Difference between revisions of "Part:BBa K4601223"

Latest revision as of 13:35, 12 October 2023

sfGFP-LVAtag expression cassette under the control of the pOmpR promoter

This part is an sfGFP-LVAtag (BBa_K2675006) expression cassette under the control of the pOmpC promoter BBa_R0083 regulated by the OmpR transcription factor.

Usage and Biology

The pOmpC promoter and its regulation

Osmoregulation in E. coli is an essential bacterial process that enables them to adjust to variations in their external environment and uphold the osmotic balance within their cells.

In this process, the EnvZ-OmpR two component system [1] is involved in sensing the variations in the osmolarity of the external medium and the consequent regulation of the expression of OmpF and OmpC which are two outer membrane pores.

EnvZ is a multidomain homodimeric protein composed of domains localized in the periplasmic space, in the inner membrane and in the cytoplasm. Notably, its cytoplasmic domain has a histidine kinase activity capable of autophosphorylation in trans with the EnvZ dimer and subsequent phosphorylation of the OmpR transcription factor. The phosphorylated OmpR dimerises and binds to specific sequences present in the promoter regions of the ompC and ompF genes thus regulating their expression [2].

Activation of this system can occur in response to changes in the surrounding medium and has been observed in connection with alterations in sugars or sodium chloride concentrations [3,4]. Additionally, the system plays crucial roles in the acid stress response in E. coli [5,6]. In this case, OmpR operates in an EnvZ-dependent manner, independent of phosphorylation, to induce cytoplasmic acidification. Notably, E. coli is capable of maintaining an acidic cytoplasm in response to both acidic and osmotic stressor [7].

Under conditions of low osmolality and a neutral pH, OmpF stands as the primary porin within the outer membrane. However, when osmolality is high, the transcription of OmpF is downregulated and that of OmpC is upregulated by the OmpR transcription factor.

This system had been widely adopted by the synthetic biology community, with a number of projects using the EnvZ histidine kinase domain fused to the protein of interest. Its activity was coupled to the expression of a reporter gene under the control of an OmpC promoter.

Analysis of pOmpC promoter in the registry

In the designing phase of our project, we realized that several versions of the OmpC promoter exist already in Parts Registry. Why? What are the differences between them? Which one to choose?

A sequence comparison (Figure 1) revealed that BBa_R0083 is the shortest (78 nucleotides), BBa_R0082, BBa_K3630015 and BBa_K199017 are around 110 ± ~10 nucleotides, while BBa_K4244006 and BBa_K199018 are longer (~180 nucleotides).

When compared to the E. coli MG1655 genome (GenBanc Acc n°U00096 / NC_000913), the longer parts cover the promoter region of the ompC gene up to the ATG, thus also including the RBS sequence. All others stop at nucleotide -81 which was shown to be the last not transcribed nucleotide (transcription initiation start +1 being at nucleotide -80 compared to the ATG codon of the ompC gene) [8]. Differences between these parts also occur in the 5’ region, but they should have no impact on the promoter regulation, as the 3 OmpR binding sites are preserved [8].

The shorter part is BBa_R0083. This truncated version corresponds to the pCD-7135 sequence described by Maeda & Mizuno [8] which only has one OmpR binding site, but that was shown to preserve the OmpR regulation while increasing the promoter strength.

For these reasons we chose BBa_R0083 as a promoter of our parts and performed characterization of its activity using sfGFP as a reporter gene (BBa_K4601223).

Figure 1. Sequence comparisons of pOmpC promoter variants present in Parts Registry. The alignment was generated using the MUSCLE algorithm implemented in SnapGene.

Experimental characterisation of this part, the pOmpC promoter

E. coli cells carrying the sfGFP gene under the control of the pOmpC promoter (BBa_K4601223) were grown overnight at 37 °C at 200 rpm in 96-deep-well plates with 1 mL of LB (Lennox) supplemented with 10 µg/mL tetracycline.

For testing the effect of NaCl concentrations, the cells were then diluted by 40 times in LB NaCl-free media with 10 µg/mL tetracycline and after 4 hours of incubation at 37°C at 200 rpm, they were further diluted by 20 times in LB NaCl-free media containing 10 µg/mL tetracycline and increasing concentrations of NaCl.

For testing the effect of sucrose concentrations, the cells were then diluted by 40 times in LB Lennox media with 10 µg/mL tetracycline and after 4 hours of incubation at 37°C at 200 rpm, they were further diluted by 20 times in the same media containing increasing concentrations of sucrose.

The opaque wall 96-well polystryrene microplate (COSTAR 96, Corning) was then incubated at 37°C at 200 rpm and the sfGFP fluorescence (λ_excitation 488 nm and λ_emission 530 nm) and optical density at 600 nm (OD600) were measured every 10 minutes for 24 hours, in a CLARIOstar (BMGLabtech) plate reader. Fluorescence values were normalized by OD600.

We have performed these characterisations in 6 different E. coli strains as a single E. coli strain is not suitable for all applications.

