Difference between revisions of "Part:BBa K4601243"

Latest revision as of 13:43, 12 October 2023

AmpR expression cassette under the control of the pOmpR promoter

This part is an expression cassette of the AmpR, an E. coli gene coding for a β-lactamase enzyme (BBa_K4601041) 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.

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

Sequence and Features