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

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<div id= "page_title"><h1><i>P<sub>araBAD</sub></i></h1>
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<div id= "page_title"><h1>Characterization and Investigation on AraC-<i>P<sub>araBAD</sub> Promoter BBa_I0500</i></h1>
 
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<h1>Investigation on <i>P<sub>araBAD</sub></i></h1>
 
<h1>Investigation on <i>P<sub>araBAD</sub></i></h1>
 
 
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<img  style="width:80%" src="https://static.igem.org/mediawiki/2015/9/9e/HKUST_Rice15histograms_comparison%28for_cover_figure%29v2.png" alt="image caption">
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<p>The 2014 HKUST iGEM team had previously submitted characterization data of Part BBa_I0500 that described an all-or-none behavior of the promoter. That is however <u>not true</u>. We discovered a few issues in reproducibility and interpretation. Essentially, our result from 2014 did not fully capture the arabinose sensing range. The all-or-none response reported on pSB3K3 was also an artifact, where dead cells from an old plate interfered with fluorescent readings.
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<h2>Download a <a href="https://static.igem.org/mediawiki/2015/b/bd/IGEM2015_HKUSTRice_Characterization_and_Investigation_of_BBa_I0500.pdf">PDF version</a> of this investigation.</h2>
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<p><i>P<sub>araBAD</sub></i> is a widely used promoter, its BioBrick, BBa_I0500, reported 392 uses to date (as of 4th Sep 2015). Yet different behaviors of <i>P<sub>araBAD</sub></i> have been reported. Cambridge 2011 iGEM team reported an All-or-None behavior whereas Groningen 2011 iGEM team reported a graded response.  
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We sincerely apologize for the misleading information that we provided last year. To remedy for that, we decided to re-characterize BBa_I0500 in a more rigorous manner, using the construct BBa_I2031. To do so within limited time, we headed for a deeper characterization instead of measuring multiple properties (e.g. induction time).
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<br><br>
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Our results demonstrated that this promoter 1) has a graded induction response, 2) has different sensing ranges on plasmids with different copy numbers, and 3) does not display all-or-none behavior on a single cell level when expressed from a low copy plasmid. We have uploaded our results to the Experience Page of BBa_I0500.
 
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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>
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<div class="project_row">
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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>Graded behavior and Plasmid copy number influenced sensing ranges</h1>
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</p>
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<div class="project_image">
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<img style="width:80%" src="https://static.igem.org/mediawiki/parts/d/d9/HKUST_RICE2015_1K3%263K3-I2031TransferCurves.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>Figure 1. Transfer curves of BBa_I2031 on pSB3K3 and pSB1K3</b></strong> The dashed lines represent auto fluorescence of cells, measured using DH10B cells harboring pSB3K3-BBa_E0240 or pSB1K3-BBa_E0240. Error bars represent SEM of 3 independent experiments on 3 different days. The molar concentration to percentage conversion was provided for comparison with previous data.</div>
 
