Difference between revisions of "Part:BBa K1766008"

Revision as of 13:32, 18 September 2015

EnvZ_V5 osmoregulatory histidine kinase from E.coli.

EnvZ is part of the EnvZ-OmpR two-component regulatory system for controlling osmotic pressure in E.coli. Structure includes the EnvZ247 allele [1] which is linked through a GGSSAAG linker to a V5 epitope tag. The full structure has a predicted size of 115.36 kDa [2]. The part was provided in the pRD400 (or pEnvZ) backbone under a lac-inducible promoter [3].

Structure

Two-component regulatory systems are a common in procaryotes for signal transduction through the membrane. In this case, EnvZ is activated by changes in osmotic pressure leading to phosphorylation of the kinase. The response regulator OmpR is then activated through phosphorylation resulting in the regulation of expression of the outer membrane porin proteins OmpF and OmpC.

Usage

EnvZ was used primarily as a scaffold for creating the bacterial antigen receptors: BAR1 (BBa_K1766009), BAR2 (BBa_K1766010) and BAR3 (BBa_K1766011). Besides this, EnvZ was also used as a control for many of the experiments with the BAR constructs.

Sequence and Features

Experiments

Is our construct expressed?

Method: To check protein expression EnvZ_V5 was cloned into a pRD400 backbone and transformed in E.coli (TOP10). Western blot was performed on lysates. Antibodies targeted the V5 tag (anti-V5) and affibody molecules (polyclonal anti-affibody, see parts K1766009, K1766010 and K1766011). The membrane were also stripped and incubated with anti-DnaK to compare expression.

Figure 1:Western blot on EnvZ wildtype and BAR constructs. pEB5 used as negative control.

Result: From the picture above the expression for IPTG induced and non-induced EnvZ in Figure 1A. It is clear that expression is increased upon induction and DnaK is seen with comparable intensities. The upper band in (A, black triangle) for induced EnvZ probably correlated to the protein homodimer at ~100 kDa while the lower is the single protein ~50 kDa. EnvZ is not seen in Figure 1B as the structure does not include the affibody molecule.

Is our construct responsive to changes in osmolarity?

EnvZ is sensible to osmolarity. In our three EnvZ-Affibody chimeras (BAR1 (K1766009), BAR2(K1766010) and BAR3(K1766011)) a part of the periplasmic region has been replaced by the affibody. Therefore it was expected that our constructs would not be reacting to changes in osmolarity. EnvZ was therefor used as a control for the constructs.
Method: For this experiment EnvZ were in the pRD400 backbone and transformed into E. coli EPB30 (strain with genomic OmpR reuglated CFP expression + constituive YFP expression). pEB5 which is an empty plasmid in the same pRD400 backbone was used as a negative control. The samples were induced in log phase with 25 µM IPTG and cultured overnight in high and low osmolarity media (0% versus 15% sucrose). Tests were performed with triplicates.

Figure 2:CFP fluorescence of the different samples in low osmolarity medium (0% sucrose) and high osmolarity medium (15% sucrose). ‘*’ signifies significant P value. ns : P > 0.05 (not significant), ‘*’ : P ≤ 0.05, ‘**’ : P ≤ 0.01, ‘***’ : P ≤ 0.001.
Results:In figure 2, the graph shows that induced EnvZ (pRD400 positice control) has a significant increase in CFT as was exprected. Non-induced EnvZ does not produce a significant increase of CFP. YFP values obtained during this experiment were off and inconclusive.

References

[1] Russo, F. D. (1992) Ph.D. thesis. Princeton University, Princeton, N.J.
[2] Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins M.R., Appel R.D., Bairoch A. (2005) Protein Identification and Analysis Tools on the ExPASy Server; (In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press
[3] Nørholm M. H. H., von Heijne G., and Draheim R. R. (2014) Forcing the Issue: Aromatic Tuning Facilitates Stimulus-Independent Modulation of a Two-Component Signaling Circuit. ACS Synth. Biol. 2015, 4, 474−481