Part:BBa_K1766005
OmpR Regulated RhlI
This part consists of the quorum synthase RhlI under control of an OmpR dependent promoter. The promoter is activated by phosphorylated OmpR protein. RhlI produces N-butyryl-homoserine lactone (BHL), a quorum sensing molecule which enables cell to cell signaling.
This part was created to convert the intracellular signal from the chimeric EnvZ-Affibody receptors, created by iGEM Stockholm 2015, into an extracellular signal that could be detected by a read-out strain.
Biology
The OmpR dependent promoter comes from Escherichia coli and has three bind sites for phosphorylated OmpR. Endogenously it is involved in osmoregulation, as it regulates expression of outer membrane porin C (OmpC) [1].
The OmpR protein is phosphorylated or dephosphorylated by the osmoreceptor EnvZ, which exhibits both kinase and phosphatase activity. At low osmolarity OmpR is dephosphorylated and expression of OmpC is low. At high osmolarity OmpR is phosphorylated by EnvZ, which increases OmpC expression [1].
In this construct OmpC has been replaced by RhlI. RhlI is a quorum synthase from Pseudomonas aeruginosa. Endogenous RhlI is involved in biofilm formation, motility and virulence [2]. However, quorum sensing can be used to induce and coordinate diverse behaviors in neighbouring cells [3].
Experiments
To show that expression of OmpR-Regulated-RhlI is controlled by OmpR we performed an osmolarity bioassay. We used Chromobacterium violaceum in soft agar as a BHL reporter. In the presence of BHL the C. violaceum lab strain CV026 produces a violet pigment called violecin.
E. coli transformed with OmpR-Regulated-RhlI was cultured overnight in high and low osmolarity media. The cultures or supernatants were then applied to separate wells on the bioassay plates.
Purple pigmentation could be seen around all wells except the negative control. There was visibly less pigmentation around the positive control compared to the OmpR-Regulated-RhlI samples. The diameter of the purple pigmentation around the high and the low osmolarity samples was measured and compared. The high osmolarity samples, on average, had a 23% bigger radius than the low osmolarity samples.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 787
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
1. 2. 3.
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