Part:BBa_K1766011

BAR construct 3

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 suitable for fusion proteins which is refereed 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 resent 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 recognized 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 protein biomarker we choose the small and stable affibody molecule. Affibody molecules consists of three helixes containing changeable residues for creating affinity to a certain protein. This has been used to create affibodies towards many different proteins such as the HER2 (human epidermal growth factor receptor) protein. This variability and the stability of the structure would mean that the affibody could be switched to detect other proteins as well.

Construct 3 was created by replacing smaller part of the PAS domain with the affibody Z:HER2:342. This resulted in 27 residues in EnvZ were exchanged for 58 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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 402
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 236
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Experiments

Expression

Method: To check protein expression BAR3_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.

Can BAR bind to SK-BR-3, a HER-2 expressing breast cancer cell line?

BARs are situated in the inner membrane as they derive from the inner membrane standing osmoregulator EnvZ. However, the integration of an Affibody molecule in the periplasmic region should allow it that the BAR expressing bacteria can bind to HER-2 expressing cancer cells when the bacterial outer membrane and cell wall has been removed. We were really gratefull that our collaborator, the iGEM Team ETH Zurich, took up on this task. The findings from these experiment represent an important checkpoint towards a functional system.

Method: They expressed all three EnvZ-Affibody chimeras (BAR1 (K1766009), BAR2(K1766010) and BAR3(K1766011)) and incubated them with a HER-2 expressing cell line (SK-BR-3). BAR expression was intiated in all three strains using ITPG induction. In order to efficiently visualize our bacteria at the cell surface, they wanted to introduce GFP into our bacteria strains which unfortuntely failed. Therefore, they continued with our unstained bacteria.

Figure 2:Testing our three BAR expressing constructs (BAR1 (BBa_K17009), BAR2 (BBa_K1766010) and BAR3 (BBa_K1766011)) for their binding capacity towards HER-2 expressing breast cancer cells. BAR expression was initiated by ITPG induction.

Results: The results represented in figure 3 show no clear interaction between the SK-BR-3 cells and our BAR-expressing bacteria. This might be due to too low contrast between bacteria and background. Therefore, we cannot conclude whether our bacteria is capable of binding membrane-bound HER-2. However, we see a stronger retainment of bacteria in all ITPG-induced cells which might hint towards a certain interaction of HER-2 and our chimeric protein. To sum it all up, we could not show an interaction between HER-2 expressing cancer cells and BAR-expressing bacteria. Other techniques will be needed to investigate on this question in order to make a final conclusion.

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