3 and pSB1K3."> | <img style="width:90%" src="https://static.igem.org/mediawiki/2015/f/f6/HKUST_Rice_ParaBAD_Fig_2.png" alt="Transfer functions for BBa_I2031 on plasmid pSB3K3 and pSB1K3."> | ||
<div class= "des"><strong><b>Figure 2. Transfer functions for BBa_I2031 on plasmid pSB3K3 and pSB1K3.</b></strong></div> | <div class= "des"><strong><b>Figure 2. Transfer functions for BBa_I2031 on plasmid pSB3K3 and pSB1K3.</b></strong></div> | ||
| − | <p>On pSB3K3, PBAD is responsive to 10-4 - 10-2 mM arabinose, whereas on pSB1K3, it senses arabinose from roughly 10-3 to 1mM (see supplementary figure 2).</p> | + | <p>On pSB3K3, PBAD is responsive to 10<sup>-4</sup> - 10<sup>-2</sup> mM arabinose, whereas on pSB1K3, it senses arabinose from roughly 10<sup>-3</sup> to 1mM (see supplementary figure 2).</p> |
<p>BBa_I2031 appeared to be giving less fluorescence in low arabinose concentrations when placed on the high copy pSB1K3 plasmid than on low copy pSB3K3 plasmid, and the value is even lower than the auto fluorescence observed from the negative control (DH10B / pSB1K3-BBa_E0240). It might be interpreted that the promoter is less leaky when placed on a high copy plasmid.</p> | <p>BBa_I2031 appeared to be giving less fluorescence in low arabinose concentrations when placed on the high copy pSB1K3 plasmid than on low copy pSB3K3 plasmid, and the value is even lower than the auto fluorescence observed from the negative control (DH10B / pSB1K3-BBa_E0240). It might be interpreted that the promoter is less leaky when placed on a high copy plasmid.</p> | ||
Revision as of 17:28, 17 September 2015
ParaBAD
Investigation on ParaBAD
ParaBAD is a widely used promoter, its BioBrick, BBa_I0500, reported 392 uses to date (as of 4th Sep 2015). Yet different behaviors of ParaBAD have been reported. Cambridge 2011 iGEM team reported an All-or-None behavior whereas Groningen 2011 iGEM team reported a graded response.
We found that ParaBAD display graded behavior across a gradient of arabinose, which is in disagreement with Cambridge 2011 iGEM team. Also, we shows that on plasmid with different copy number, ParaBAD is sensitive to different range of arabinose.
Since this year our team will also use this promoter in the project, we wish to further characterize this promoter. Through this investigation, our team hope to caution users in choosing this promoter for future construction.
Graded behavior across a gradient of arabinose
Cambridge 2011 reported a threshold of all-or-none between 0.001mM and 0.01mM of arabinose and Groningen 2011 reported an input dynamic range from 0.05% to 1% of arabinose. Our result did not agree with either of theirs.
Our result shows that the sensing range for ParaBAD is graded and roughly spans from 10-4 mM (or 0.0001mM) to 10-2 mM (or 0.01mM), and the production of GFP saturates beyond these concentrations.
In comparison to result by Cambridge 2011, the claimed call all-or-none response was not observed and the fluorescence intensity on a population level can be tuned incrementally.
In comparison to result by Groningen 2011, they reported an input dynamic range from 0.05% to 1% arabinose (corresponding to 3.33 mM and 66.7 mM respectively). That reported range lies in our induction saturated range( the red zone).
Plasmid copy number and range of sensitivity
On pSB3K3, PBAD is responsive to 10-4 - 10-2 mM arabinose, whereas on pSB1K3, it senses arabinose from roughly 10-3 to 1mM (see supplementary figure 2).
BBa_I2031 appeared to be giving less fluorescence in low arabinose concentrations when placed on the high copy pSB1K3 plasmid than on low copy pSB3K3 plasmid, and the value is even lower than the auto fluorescence observed from the negative control (DH10B / pSB1K3-BBa_E0240). It might be interpreted that the promoter is less leaky when placed on a high copy plasmid.