Team:Aix-Marseille/pIII

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Ingeneering M13

Bacteriophages play a special role in nanoscale cargo-delivery developments, because they can be regarded as naturally occurring nanomaterials. Viral nanoparticles (VNPs), in particular bacteriophages, are attractive options for cargo-delivery as they are biocompatible, biodegradable, and non-infectious to mammals.[1]

Phage systems, like M13, have been employed in biotechnological applications, most prominently in the identification and maturation of medically-relevant binding molecules through phage display. The application of phages in materials and nanotechnology is mainly due to their nanoscale size and simple life cycles. We choose to use those application in our advantage in order to target Xylella fastidiosa and other pathogenic bacteria.

M13 is a filamentous phage that infects E. coli that carry the F-episome. Active infection with M13 does not kill the host cell. The M13 phage particle consists of a single-stranded DNA (ssDNA) genome encased in approximately 2700 copies of a major coat protein protein VIII.[2]

T--Aix-Marseille--M13pIII-explication-LC.png
M13 Lifecycle[3]

The M13 life cycle begins with passage of the phage genome into a host cell in a process induced by protein III (pIII). After a while, as the concentrations of phage proteins increase, the protein V (pV) binds to the ssDNA genomes for packaging into progeny phages. pV recognise the single stranded M13 origin of replication. The pV-sequestered ssDNA is recognized by the membrane spanning phage assembly complex. [2]

Protein III

The molecular interactions that mediate the entry of Escherichia coli derived filamentous phages into their hosts have been studied in considerable detail. The 424-amino-acid protein III is thought to consist of a leader sequence and three domains, separated by glycine-rich regions, that serve distinct roles in phage entry and release. The first two pIII domains, D1 and D2, are required for M13 adsorption and entry, while the third domain D3 is required for the assembly and release of M13 particles from host.[4]

T--Aix-Marseille--M13pIII-explication-1.png

Our goal is to create a engineered M13 phage that will be specific to an other bacteria. Thus we started to look in the bibliography and in the NCBI data base, filamentous phages that were able to infect various pathogens.

Pathogene Filamentous phage gene ID
Escherichia coli M13 (fd,ff) 927334
Neisseria gonorrheae NgoΦ6[5] 1260906
Pseudomonas aeruginosa Pf3 1260906
Ralstonia solanacearum RSM1Φ[6] 5179368
RSS1Φ[6] 4525385
Vibrio Cholerea CTXΦ[4] 26673076
VFJΦ(fs2) 1261866
VGJΦ[7] 1260523
Xanthomonas campestris ΦLf[8] 3730653
Xanthomonas fucans XacF1 17150318
Xylella fastidiosa XfasM23 6203562

D3 and the signal sequence are both the best conserved part from the attachment protein. So with protein global alignment (Needleman-Wunsch alignment), from two or three sequence at one time, we were eventually able to determinate D1 and D2.

Genomic modification of M13

In order to engineered multiple phages to infect various pathogenes we decided to remove D1 and D2. As we wanted to insert those two domains in the p3 of the M13 genome. Thus we use M13KO7 from New England BioLab. M13KO7 is an M13 derivative which carries the mutation Met40Ile in gII. M13KO7 is able to replicate in the absence of phagemid DNA. In our design we wanted to keep the signal sequence and D3 of M13, because their are crucial for the formation of the phage. We just want to insert D1 and D2 from another phages (we’ll call it X).

T--Aix-Marseille--M13pIII-explication-01.png

In M13KO7 we manage to insert two restriction site (AvrII and BspI) which are compatible with XbaI and AgeI. Thus, we will create two types of biobrick, one with the signal sequence of M13, and the other one with D1 and D2 of another p3 from another filamentous phages.

T--Aix-Marseille--M13pIII-explication-3.png

Signal sequence

The signal sequence is crucial for the excretion of p3 in the periplasm.[4] As we remove it with our construction, we must put another one. We choose to use the one coming from M13 as we use E. coli to produce our phage. In order to be functional, the signal peptide must be cut down from the rest of the protein. Thus, we must add the cleavage site. Using the logiciel SignalP 4.1, we saw that the cleavage is made between the alanine and the glutamate.

T--Aix-Marseille--M13pIII-explication-6.png

In order to gain flexibility, which will help the enzyme to cleave the signal sequence, we add two glycine and one serine residue. Which we retrotranslate, with the codon biais of E. coli K12.

The signal sequence and D1-D2 sequence are designed to make fusion protein, thus we choose to make them Freiburg assembly standard with Rfc25 prefix and sufix. This will be helpful in order to assemble our biobrick.

Phages-like particules

Bacteriophages are capable of expressing their genomes, and generating new copies of themselves. We choose to limit the phage ability to reproduce it-self in order to contain it. As it is possible to produce recombinant viruses that express foreign proteins, it is possible to restrain their capacity to reproduce them self. [9] Virus-like particles (VLPs) are multiprotein structures that mimic the organization and conformation of authentic native viruses but lack the viral genome. They have been applied not only as prophylactic and therapeutic vaccines but also as vehicles in drug and gene delivery and, more recently, as tools in nanobiotechnology. [9]


References

  1. ↑ Czapar, A. E. & Steinmetz, N. F. Plant viruses and bacteriophages for drug delivery in medicine and biotechnology. Current Opinion in Chemical Biology 38, 108–116 (2017).
  2. ↑ 2.0 2.1 Smeal, S. W., Schmitt, M. A., Pereira, R. R., Prasad, A. & Fisk, J. D. Simulation of the M13 life cycle I: Assembly of a genetically-structured deterministic chemical kinetic simulation. Virology 500, 259–274 (2017).
  3. ↑ Mustafa Gungormus. Controlled Biomineralization Towards Tissue Engineering Using Genetically Engineered Hydroxyapatite Binding Peptides. Thesis of Yıldırım Beyazıt Üniversitesi(2006)
  4. ↑ 4.0 4.1 4.2 Heilpern, A. J. & Waldor, M. K. pIIICTX, a predicted CTXphi minor coat protein, can expand the host range of coliphage fd to include Vibrio cholerae. J. Bacteriol. 185, 1037–1044 (2003).
  5. ↑ Piekarowicz, A. et al. Neisseria gonorrhoeae Filamentous Phage NgoΦ6 Is Capable of Infecting a Variety of Gram-Negative Bacteria. J Virol 88, 1002–1010 (2014).
  6. ↑ 6.0 6.1 T, K. et al. Genomic characterization of the filamentous integrative bacteriophages {phi}RSS1 and {phi}RSM1, which infect Ralstonia solanacearum., Genomic Characterization of the Filamentous Integrative Bacteriophages φRSS1 and φRSM1, Which Infect Ralstonia solanacearum. J Bacteriol 189, 189, 5792, 5792–5802 (2007).
  7. ↑ Campos, J. et al. VGJφ, a Novel Filamentous Phage of Vibrio cholerae, Integrates into the Same Chromosomal Site as CTXφ. J. Bacteriol. 185, 5685–5696 (2003).
  8. ↑ Tseng, Y.-H., Lo, M.-C., Lin, K.-C., Pan, C.-C. & Chang, R.-Y. Characterization of filamentous bacteriophage ΦLf from Xanthomonas campestris pv. campestris. Journal of general virology 71, 1881–1884 (1990).
  9. ↑ 9.0 9.1 Roldão, A., Silva, A. C., Mellado, M. C. M., Alves, P. M. & Carrondo, M. J. T. Viruses and Virus-Like Particles in Biotechnology: Fundamentals and Applications. in Reference Module in Life Sciences (Elsevier, 2017).