Coding

Part:BBa_K4307025

Designed by: Chi Zhang   Group: iGEM22_Tsinghua   (2022-09-30)
Revision as of 16:51, 13 October 2022 by Oldluckyzhang (Talk | contribs)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)


AffiPmrB

This part is modified from PmrB receptor in PmrB/A two-component system in E.coli. PmrB uses an iron (III)-binding motif to sense the environment(Chen & Groisman, 2013). The extracellular iron (III)-binding motif of pmrB receptor is replaced with another protein named affibody to construct this part. Affibody is a protein that can recognize Fc fragment of antibody(Stahl et al., 2017). This part is used to recognize the extracellular antibody signal and start the downstream signal transduction system.
In Tsinghua 2022 project, it was used in affipmrABC(BBa_K4307042) system.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 855
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

The following figure demonstrates our successful construction.


Figure 1: The construction results of AffiPmrB.

In order to determine the feasibility of our modification, we first used AlphaFold2 software to predict the structure of the receptor Affi-PmrB that we constructed. Compared with the original receptor PmrB (uniport ID: P36557 · BASS_SALTY), the predicted structure of Affi-PmrB according to our modified sequences are highly similar. Almost identical structure is displayed in the recognition area of the receptor and ligand, confirming the feasibility of our modification.


Figure 2: (A)The predicted structure of Affi-PmrB by AlphaFold2. (B) The structure of PmrB.

Molecular docking was simulated between the modified receptor Affi-PmrB and the Fc peptide of IgG antibody to further determine the feasibility of our modification scheme. We downloaded the Fc peptide structure (PDB DOI: 10.2210/pdb5VGP/pdb) from the PDB database, and then used the online protein molecular docking software Vasklab. Simulation results demonstrated that the Fc peptide could still bind to the ligand recognition region of our modified receptor with hydrophobic bonds, indicating that our modification did not affect the binding of Fc peptide with affibody.


Figure 3: Molecular docking simulation results. (A) The predicted structure of Affi-PmrB. (B) The Fc region of IgG. (C) The simulation of molecular docking between Fc region and Affi-PmrB predicted by Vasklab.

In summary, the strutural prediction and molecular docking simulation results have verified the feasibility of our modification design, confirming that the modified receptor Affi-Pmr still possesses the correct structure capable of binding and interactering with the Fc region of IgG antibody. Therefore, we have demonstrated that theoretically our modified receptor Affi-Pmr can serve as a protein signal detector.

We then transformed plasmid pAffi-PmrB/A into E. coli for verification. IPTG was used to induce the expression of Affi-PmrB receptor and PmrA protein, and human IgG antibody was added to the experimental group. The fluorescence of EGFP was observed under fluorescence microscope. Our results demonstrated that the fluorescence signal was much stronger with antibody induction, indicating that our Affi-PmrB/A system can efficiently detect and respond to target protein signals.


Figure 4: Fluorescence microscopy of Affi-PmrBA system. Plasmid pAffi-PmrB/A was induced with IPTG (final concentration 0.24 mg/mL). The experimental group (B and D) was then induced with 100 mM human IgG antibody, while the control group (A and C) did not add any antibody. Photos were taken under confocal fluorescence microscope (60×).

References

[1] Chen, H. D., & Groisman, E. A. (2013). The biology of the PmrA/PmrB two-component system: the major regulator of lipopolysaccharide modifications. Annu Rev Microbiol, 67, 83-112.

[2] Stahl, S., Graslund, T., Eriksson Karlstrom, A., Frejd, F. Y., Nygren, P. A., & Lofblom, J. (2017). Affibody Molecules in Biotechnological and Medical Applications. Trends Biotechnol, 35(8), 691-712.


[edit]
Categories
Parameters
None