Team:SDU-Denmark/magisk-regnbue-enhjoerning


Determination of Noise Levels in Constitutive Promoter Family Members


Fluorescence microscopy and flow cytometry revealed that a strong constitutive promoter was suitable for the expression of the photocontrol device.
To assess which of the constitutive promoters would be suitable for a uniform expression of the photocontrol device, BBa_K519030, and the antitoxin RelB, BBa_K2449028, the expression levels and the noise of four different members of the Anderson promoter collection and their RFP reporter systems, were studied by fluorescence microscopy. These were, in increasing promoter strength, BBa_J23114, BBa_J23110, BBa_J23106, and BBa_J23102.
Additionally, the change in RFP expression levels and noise during growth were tested for the promoters with the highest and lowest relative promoter strength by flow cytometry and qualitative analysis by fluorescence microscopy. Combining these two techniques, the expression and noise levels for the promoters were determined as follows:

  • The weak promoter, BBa_J23114, exhibited a relatively low expression of RFP, indicating low gene expression and an increasing high level of noise throughout growth.
  • Both medium strength promoters, BBa_J23110 and BBa_J23106, displayed a moderate level of both noise and protein expression of the RFP reporter.
  • The strong promoter, BBa_J23102, exhibited a comparatively high expression of the reporter RFP and an increasing high level of noise throughout growth.

For further information about the experiments and a proposed cause for the increasing noise level, read here.
As the strong constitutive promoter exhibited the most uniform expression, this was chosen to regulate the expression of the photocontrol device genes. With the modelling results in mind, it was decided that the relB gene should be regulated by a tightly controllable uniform promoter, thereby ruling out the constitutive promoter family members as a possibility.


Expression Level of the Mnt- and Penl-Regulated Promoters


The PenI-regulated promoter was chosen to control the expression of the photocontrol device, as it mediated a stronger expression than the Mnt-regulated promoter.
The cloning of the strong constitutive promoter, BBa_J23102, and the photocontrol device, BBa_K519030, emerged problematic. For further information about these difficulties, read here.
Consequently, the applicability of two different promoters was studied. These were the PenI-regulated, BBa_R0074, and the Mnt-regulated promoters, BBa_R0073, whose repressors are not found in E. coli, making the gene expression constitutive in this organism. The relative expression and noise levels were quantitatively assessed using fluorescence microscopy of a YFP reporter system expressed on pSB1C3 in MG1655 at OD600=0.3-0.5. For this purpose an Olympus IX83 with a photometrics prime camera was used with exposure time for YFP at 200 ms.


Figure 16. Fluorescence microscopy of YFP under the control of PenI-regulated and Mnt-regulated promoters on pSB1C3 in E. coli MG1655 at OD600=0.3-0.5.

From the data obtained, it was evident that the PenI-regulated promoter mediated a substantially higher level of YFP expression than the Mnt-regulated promoter, as seen in Figure 16. Furthermore, the PenI-regulated promoter displayed a notably lower level of noise. On the basis of these results, the PenI-regulated promoter was chosen to control the expression of the photocontrol device.


Leaky Expression by the OmpR-Regulated Promoter on Different Vectors


The leaky expression by the OmpR-regulated promoter is reduced when cloned into a low copy vector compared to a high copy vector.
Proper regulation of the OmpR-dependent promoter, BBa_R0082, is necessary for the implementation of a functional dormancy system, as the balance between RelE and RelB is imperative. To verify that the OmpR-regulated promoter is up to the task, a reporter system containing RFP under control of the OmpR-regulated promoter, BBa_M30011, was cloned into E. coli strain SØ928 ΔOmpR, lacking the OmpR transcription factor, on a high copy vector. By using a ΔOmpR strain, the background generated by stimulation of the intrinsic OmpR system is removed, and the strain functions as a negative control.
RFP expression was assessed by fluorescence microscopy using an Olympus IX83 with a photometrics prime camera, with exposure time for RFP at 200 ms. Assessing the RFP expression by fluorescence microscopy, it was discovered that the OmpR-regulated promoter mediated gene expression even in the absence of its transcription factor, see Figure 17. This observation was confirmed by going through the literatureLevskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, Levy M, et al. Synthetic biology: engineering Escherichia coli to see light. Nature. 2005;438(7067):441-2..


Figure 17. Fluorescence microscopy of RFP controlled by the OmpR-regulated promoter on a high copy vector in E. coli strain SØ928 ΔOmpR.

On the basis of this finding, the relE gene controlled by the OmpR-regulated promoter required a low copy plasmid or insertion into the chromosome. Protein expression of RFP in pSB1C3 with a copy number of 100-300 plasmids per cell, and pSB3K3 with a copy number of 10-12 plasmids per cell, was studied by flow cytometry. As for the determination of noise levels in the weak, BBa_J23114, and strong BBa_J23102 constitutive promoters, , the experiment was carried out in both LB medium and M9 minimal medium, the latter supplemented with 0.2% glycerol. In the LB medium, selection was carried out by the addition of 30 µg/mL chloramphenicol, 30 µg/mL kanamycin, or 50 µg/mL ampicillin, depending on the resistance, and for M9 minimal medium, the concentrations used were 60 µg/mL chloramphenicol, 60 µg/mL kanamycin, and 100 µg/mL ampicillin. Excitation of RFP was at 561 nm, and emission was measured around 580 nm. Expression levels in both E. coli MG1655 and E. coli MG1655 ΔOmpR were studied to determine the baseline of the leaky expression not influenced by intrinsic pathways including the OmpR transcription factor.


