Difference between revisions of "Team:HKUST-Rice/Expression/ParaBAD"

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We found that <i>P<sub>araBAD</sub></i> 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, <i>P<sub>araBAD</sub></i> is sensitive to different range of arabinose.
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We found that <i>P<sub>araBAD</sub></i> 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, <i>P<sub>araBAD</sub></i> is sensitive to different range of arabinose. Besides that, we found that <i>P<sub>araBAD</sub></i> on a low copy plasmid does not display all-or-none behaviour on a single cell level.
 
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<p>
 
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.
 
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.
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<h1>Plasmid copy number and range of sensitivity</h1>
 
<h1>Plasmid copy number and range of sensitivity</h1>
 
<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.">
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<div class= "des"><strong><b>Figure 2. Transfer functions for BBa_I2031 on plasmid pSB3K3 and pSB1K3.</b></strong></div>
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<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<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>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>
 
 
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<h1>All-or-none behaviour  NOT on a single cell level</h1>
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<img style="width:90%" src="https://static.igem.org/mediawiki/2015/2/27/HKUST_Rice_ParaBAD_Fig_1.png" alt="Comparison of our result to those by Cambridge 2011 and Groningen 2011">
 +
<div class= "des"><strong><b>Figure 4 from literature showing all-or-none behavior of PBAD on a single cell level. </b></strong>As shown in previous studies, arabinose induction at a sub-saturating concentration leads to heterogeneously activated population. Figure was directly cropped from figure 3 in the paper “Regulatable Arabinose-Inducible Gene Expression System with Consistent Control in All Cells of a Culture” (Khlebnikov et al., 2000). </div>
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<p>
 +
The all or none behavior of PBAD on a single cell level has been reported in a number of literatures (Fritz et al., 2014; Khlebnikov et al., 2000; Khlebnikov et al., 2001; Siegele and Hu, 1997). It describes that an increase in arabinose concentration does not result in an increase in promoter / gene activity per se, but rather, increases the proportion of population that are fully induced (Figure 4). According to Khelbnikov et al., the all-or-none behavior observed is due to the autocatalytic behavior of the AraE transporter, creating a positive feedback mechanism on a single cell level. Driving expression of the AraE by a constitutive promoter abrogates the feedback and transforms the all-or-none behavior into homogenous expression of PBAD. (Khlebnikov et al., 2001)</p>
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 +
<p>Nonetheless, as revealed by our histograms for intermediate level of induction, the situation described above is not always true. The all-or-none behavior applied only when BBa_I0500 is placed on a high copy plasmid, where induced cells were mostly distributed among two bins of fluorescence (Figure 5). Yet, when the low copy pSB3K3 plasmid was used, the all-or-none behavior no longer holds and the populations remain homogenous along the arabinose concentration gradient. </p>
 +
 +
<p>Going back to the literature, we confirmed that all of them were studying PBAD on plasmids with high copy origins of replication (Table 1). Thus, we believe that the homogenous expression of PBAD promoter from a low copy plasmid has been a long overlooked issue.</p>
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<table>
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<tr><td>Reference</td><td>Plasmid harboring PBAD-reporter</td><td>Plasmid lineage</td><td>Traced origin of replication</td></tr>
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<tr><td>Khlebnikov et al., 2000
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Khlebnikov et al., 2001
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</td><td>pCSAK50</td><td>pBAD24 &#8594; pTC40 &#8594; pCSAK50</td><td>High copy
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pBR322 origin *
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</td></tr>
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<tr><td>Siegele and Hu, 1997</td><td>pDS439</td><td>pBAD18 &#8594; pDS439</td><td>High copy
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pBR322 origin *
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</td></tr>
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<tr><td>Fritz et al., 2014</td><td>pBAD24-GFP</td><td>pBAD24 &#8594; pBAD24-GFP</td><td>High copy
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pBR322 origin *
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</td></tr>
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</table>
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<p>* The paper by Guzman et al. described the origins as pBR origins (Guzman et al., 1995). However, they lack the rop gene that maintains low copy number. Thus they are high copy origins (Cronan, 2006). The origin of pBAD24 in ATCC (ATCC® 87399™) was also documented to have the pMB1 origin, which should be the same origin in pSB1C3. </p>
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<p>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). </p>
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<img style="width:90%" src="https://static.igem.org/mediawiki/2015/5/5b/HKUST_RICE2015_1K3_3K3-I2031HistogramsComparison.png" alt="Histogram plots for sensing ranges of BBa_I0500 on high and low copy plasmid">
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<div class= "des"><strong><b>FFigure 5. Histogram plots for sensing ranges of BBa_I0500 on high and low copy plasmid.  </b></strong>Concentrations of arabinose for high copy pSB1K3 plasmid: 0.488µM – 0.25mM. For low copy pSB3K3 plasmid: 0.0610µM – 0.03125mM. Only 1 set of experiment result from 3 replicates is presented. </div>
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Revision as of 18:47, 17 September 2015

