Difference between revisions of "Part:BBa K1766005"

Revision as of 20:46, 18 September 2015

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In this construct the ompC gene has been replaced by RhlI.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 violacein.

E. coli transformed with OmpR-Regulated-RhlI was cultured overnight in high (15% sucrose) and low (0% sucrose) osmolarity media. The cultures were then applied to separate wells on the bioassay plates, together with a negative and positive control.

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 (see Figure 1).

alt text

Figure 1. A: Bacterial cultures applied to plates containing C. violaceum in soft agar. B: Violacein induction in C. violaceum. Comparison between E. coli transformed with OmpR-Regulated-RhlI, cultured at high and low osmolarity. ‘*’ signifies significant P value. ns : P > 0.05 (not significant), ‘*’ : P ≤ 0.05, ‘**’ : P ≤ 0.01, ‘***’ : P ≤ 0.001

Statistical analysis was performed by Student's t-test on the average of the biological replicates. The total number of biological and technical replicates were three and four, respectively. The obtained P value was 0.00053 which is statistically significant.

These results show that OmpR-Regulated-RhlI expresses functional RhlI and synthesizes BHL in an OmpR dependent manner.

For more details about this part, please visit the [http://2015.igem.org/Team:Stockholm/Results 2015 iGEM Stockholm wiki page]

Sequence and Features

References

1. Cai SJ, & Inouye M. (2002). EnvZ-OmpR interaction and osmoregulation in Escherichia coli. Journal of Biological Chemistry, 277(27), 24155-24161. doi: 10.1074/jbc.M110715200

2. Rutherford ST, Bassler BL. (2012). Bacterial quorum sensing: its role in virulence and possibilities for its control. Cold Spring Harbor Perspect. Med. 2, 1–26. doi: 10.1101/cshperspect.a012427

3. Davis RM, Muller RY and Haynes KA. (2015). Can the natural diversity of quorum-sensing advance synthetic biology? Front. Bioeng. Biotechnol. 3:30. doi:10.3389/fbioe.2015.00030