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

Line 142: Line 142:
 
<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>
 
<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>
 
 
−
 
−
<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>
 
−
 
−
 
−
<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">
 
−
<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>
 
 
<hr class="para">
 
<hr class="para">
 
</div>
 
</div>

Revision as of 13:51, 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)

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.