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<p>BBa_I0500 has a graded input-output response when put on a low copy or high copy plasmid. On low copy plasmid pSB3K3, BBa_I0500 is responsive to 10<sup>-4</sup> - 10<sup>-2</sup> mM arabinose, whereas on high copy plasmid pSB1K3, it senses arabinose from roughly 10<sup>-3</sup> to 1mM.
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<h1>Graded Behavior Across a Gradient of Arabinose</h1>
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<br><br>
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<img style="width:80%" 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">
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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 BBaI0500 is less leaky when placed on a high copy plasmid.
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<div class= "des"><strong><b>Figure 1. Comparison of our result to those by Cambridge 2011 and Groningen 2011.</b></strong></div>
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<br><br>
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<p>
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Cambridge 2011 used BBa_I0500 on pSB3K3 to drive their ReflectinA1-sfGFP and reported a threshold of all-or-none between 0.001mM and 0.01mM of arabinose. Groningen 2011 measured BBa_I0500 on pSB1C3 and reported an input sensing range from 0.05% to 1% of arabinose. Our result did not agree with either of theirs.</p>
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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>
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<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>
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<p>
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<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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<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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<div class="project_row">
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<h1>Plasmid Copy Number and Range of Sensitivity</h1>
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<img style="width:80%" 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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<p>On pSB3K3, <i>P<sub>araBAD</sub></i> 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>
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<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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<div class="project_row">
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<h1>All-or-none Behavior NOT on a Single Cell Level</h1>
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<img style="width:80%" src="https://static.igem.org/mediawiki/2015/d/d5/HKUST_Rice_15Literature_Figure_of_all-or-none_behavior.png" alt="Comparison of our result to those by Cambridge 2011 and Groningen 2011">
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<div class= "des"><strong><b>Figure 4 from literature showing all-or-none behavior of <i>P<sub>araBAD</sub></i> 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>
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The all or none behavior of <i>P<sub>araBAD</sub></i> 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 <i>P<sub>araBAD</sub></i>. (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>
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<p>Going back to the literature, we confirmed that all of them were studying <i>P<sub>araBAD</sub></i> on plasmids with high copy origins of replication (Table 1). Thus, we believe that the homogenous expression of <i>P<sub>araBAD</sub></i> promoter from a low copy plasmid has been a long overlooked issue.</p>
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<table class="catalog_table">
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<tr><td>Reference</td><td>Plasmid harboring <i>P<sub>araBAD</sub></i>-reporter</td><td>Plasmid lineage</td><td>Traced origin of replication</td></tr>
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<tr><td>Khlebnikov et al., 2000<br>
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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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<div class= "des">* 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. </div>
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<img style="width:80%" 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>Figure 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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<hr class="para">
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<div class="project_row">
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<h1>Misuse of BBa_I0500 and Part Documentation Issue</h1>
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<p>BBa_I0500 has been a popular part with 329 uses to date (as of 4th Sep 2015), and has functioned as expected in many cases. Unfortunately, we have had unpleasant experiences with BBa_I0500 because we were unaware of one of its properties, which was not documented on its Registry Page.</p>
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<p>In the year 2014, our iGEM team was trying to reproduce previous riboregulators results from different iGEM teams, as well as the very first versions of riboregulators, crR12 and taR12 (Isaacs et al., 2004). Isaacs et al. has used a non-BioBricked version of <i>P<sub>araBAD</sub></i> to control expression of taR12. To follow the setup as close as possible, we employed BBa_I0500 to drive expression of key1 (<a  href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008</a>) in presence of constitutively expressed lock1-RBS-GFP (<a  href="http://parts.igem.org/Part:BBa_K1379020">BBa_K1379020</a>). However, <a  href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterization">our results</a> did not show a significant upregulation of gene expression upon induction with arabinose.</p>
