Difference between revisions of "Team:HKUST-Rice/Expression/ParaBAD"
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| − | <h1>Characterization and Investigation on <i>P<sub>araBAD</sub></i> BBa_I0500</h1> | + | <h1>Characterization and Investigation on AraC-<i>P<sub>araBAD</sub></i> BBa_I0500</h1> |
<p>The 2014 HKUST iGEM team had previously submitted <a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterization">characterization data</a> for part <a href="http://parts.igem.org/Part:BBa_I0500"> BBa_I0500</a> 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. | <p>The 2014 HKUST iGEM team had previously submitted <a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterization">characterization data</a> for part <a href="http://parts.igem.org/Part:BBa_I0500"> BBa_I0500</a> 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>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> | <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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Latest revision as of 14:32, 18 September 2015
AraC-ParaBAD Promoter BBa_I0500
Characterization and Investigation on AraC-ParaBAD BBa_I0500
The 2014 HKUST iGEM team had previously submitted characterization data for 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 Pages of BBa_I0500 and that of BBa_I2031.
Graded behavior and plasmid copy number influenced sensing ranges
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
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 refers to the phenomenon 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.
In concordance with literatures, we observed an all-or-none induction when BBa_I0500 is placed on a high copy plasmid, where induced cells were mostly distributed among two bins of fluorescence. Yet on low copy plasmid, the induced populations remain homogenous along the arabinose concentration gradient. Since all literatures before have been using high copy plasmids for the study of ParaBAD, we believe the homogenous expression of ParaBAD promoter from a low copy plasmid has been a long overlooked issue.
Our misuse of BBa_I0500 and its part documentation issue
Lastly, we would like to warn potential users of this part not to drive expression of riboregulators (Isaacs et al., 2004) using BBa_I0500. This ParaBAD promoter promoter contains a 19bp sequence after the transcription start site, which would produce a taRNA with additional nucleotides at its 5’ end, and rendering it incapable to activate crRNA. Relative Promoter Unit measurement (Kelly et al., 2009) for this BioBrick is also inappropriate because the mRNA produced from BBa_I2031 would contain these extra 19nt when compared to that from BBa_I20260. To avoid misuse of this part due to incognizance of its sequence information, we recommend users to look up BBa_K1067007 for annotations.
For a detailed doucmentation of this characterization and the materials or methods used, please refer to our PDF version of this investigation.
Reference
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.
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.