Team:HKUST-Rice/Expression/ParaBAD
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. 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
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, ParaBAD 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
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)
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 ParaBAD on plasmids with high copy origins of replication (Table 1). Thus, we believe that the homogenous expression of ParaBAD promoter from a low copy plasmid has been a long overlooked issue.
| Reference | Plasmid harboring ParaBAD-reporter | Plasmid lineage | Traced origin of replication |
| Khlebnikov et al., 2000 Khlebnikov et al., 2001 | pCSAK50 | pBAD24 → pTC40 → pCSAK50 | High copy pBR322 origin * |
| Siegele and Hu, 1997 | pDS439 | pBAD18 → pDS439 | High copy pBR322 origin * |
| Fritz et al., 2014 | pBAD24-GFP | pBAD24 → pBAD24-GFP | High copy pBR322 origin * |
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).
Misuse of BBa_I0500 and part documentation issue
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.
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 ParaBAD to control expression of taR12. To follow the setup as close as possible, we employed BBa_I0500 to drive expression of key1 (BBa_J01008) in presence of constitutively expressed lock1-RBS-GFP (BBa_K1379020). However, our results did not show a significant upregulation of gene expression upon induction with arabinose.
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.
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.
The lack of sequence feature information might also have impacted the design of BBa_I0500’s derivative BBa_I13453, which is a truncated form of BBa_I0500. It was created with the goal of decoupling the ParaBAD promoter from the araC operon. In its experience page, 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.
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.
Materials and methods
Strain and Medium
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.
Measurement
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.
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.
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.