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<p>A year later, now in 2015, we tried to troubleshoot and we identified the design of BBa_I0500 as a possible cause of failure. Issacs et al. demonstrated in their paper that a precise 5’ end for the taRNA is indispensable and additional nucleotides can jeopardize its ability to open up the crRNA hairpin structure (Isaacs et al., 2004). BBa_I0500 contains 19 additional nucleotides after its native transcription start site, and thus would add them to the 5’ end of taRNA and should render it functionless. </p>
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<p>In addition, the extra nucleotides also invalidated our attempt to characterize the Relative Promoter Unit (RPU) for BBa_I0500, because one of the prerequisites for promoter-BBa_E0240 to be made comparable to the standard promoter BBa_I20260 is that they drive transcription of mRNA with identical sequences (Kelly et al., 2009). With the extra nucleotides, BBa_I0500 is not a valid promoter for standard promoter measurement.</p>
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<p>The lack of sequence feature information might also have impacted the design of BBa_I0500’s derivative <a href="http://parts.igem.org/Part:BBa_I13453">BBa_I13453</a>, which is a truncated form of BBa_I0500. It was created with the goal of decoupling the <i>P<sub>araBAD</sub></i> promoter from the araC operon. In its <a href= "http://parts.igem.org/Part:BBa_I13453:Experience">experience page</a>, iGEM 2012 Michigan analyzed the sequence and reported that the O2 binding site is missing. Theoretically, AraC mediated repression in absence of arabinose should not happen. Interestingly though, when put into MG1655, where an endogenous copy of AraC is present, the promoter appears to be inducible (British_Columbia 2009, K.U.Leuven 2011, SDU-Denmark 2012). However, if the information of the O2 binding site was available earlier, we believe the part could have been more carefully designed to fully utilize the regulation by AraC.</p>
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<p>In summary, our experience of failure is due to the incognizance of sequence features in BBa_I0500, leading to its misuse, and we believe this can be improved with proper annotation. Yet due to access authority settings, we could not add back the sequence features to BBa_I0500. In view of this, we strongly recommend future users to cross check the sequence features of BBa_I0500 with that of BBa_K1067007, which has most, if not all, of the important sequence features annotated.</p>
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<hr class="para">
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<h1>Materials and Methods</h1>
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<h2>Strain and Medium</h2>
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<p>All measurements were done in E. coli DH10B cells growing in M9 medium supplemented with 0.2% casamino acids, 4% glycerol and 50µg/mL kanamycin (hereto referred as M9s, please refer to our protocol page in wiki for the recipe) with various concentrations of L-(+)-Arabinose (Sigma #A3256). Graded concentrations of M9s medium with arabinose were prepared by serial dilution from 16mM M9s+arabinose in a 2-fold manner.</p>
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<h2>Measurement</h2>
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<p>For each independent experiment, a single colony of each strain was picked and inoculated in M9s until a turbid culture was obtained. 50 µL starter cultures were then inoculated in 500 µL of different concentrations of fresh M9s+arabinose for an overnight period at 37˚C with 200rpm shaking, in a 96 well deep plate (Sigma #CLS3960) sealed with a breathable tape (Sigma #CLS3345). This step is to minimize the lag time by arabinose induction so that cells do not need to re-adjust to new arabinose concentrations, and hopefully that they would be attaining induction equilibrium when reaching log phase. The next day, the culture was diluted 20-fold into a new plate with fresh M9s+arabinose medium, each into their corresponding and identical arabinose concentration. The new plate was then sealed and inoculated in an identical manner as the overnight culture. OD600 was monitored by removing a 200 µL aliquot from the culture and then reading in a plate reader. When most culture in the wells reached OD600 from 0.25 to 0.5, 300 µL of cells would then be fixed by diluting into 300 µL of 1% paraformaldehyde in 1X PBS.</p>
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<p>We used the BD FACSAria IIIu Cell Sorter for all measurements, with the following settings: FSC 300V, SSC 300V, FITC 500V. Samples were run at 200-400 events per second on a flow rate of 10 and 10000 events were collected for each sample. Noise was distinguished from background fluorescence using DH10B / pSB3K3-BBa_E0240 and was gated away. The remaining population geometric mean fluorescence values and the histograms were then exported for analysis.</p>
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</div>
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<hr class="para">
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<div class="project_row">
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<h1>Reference</h1>
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<p>Cronan, J.E. (2006). A family of arabinose-inducible Escherichia coli expression vectors having pBR322 copy control. Plasmid 55, 152-157.</p>
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<p>Fritz, G., Megerle, J.A., Westermayer, S.A., Brick, D., Heermann, R., Jung, K., Rädler, J.O., and Gerland, U. (2014). Single Cell Kinetics of Phenotypic Switching in the Arabinose Utilization System of <i>E. coli</i>. PLoS ONE 9, e89532.</p>
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<p>Guzman, L.M., Belin, D., Carson, M.J., and Beckwith, J. (1995). Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. Journal of Bacteriology <i>177</i>, 4121-4130.</p>
