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Improvement of an Existing Part

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Improvement of T7's tail fiber (GP17)

BBa_K3211602


Wildtype T7

 Our project worked with the gp17 protein in the lytic T7 bacteriophage. This part was previously documented by the ETH_Zurich team in 2019, and functions in T7 recognition of lipopolysaccharides on the bacterial cell wall of Escherichiacoli (E. Coli). Gp17 forms homotrimers, six of which are attached at the tail end of T7 [1]. We worked to re-engineer this protein to expand host range and therefore increase the phage's applicability in therapeutics and medicine.

Fig. 1 - WT_Control_T7
Fig. 2 - Truncated_Control

Truncation of T7

 The C-terminus of the wild type gp17 is oriented towards the icosahedral phage capsid. As part of our proof-of-concept experiments, we produced a phage with a truncated tail fiber in order to expose the C-terminus. We hypothesized that this would promote a more efficient attachment of target moieties and functional groups.
 AlphaFold 3D modeling suggests that the truncated gp17 undergoes complex unfolding. Intriguingly, despite these predictions, we have evidence to support that infectivity may still be maintained. This conclusion stems from the more robust plaquing results produced by truncated T7 phage in comparison to the wild type. Results of plaquing assays (Figure 3) show significantly more plaquing with truncated T7 as opposed to regular (non-truncated) T7 and T3.

Fig. 3: plaquing of two T7 variants in comparison to T3 on two bacterial strains.

 From there we proceeded with our experiments and sought to engineer a fusion between the tail fiber and a nanobody. AlphaFold modeling once again predicted unfolding for this construct. Regardless, we chose to synthesize the tail fiber- nanobody fusion for later comparison with the same protein produced by enzymatic catalysis.
 In preparation for post-iGEM activity assays, the peptide sequence, LPETGG, was added to both the wild type and truncated version of T7 bacteriophage tail fiber. Sortase will recognize this sequence and ligate it to the nanobody expressing a polyGlycine peptide sequence on its N-terminus.

Expanding Nanobody Application

BBa_K3410001


Addition of the nanobody to T7 (from pDSG289)

 Nanobodies are miniature antibodies that are naturally found in camels. These nanobodies have two heavy chains but are missing the light chains, but still possess strong antigen binding capabilities [2]. The addition of the nanobody to the T7 bacteriophage allows T7 to infect a wider range of harmful bacteria [3]. We were fortunate to have guidance from previous documentation by Bielefeld-CeBi in 2020. This allows us to further pursue T7 as a viable delivery method for various drugs, peptides, and even CRISPR-Cas based antimicrobials.
 In addition, incorporating the nanobody directly to the T7 tail fiber provides the opportunity for future editing of its complementarity-determining regions (CDRs), three of which (CDR1, CDR2, and CDR3) are found in the nanobody and are important for antigen binding [2]. Modifying these regions can potentially alter what the nanobody targets. With directed editing of the nanobody structure, therefore, it is possible to selectively change which bacterial strains are attacked by the bacteriophage. The nanobody from pDSG289 has been shown to target the antigen from pDSG419 [3], which we hope to take advantage of in our design of modular therapeutics.

Design Considerations

 To achieve incorporation of the nanobody into our T7 system, a g-block was designed in order to add two different functional sequences. The first sequence was the five glycine tag for the Sortase reaction while the second sequence is a histidine purification tag. The histidine tag will be used for purification via Fast Protein Liquid Chromatography (FPLC).

Part Modified Plasmid Name Modification Base Pairs
BBa_K4514048 pGEM3RCF - T7 TailFiber Plasmid backbone No modification
BBa_K4514045 pGEM3RCF-T7 LEPTGG tail fiber truncated Truncated GP17 and added the sortase motif 252 nucleotides removed and 18 added
BBa_K4514046 pGEM3RCF-T7-LPETGG Tail fiber Added the sortase motif 18 nucleotides added
BBa_K4514053 pMGP4185-T3-tail fiber Plasmid No modification
BBa_K4514054 pMGP4185-T3-LPETGG tail fiber Added the sortase motif to T-3 18 nucleotides added
Nanobody
BBa_K4514049 pDSG289-nanobody 2 intimin Plasmid backbone No modification
BBa_K4514044 Nanobody 2 (Nbdy2) in pET29 Nanobody gene was added to sortase backbone. The sortase N-terminus motif was added and a histidine purification tag. 463 nucleotides replacing the sortase gene
T7 nanobody fusion
BBa_K4514050 pGEM3RCF-T7 LPETGG-GGG-Nanobody Fusion Added the nanobody from pDSG289 to BBa_K4514045 393 nucleotides added

References

  1. Garcia-Doval, C., & van Raaij, M. J. (2012). Structure of the receptor-binding carboxy-terminal domain of bacteriophage T7 tail fibers. Proceedings of the National Academy of Sciences, 109(24), 9390-9395.
    2. https://doi.org/10.1073/pnas.1119719109
  2. Muyldermans, S., Baral, T. N., Retamozzo, V. C., De Baetselier, P., De Genst, E., Kinne, J., Leonhardt, H., Magez, S., Nguyen, V. K., Revets, H., Rothbauer, U., Stijlemans, B., Tillib, S., Wernery, U., Wyns, L., Hassanzadeh-Ghassabeh, G., & Saerens, D. (2009). Camelid immunoglobulins and nanobody technology. Veterinary Immunology and Immunopathology, 128(1-3), 178-183.
    3. https://doi.org/10.1016/j.vetimm.2008.10.299
  3. Glass, D. S., & Riedel-Kruse, I. H. (2018). A synthetic bacterial cell-cell adhesion toolbox for programming multicellular morphologies and patterns. Cell, 174(3).
    4. https://doi.org/10.1016/j.cell.2018.06.041
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