Difference between revisions of "Part:BBa K1766009"

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BAR construct 1

The bacterial antigen receptor (BAR) was created in the hope of making a receptor able to signal binding to an antigen. By making a chimera between a histidine kinase (EnvZ) and a affibody molecule we would have a receptor capable of detecting protein biomarkers. The construct would then use the use the EnvZ-OmpR two-component regulatory system to signal biomarker binding and activation of the BAR.

Structure

The osmoregulator EnvZ was chosen a the scaffold for the BAR as it is a well characherized protein that has been used in other many iGEM projects. One particularly interesting aspect of EnvZ is that functional chimeras between it and other histidine kinases have been made. Specifically protein containing a HAMP domain responsible for signal progression seem to be suitalble for fusion proteins which is refered to as a “control cable”[1]. For example, Cph8 (BBa_K1017301 and BBa_I15010), a Cph1-EnvZ chimera is already available in the registry. Also in a reasent article [2] the activation and inactivation of EnvZ by moving aromatic residues was studied and later employed on a Tar-EnvZ chimera [3].

One troubling aspect on EnvZ is that the periplasmic domain has not been properly characherized or the structure determined. Structure prediction software recoognized what seems to be a PAS domain that could be involved in the signaling of EnvZ. In this PAS domain we found regions that seemed suitable for inserting a biomarker binding protein.

For binding the portein biomarker we choosed the small and stable affibody molecule. Affibody molecules consists of three helixes containging changeable residues for creating affinity to a certain protein. This has been used to create affibodies towards manny different proteins such as the HER2 (human epidermal growth factor receptor) protein. This variablilty and the stability of the structure would mean that the affibody could be switched to detect other proteins as well.

Construct 1 was created by replacing part of the PAS domain with the affibody Z:HER2:342. This resulted in 62 residues in EnvZ were exchanged for 52 residues in the affibody. The PAS Beta sheet/Coil I region was kept as this was thought to effect dimerization between constructs.

Vision

BAR construct 3 was created to respond to presence of human epidermal growth factor receptor (HER2). This would lead to activation of the responce regulator OmpR inherent in E.coli. In practice this has not yet been proven.

Sequence and Features

Experiments

Expression

Method: To check protein expression BAR1_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). 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: The construct can be observed in figure 1 as a homodimer with an approximate size of 100kDa (black triangle) and monomer with an approximate size of 50kDa (black circle). Further analysis, using an anti-Affibody antibody, confirmed that our construct strains are expressing the desired BAR constructs (black star) (Figure 1B). The faint bands at around 25kDa (black square) are presumably caused by some unspecific binding. The loading control DnaK shows in all WB lanes proving that all wells have been loaded equally with whole cell lysates of the different strains.

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 BAR 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 and EnvZ (K1766008) as a positve 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 BAR1 has a highly significant increase in CFT fluorescence. This proves that our construc is still sensitive to changes in osmolarity. YFP values obtained during this experiment were off and inconclusive.

References

[1] Parkinson, J. S. (2010) Signaling mechanisms of HAMP domains in chemoreceptors and sensor kinases. Annu. Rev. Microbiol. 64, 101− 122
[2] 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
[3] Yusuf R. and Draheim R. R. (2015) Employing aromatic tuning to modulate output from two-component signaling circuits. Journal of Biological Engineering, 9:7