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<p>Isaacs, F.J., Dwyer, D.J., Ding, C., Pervouchine, D.D., Cantor, C.R., and Collins, J.J. (2004). Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotech 22, 841-847.</p>
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<p>Kelly, J., Rubin, A., Davis, J., Ajo-Franklin, C., Cumbers, J., Czar, M., de Mora, K., Glieberman, A., Monie, D., and Endy, D. (2009). Measuring the activity of BioBrick promoters using an in vivo reference standard. Journal of Biological Engineering 3, 4.</p>
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<p>Khlebnikov, A., Datsenko, K.A., Skaug, T., Wanner, B.L., and Keasling, J.D. (2001). Homogeneous expression of the PBAD promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter. Microbiology <i>147</i>, 3241-3247.</p>
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<p>Khlebnikov, A., Risa, Ø, Skaug, T., Carrier, T.A., and Keasling, J.D. (2000). Regulatable Arabinose-Inducible Gene Expression System with Consistent Control in All Cells of a Culture. Journal of Bacteriology <i>182</i>, 7029-7034.</p>
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<p>Siegele, D.A., and Hu, J.C. (1997). Gene expression from plasmids containing the araBAD promoter at subsaturating inducer concentrations represents mixed populations. Proceedings of the National Academy of Sciences <i>94</i>, 8168-8172.</p>
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<p style = "font-size:110%">*For a more detailed comparison between our results and previous characterizations, please refer to our detailed report.</p>
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{{HKUST-Rice Directory}}
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<div class="project_row">
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<h1>All-or-none behavior on a single cell level</h1>
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<div class="project_image">
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<img style="width:80%" src="https://static.igem.org/mediawiki/parts/9/9e/HKUST_RICE2015_1K3%263K3-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>Figure 2. Histogram plots for sensing ranges of BBa_I0500 on high and low copy plasmid.</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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</div>
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<p>The all or none behavior of <i>P<sub>araBAD</sub></i> 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 <i>P<sub>araBAD</sub></i>. (Khlebnikov et al., 2001)</p>
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<div class= "des">* 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. </div>
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<img style="width:80%" 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" alt="Histogram plots for sensing ranges of BBa_I0500 on high and low copy plasmid">
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<div class= "des"><strong>Figure 5. Histogram plots for sensing ranges of BBa_I0500 on high and low copy plasmid.</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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<hr class="para">
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</div>
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<div class="project_row">
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<h2>For a detailed doucmentation of this characterization and the materials or methods used, please refer to our <a href="https://static.igem.org/mediawiki/2015/b/bd/IGEM2015_HKUSTRice_Characterization_and_Investigation_of_BBa_I0500.pdf">PDF version</a> of this investigation.</h2>
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</div>
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<div class="project_row">
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<h1>Reference</h1>
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<p>Cronan, J.E. (2006). A family of arabinose-inducible Escherichia coli expression vectors having pBR322 copy control. Plasmid 55, 152-157.</p>
 +
<p>Fritz, G., Megerle, J.A., Westermayer, S.A., Brick, D., Heermann, R., Jung, K., Rädler, J.O., and Gerland, U. (2014). Single Cell Kinetics of Phenotypic Switching in the Arabinose Utilization System of <i>E. coli</i>. PLoS ONE 9, e89532.</p>
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<p>Guzman, L.M., Belin, D., Carson, M.J., and Beckwith, J. (1995). Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. Journal of Bacteriology <i>177</i>, 4121-4130.</p>
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<p>Isaacs, F.J., Dwyer, D.J., Ding, C., Pervouchine, D.D., Cantor, C.R., and Collins, J.J. (2004). Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotech 22, 841-847.</p>
 +
<p>Kelly, J., Rubin, A., Davis, J., Ajo-Franklin, C., Cumbers, J., Czar, M., de Mora, K., Glieberman, A., Monie, D., and Endy, D. (2009). Measuring the activity of BioBrick promoters using an in vivo reference standard. Journal of Biological Engineering 3, 4.</p>
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<p>Khlebnikov, A., Datsenko, K.A., Skaug, T., Wanner, B.L., and Keasling, J.D. (2001). Homogeneous expression of the PBAD promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter. Microbiology <i>147</i>, 3241-3247.</p>
 +
<p>Khlebnikov, A., Risa, Ø, Skaug, T., Carrier, T.A., and Keasling, J.D. (2000). Regulatable Arabinose-Inducible Gene Expression System with Consistent Control in All Cells of a Culture. Journal of Bacteriology <i>182</i>, 7029-7034.</p>
 +
<p>Siegele, D.A., and Hu, J.C. (1997). Gene expression from plasmids containing the araBAD promoter at subsaturating inducer concentrations represents mixed populations. Proceedings of the National Academy of Sciences <i>94</i>, 8168-8172.</p>
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</div>
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</body>
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</html>
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{{HKUST-Rice Directory}}