Figure 18. Flow cytometric fluorescence measurements in arbitrary units as a function of time. Left: Cultures were grown in LB medium. Right: Cultures were grown in M9 minimal medium supplemented with 0.2% glycerol. Fluorescence of RFP expressed by the the OmpR-regulated promoter on the high copy vector, pSB1C3, and the low copy vector, pSB3K3, in MG1655 WT and ΔOmpR MG1655 strain. All fluorescence levels were measured relative to the negative control WT E. coli MG1655, and the weak and strong constitutive promoters are included as references. Standard error of mean is shown, but are in several cases indistinguishable from the graph.

Fluorescence levels in the two different media display similar behavior, as seen in Figure 18. The main difference observed, was that the decrease in fluorescence over time was faster in LB medium than in M9 minimal medium, in concordance with the observations made in previous experiments. On a general level, the data revealed, that MG1655 cloned with the POmpR-RFP reporter system on the high copy vector exhibited a fluorescence level, equivalent to that mediated by the strong constitutive promoter. On the low copy vector, the POmpR-RFP reporter system yielded a fluorescence level comparable to the gene expression mediated by the weak constitutive promoter. On the other hand, expression levels in the MG1655 ΔOmpR strain were markedly reduced compared to MG1655, indicating that pathways including the transcription factor OmpR interfere with RFP expression under these conditions. Again, the fluorescence levels observed for the POmpR-RFP reporter system on the low copy vector were distinctly lower than for the high copy vector.
All things considered, the OmpR-regulated promoter was found to exhibit leaky expression comparable to the expression levels mediated by the constitutive promoters. When cloned into a low copy vector, the leaky expression was reduced prominently. Thus, to obtain proper regulation of RelE expression by the OmpR-dependent promoter, a low copy vector is required.


Transposon Hotspot Formation in LacI-Regulated lambda pL Hybrid Promoter Reporter System


During cloning, formation of a transposon hotspot between the LacI-regulated lambda pL hybrid promoter and a reporter system was observed, making this promoter inapt for the dormancy system
To control the gene expression of RelB, the LacI-regulated lambda pL hybrid promoter, BBa_R0011, was chosen. Due to a putative formation of a transposon hotspot between the promoter sequence and the GFP reporter, which is described further here, another inducible promoter was chosen to regulate the expression of RelB.


Induction and Subsequent Inhibition of the pBAD Promoter


Expression by the pBAD promoter can be regulated tightly by induction and subsequent inhibition.
The pBAD promoter holds great potential to regulate the expression of the relB gene in our system, as it is capable of both an induction and repression. The HKUST-Rice iGEM team from 2015 found that the pBAD promoter exhibits an almost all-or-none behaviour upon induction with arabinose when located on a high copy vector, but allows for gradual induction when cloned into a low copy vector. Thus, it was evident that this promoter on a high copy vector would be inappropriate for the regulation of RelB expression. Based on these findings, a low copy vector was used to investigate the ability to inhibit gene expression subsequent to induction of pBAD.
RelB expression was simulated by fluorescence microscopy using a pBAD-YFP reporter system, BBa_I6058. For this purpose, an Olympus IX83 with a photometrics prime camera was used with an exposure time for YFP at 200 ms. Transformed E. coli MG1655 cells were cultured in M9 minimal medium supplemented with 0.2% glycerol and 30 µg/mL chloramphenicol, to avoid catabolite repression from glucose residues present in LB medium. Two cultures were incubated, of which one was induced with 0.2 % arabinose from the beginning. At OD600=0.1, designated time 0, the cultures were split in two and 0.2 % glucose was added to one of each pair. Samples were obtained at time 0, before division of the cultures, and at 30 min, 60 min, and 120 min. The resulting images revealed, that the inducer arabinose was required to stimulate expression of YFP, and that the addition of the repressor glucose to a uninduced culture had no effect. Furthermore, it was evident that addition of arabinose induced expression of YFP, and that subsequent addition of glucose terminated the pBAD regulated gene expression on a low copy vector, resulting in a reduced fluorescence level. 30 minutes after inhibition this reduction was already evident, and after 120 minutes the gene expression controlled by pBAD was even further decreased, as seen in Figure 19.



Figure 19. YFP fluorescence levels in E. coli MG1655 transformed with the pBAD-YFP reporter system on pSB3K3. Left: Cultures with the inducer arabinose added. Right: Cultures not induced with arabinose. Both cultures were split up at OD600=0.1, designated time 0, and the inhibitor glucose was added to one half of each culture. Images were obtained at 0, 30, 60, and 120 minutes.

This experiment made it clear, that gene expression controlled by the pBAD promoter is both inducible and repressible as required when cloned into the low copy vector pSB3K3. Consequently, the pBAD promoter was found to be suitable for controlling gene expression of the antitoxin RelB in the implemented dormancy system.