Engineering M13

In order to engineered multiple phages to infect various pathogenes we first 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 , with the origin of replication from P15A and the kanamycin resistance gene from Tn903 both inserted within the M13 origin of replication.

In M13KO7 we wanted to insert two restriction site (AvrII and BspI) which are compatible with XbaI and AgeI. Thus, we 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.

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).

Another way to engineered M13, is to remove entierely the protein III from the phage genome and to reconstruct it in another plasmid. Thus, we create another part : p3_D3, which is the domain involved in the assembly and release of M13 particles.

Attachment protein

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. 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.

Pathogene	Filamentous phage	gene ID
Escherichia coli	M13 (fd,ff)[1]	927334
Neisseria gonorrheae	NgoΦ6[2]	1260906
Pseudomonas aeruginosa	Pf3[3]	1260906
Ralstonia solanacearum	RSM1Φ[4]	5179368
	RSS1Φ[4]	4525385
Vibrio Cholerea	CTXΦ[5]	26673076
	VFJΦ(fs2)[6]	1261866
	VGJΦ[7]	1260523
Xanthomonas campestris	ΦLf[8]	3730653
Xanthomonas fucans	XacF1[9]	17150318
Xylella fastidiosa	XfasM23[10]	6203562

Table showing the attachment proteins from various filamentous phages.

Signal sequence

The signal sequence is crucial for the excretion of p3 in the periplasm.^[5] 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.

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.

Phagemid

As M13KO7 genome is not capable to be used for the phage assemblage, we will use a phagemid which will carry a toxin to assemble our ingeneered M13 phage.

We thought about using the Super Nova toxin (BBa_K1491017), made by the iGEM team Carnegie Mellon in 2014. This toxin has multiple benefits, because of the production of ROS occuring only in a yellow - orange light, we can produce phages-like particules carring this toxin without killing E.coli. However, when this toxin will be produced in X. fastidiosa with the light coming from the sun, the bacterium will be harmed, even if it is in the Xylem vessels. To optimise the production of this toxin in X. fastidiosa we tried to find a strong and constitutive promoter of this bacterium.

We thought about multiple ways to engineer our phagemid.

To deliver our toxin, either we created a phagemid that contain the oriM13 (BBa_K1445000) which will gives it the opportunity to be used in the phage construction, or we used the phagemid pBluescript II KS(+).

Both of those phagemid contain a M13 origin of replication and a gene for antibiotic resistance. We insert in both, a E.coli or X. fastidiosa promoter along with SuperNova gene.

X. fastidiosa promoter

Firstly, we found best bidirectional hit (BBH) between Escherichia coli str. K-12 substr. MG1655 genes and Xylella fastidiosa 9a5c ones. In order to have a strong constitutive promoter we look at highly expressed genes from E.coli.^[11]

Secontly, with the tool rsat, for each gene selected we take the upstream sequence from the previous gene to the ATG And with the tool BPROM we choose the sequence with predicted box with the best score. We choose XF_RS01885 which is the BBH of purA, which code for an adenylosuccinate synthetase.

Finaly, we tried to find the ribosome binding site (RBS) consensus in Xylella fastidiosa. To do so we search for the anti-Shine dalgarno sequence with Xylella fastidiosa 16S ribosomal RNA gene (accession number : NR_041779). The consus found is : AGGAGG. The RBS is supposed to be 6 to 12 nucleotide upstream the ATG. So we modified the sequence. And we added Rfc10 prefix and suffix region.

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).
2. ↑ 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).
3. ↑ Luiten, R. G., Schoenmakers, J. G. & Konings, R. N. The major coat protein gene of the filamentous Pseudomonas aeruginosa phage Pf3: absence of an N-terminal leader signal sequence. Nucleic Acids Res 11, 8073–8085 (1983).
4. ↑ ^{4.0 4.1} 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).
5. ↑ ^{5.0 5.1} 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).
6. ↑ Ikema, M. & Honma, Y. A novel filamentous phage, fs-2, of Vibrio cholerae O139. Microbiology 144, 1901–1906 (1998).
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. ↑ Ahmad, A. A., Askora, A., Kawasaki, T., Fujie, M. & Yamada, T. The filamentous phage XacF1 causes loss of virulence in Xanthomonas axonopodis pv. citri, the causative agent of citrus canker disease. Front. Microbiol. 5, (2014).
10. ↑ Chen, J. & Civerolo, E. L. Morphological evidence for phages in Xylella fastidiosa. Virology Journal 5, 75 (2008).
11. ↑ S, K., J, M., A, C. & D, K. Characterizations of highly expressed genes of four fast-growing bacteria., Characterizations of Highly Expressed Genes of Four Fast-Growing Bacteria. J Bacteriol 183, 183, 5025, 5025–5040 (2001).