Our results presented in figures 2 and 3 show similar behavior of the pOmpC promoter in all tested strains, but also differences. We first noticed that the bacterial growth was changed in different media, with NaCl having a less important effect than sucrose. Bacteria were growing faster and reaching a higher OD in the stationary phase in the media containing 200 mM NaCl, which is a value close to the 10 g/L present in LB Luria-Miller media (Figure 2A). Increasing NaCl concentration had the same effect as decreasing it. In the media prepared without NaCl (only 10 g/L tryptone and 5 g/L yeast extract) growth was the slowest. In our conditions we are aware that the NaCl concentration is not zero, as both tryptone and yeast extract bring a certain amount of NaCl.

When bacteria were grown in the presence of increasing sucrose concentrations, the growth was gradually affected (Figure 3A), at the highest sucrose concentration, upon overnight culture, the final OD600 being nearly a third of the culture performed in the absence of sucrose.

sfGFP fluorescence values were measured in parallel (Figure 2B and 3B) and normalized by the OD600 values in order to be able to compare the different conditions.

The results presented in Figure 2C show no significant influence of NaCl concentration on the fluorescent output, One can only notice a much lower intensity in the C43(DE3) strain. Moreover, when the pOmpC promoter activity was tested in the ΔenvZ strains we constructed, the activity was drastically reduced as expected as the EnvZ-OmpR signaling cascade was disrupted (Figure 2D and E). In the presence of increasing sucrose concentrations we first noticed that the fluorescence values were increasing during the exponential growth phase, then they were decreasing upon cells entering the stationary growth phase (Figure 3B). When comparing the Fluorescence / OD600 values at this transition, we observe a positive relationship between the increase of the sucrose concentration and the gain of fluorescence (Figure 3C). Moreover, as in the case of NaCl, when the pOmpC promoter activity was tested in the ΔenvZ strains, the activity was drastically reduced as expected.

Figure 2. In vivo characterization of sfGFP expression under the control of the pOmpC promoter (BBa_K4601223) in E. coli cells in the presence of increasing concentrations of NaCl. Example of growth curves of E. coli NEB^® 5-alpha (A) and the corresponding sfGFP fluorescence values (B). Fluorescence/OD_600nm values obtained in the late exponential growth phase for six E. coli cells (C, D, E). The data and error bars are the mean and standard deviation of at least three measurements on independent biological replicates.

Figure 3. In vivo characterization of sfGFP expression under the control of the pOmpC promoter (BBa_K4601223) in E. coli cells in the presence of increasing concentrations of sucrose. Example of growth curves of E. coli NEB^® 5-alpha (A) and the corresponding sfGFP fluorescence values (B). Fluorescence/OD600nm values obtained in the late exponential growth phase for six E. coli cells (C, D, E). The data and error bars are the mean and standard deviation of at least three measurements on independent biological replicates.

Our contribution was based on the characterization of this part's activity in E. coli cells grown in different conditions. Promoters are used to drive the expression of genes of interest and, by characterizing its behavior, researchers can select the most suitable one for their specific application. This is especially important when engineering synthetic biological systems.

References

[1] Mizuno T, Mizushima S. Signal transduction and gene regulation through the phosphorylation of two regulatory components: the molecular basis for the osmotic regulation of the porin genes. Molecular Microbiology (1990) 4: 1077–1082.

[2] Yoshida T, Qin L, Egger LA, Inouye M. Transcription regulation of ompF and ompC by a single transcription factor, OmpR. The Journal of Biological Chemistry (2006) 281: 17114–17123.

[3] Cai SJ, Inouye M. EnvZ-OmpR Interaction and Osmoregulation in Escherichia coli. Journal of Biological Chemistry (2002) 277: 24155–24161.

[4] Wang LC, Morgan LK, Godakumbura P, Kenney LJ, Anand GS. The inner membrane histidine kinase EnvZ senses osmolality via helix-coil transitions in the cytoplasm. The EMBO Journal (2012) 31: 2648–2659.

[5] Chakraborty S, Winardhi RS, Morgan LK, Yan J, Kenney LJ. Non-canonical activation of OmpR drives acid and osmotic stress responses in single bacterial cells. Nature Communications (2017) 8: 1587.

[6] Chakraborty S, Kenney LJ. A New Role of OmpR in Acid and Osmotic Stress in Salmonella and E. coli. Frontiers in Microbiology (2018) 9: 2656.

[7] Kenney LJ, Anand GS. EnvZ/OmpR two-component signaling: an archetype system that can function non-canonically. EcoSal Plus (2020) 9: 10.1128/ecosalplus.ESP-0001–2019.

[8] Maeda S, Mizuno T. Evidence for multiple OmpR-binding sites in the upstream activation sequence of the ompC promoter in Escherichia coli: a single OmpR-binding site is capable of activating the promoter. Journal of Bacteriology (1990) 172: 501–503.

Sequence and Features