Revision as of 13:49, 18 September 2015

Characterization and Investigation on AraC-ParaBAD Promoter BBa_I0500

Investigation on ParaBAD

The 2014 HKUST iGEM team had previously submitted characterization data of Part BBa_I0500 that described an all-or-none behavior of the promoter. That is however not true. We discovered a few issues in reproducibility and interpretation. Essentially, our result from 2014 did not fully capture the arabinose sensing range. The all-or-none response reported on pSB3K3 was also an artifact, where dead cells from an old plate interfered with fluorescent readings.

We sincerely apologize for the misleading information that we provided last year. To remedy for that, we decided to re-characterize BBa_I0500 in a more rigorous manner, using the construct BBa_I2031. To do so within limited time, we headed for a deeper characterization instead of measuring multiple properties (e.g. induction time).

Our results demonstrated that this promoter 1) has a graded induction response, 2) has different sensing ranges on plasmids with different copy numbers, and 3) does not display all-or-none behavior on a single cell level when expressed from a low copy plasmid. We have uploaded our results to the Experience Page of BBa_I0500.

Graded behavior and Plasmid copy number influenced sensing ranges

Histogram plots for sensing ranges of BBa_I0500 on high and low copy plasmid..
Figure 1. Transfer curves of BBa_I2031 on pSB3K3 and pSB1K3 The dashed lines represent auto fluorescence of cells, measured using DH10B cells harboring pSB3K3-BBa_E0240 or pSB1K3-BBa_E0240. Error bars represent SEM of 3 independent experiments on 3 different days. The molar concentration to percentage conversion was provided for comparison with previous data.

BBa_I0500 has a graded input-output response when put on a low copy or high copy plasmid. On low copy plasmid pSB3K3, BBa_I0500 is responsive to 10-4 - 10-2 mM arabinose, whereas on high copy plasmid pSB1K3, it senses arabinose from roughly 10-3 to 1mM.

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 BBaI0500 is less leaky when placed on a high copy plasmid.

Cambridge 2011 used BBa_I0500 on pSB3K3 to drive their ReflectinA1-sfGFP and reported a threshold of all-or-none between 0.001mM and 0.01mM of arabinose. Groningen 2011 measured BBa_I0500 on pSB1C3 and reported an input sensing range from 0.05% to 1% of arabinose. Our result did not agree with either of theirs.

*For a more detailed comparison between our results and previous characterizations, please refer to our detailed report.

All-or-none behavior on a single cell level

Histogram plots for sensing ranges of BBa_I0500 on high and low copy plasmid.
Figure 2. 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.

The all or none behavior of ParaBAD 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 ParaBAD. (Khlebnikov et al., 2001)

* 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.
Histogram plots for sensing ranges of BBa_I0500 on high and low copy plasmid
Figure 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.

For a detailed doucmentation of this characterization and the materials or methods used, please refer to our PDF version of this investigation.

Reference

Cronan, J.E. (2006). A family of arabinose-inducible Escherichia coli expression vectors having pBR322 copy control. Plasmid 55, 152-157.

Fritz, G., Megerle, J.A., Westermayer, S.A., Brick, D., Heermann, R., Jung, K., Rädler, J.O., and Gerland, U. (2014). Single Cell Kinetics of Phenotypic Switching in the Arabinose Utilization System of E. coli. PLoS ONE 9, e89532.

Guzman, L.M., Belin, D., Carson, M.J., and Beckwith, J. (1995). Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. Journal of Bacteriology 177, 4121-4130.

Isaacs, F.J., Dwyer, D.J., Ding, C., Pervouchine, D.D., Cantor, C.R., and Collins, J.J. (2004). Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotech 22, 841-847.

Kelly, J., Rubin, A., Davis, J., Ajo-Franklin, C., Cumbers, J., Czar, M., de Mora, K., Glieberman, A., Monie, D., and Endy, D. (2009). Measuring the activity of BioBrick promoters using an in vivo reference standard. Journal of Biological Engineering 3, 4.

Khlebnikov, A., Datsenko, K.A., Skaug, T., Wanner, B.L., and Keasling, J.D. (2001). Homogeneous expression of the PBAD promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter. Microbiology 147, 3241-3247.

Khlebnikov, A., Risa, Ø, Skaug, T., Carrier, T.A., and Keasling, J.D. (2000). Regulatable Arabinose-Inducible Gene Expression System with Consistent Control in All Cells of a Culture. Journal of Bacteriology 182, 7029-7034.

Siegele, D.A., and Hu, J.C. (1997). Gene expression from plasmids containing the araBAD promoter at subsaturating inducer concentrations represents mixed populations. Proceedings of the National Academy of Sciences 94, 8168-8172.