Part:BBa_K4810013

M13KE vector modified for production of M13 Phages detecting "Eicosane"

M13 phage is a type of non-lytic filamentous bacteriophage containing single-stranded DNA and replicating within E.coli cells. M13 bacteriophage has gained a lot of attention due to its usage in tissue-regeneration, drug delivery and biodetection. Many of those applications involve the genetic modification of M13 pVIII capsid protein.[1]

For the genetic engineering of the bacteriophage, phage vectors can be used since they contain closed double-stranded circular DNA . A phage vector successfully transformed into a host cell replicates to produce a complete phage particle using general protocols[2]

M13KE is a phage vector that can be genetically engineered to produce phage particles with modified pVIII protein able to bind to “Eicosane”, a volatile organic compound found in increased concentrations in the sebum of patients with Parkinson’s disease. Engineered M13 phage can operate as a biosensor for “Eicosane” since protein-compound interactions cause structural changes in the phage that can be translated into colorimetric output when phage particles are self-assembled in thin films, usually on gold substrates.[3]

In order to build the “Eicosane”-detecting phages, M13KE DNA was genetically modified to express a specific hexapeptide (ADEFYQ) in pVIII N-terminal by replacing the respective codons of pVIII protein. The 23 amino acids of the N-terminal of pVIII protein undergo cleavage by a signal peptidase inside the bacterial cell, resulting in a functional protein of 50 amino acids. So the specific modification occurred by deletion of the codons #2 to #5 of the functional protein (codon #1 already translates to amino acid "A") and insertion of the respective codons of the desired peptide. Thus, the newly introduced peptide sequence is incorporated in the N-terminal of the protein.

The sequence of the specific peptide (ADEFYQ) is determined by molecular docking simulations using Autodock Vina, opting for peptides with the highest affinity with "Eicosane" and simultaneously the lowest affinity with other common organic compounds found in human sebum.

A new HindIII restriction site is introduced at the codons of K3 and L4 of M13geneIII to be used as verification checkpoint of the modified M13KE vector from the unmodified one by restriction digestion.

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References:

[1]-Chang, C., Guo, W., Yu, X., Guo, C., Zhou, N., Guo, X., Huang, R., Li, Q., & Zhu, Y. (2023). Engineered M13 phage as a novel therapeutic bionanomaterial for clinical applications: From tissue regeneration to cancer therapy. Materials Today Bio, 20, 100612. https://doi.org/10.1016/j.mtbio.2023.100612

[2] - Wang, R., Li, HD., Cao, Y. et al. M13 phage: a versatile building block for a highly specific analysis platform. Anal Bioanal Chem 415, 3927–3944 (2023). https://doi.org/10.1007/s00216-023-04606-w

[3] - Lee, J., Warner, C., Jin, HE. et al. Production of tunable nanomaterials using hierarchically assembled bacteriophages. Nat Protoc 12, 1999–2013 (2017). https://doi.org/10.1038/nprot.2017.085