ParaBAD



Investigation on ParaBAD

image caption

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. Besides that, we found that ParaBAD on a low copy plasmid does not display all-or-none behaviour on a single cell level.

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

Comparison of our result to those by Cambridge 2011 and Groningen 2011
Figure 1. Comparison of our result to those by Cambridge 2011 and Groningen 2011.

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

Transfer functions for BBa_I2031 on plasmid pSB3K3 and pSB1K3.
Figure 2. Transfer functions for BBa_I2031 on plasmid pSB3K3 and pSB1K3.

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.

All-or-none behaviour NOT on a single cell level

Comparison of our result to those by Cambridge 2011 and Groningen 2011
Figure 4 from literature showing all-or-none behavior of PBAD on a single cell level. As shown in previous studies, arabinose induction at a sub-saturating concentration leads to heterogeneously activated population. Figure was directly cropped from figure 3 in the paper “Regulatable Arabinose-Inducible Gene Expression System with Consistent Control in All Cells of a Culture” (Khlebnikov et al., 2000).

The all or none behavior of PBAD on a single cell level has been reported in a number of literatures (Fritz et al., 2014; Khlebnikov et al., 2000; Khlebnikov et al., 2001; Siegele and Hu, 1997). It describes that an increase in arabinose concentration does not result in an increase in promoter / gene activity per se, but rather, increases the proportion of population that are fully induced (Figure 4). According to Khelbnikov et al., the all-or-none behavior observed is due to the autocatalytic behavior of the AraE transporter, creating a positive feedback mechanism on a single cell level. Driving expression of the AraE by a constitutive promoter abrogates the feedback and transforms the all-or-none behavior into homogenous expression of PBAD. (Khlebnikov et al., 2001)

Nonetheless, as revealed by our histograms for intermediate level of induction, the situation described above is not always true. The all-or-none behavior applied only when BBa_I0500 is placed on a high copy plasmid, where induced cells were mostly distributed among two bins of fluorescence (Figure 5). Yet, when the low copy pSB3K3 plasmid was used, the all-or-none behavior no longer holds and the populations remain homogenous along the arabinose concentration gradient.

Going back to the literature, we confirmed that all of them were studying PBAD on plasmids with high copy origins of replication (Table 1). Thus, we believe that the homogenous expression of PBAD promoter from a low copy plasmid has been a long overlooked issue.

ReferencePlasmid harboring PBAD-reporterPlasmid lineageTraced origin of replication
Khlebnikov et al., 2000 Khlebnikov et al., 2001 pCSAK50pBAD24 → pTC40 → pCSAK50High copy pBR322 origin *
Siegele and Hu, 1997pDS439pBAD18 → pDS439High copy pBR322 origin *
Fritz et al., 2014pBAD24-GFPpBAD24 → pBAD24-GFPHigh copy pBR322 origin *

* The paper by Guzman et al. described the origins as pBR origins (Guzman et al., 1995). However, they lack the rop gene that maintains low copy number. Thus they are high copy origins (Cronan, 2006). The origin of pBAD24 in ATCC (ATCC® 87399™) was also documented to have the pMB1 origin, which should be the same origin in pSB1C3.

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).

Histogram plots for sensing ranges of BBa_I0500 on high and low copy plasmid
FFigure 5. Histogram plots for sensing ranges of BBa_I0500 on high and low copy plasmid. Concentrations of arabinose for high copy pSB1K3 plasmid: 0.488µM – 0.25mM. For low copy pSB3K3 plasmid: 0.0610µM – 0.03125mM. Only 1 set of experiment result from 3 replicates is presented.