Difference between revisions of "Part:BBa K1766008"

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<p><&nbsp><&nbsp>Figure 1:Western blot on EnvZ wildtype and BAR constructs. pEB5 used as negative control. </p>
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<p>Figure 1:Western blot on EnvZ wildtype and BAR constructs. pEB5 used as negative control. </p>
 
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<p><b>Result:</b> 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.</p>
 
<p><b>Result:</b> 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.</p>

Revision as of 10:37, 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


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 316
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 316
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 316
    Illegal BglII site found at 236
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 316
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 316
  • 1000
    COMPATIBLE WITH RFC[1000]

Experiments

Expression

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.

STHLM recog results hyp1 fig1.PNG

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.



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