Engineering M13

Our goal is to create a engineered M13 phage that will be specific to Xylella fastidiosa. To do so we look into the attachment protein of M13. This protein contain three domaine (D1, D2 and D3) and a signal sequence. In filamentous phages, only D1 and D2 are crucial for target attachment.The signal sequence and D3 are important for phage formation.

In our design we choose to 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 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 insert two restriction site (AvrII and BspI) in the p3 gene which are compatible with XbaI and AgeI.

But with this modification we remove multiple domains from p3 gene : the sequence signal, the domain 1 (D1) and the domain 2 (D2). In our design we wanted to keep the signal sequence and D3 of M13, because their are crucial for the formation of the phage.

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 : [http://parts.igem.org/Part:BBa_K2255005 p3_D3], which is the domain involved in the assembly and release of M13 particles.

Attachment protein

To create our Xylella fastidiosa filamentous phage, we started to look in the bibliography and in the NCBI data base, filamentous phages that were able to infect this pathogens. D3 and the signal sequence are both the best conserved part from the attachment protein. ^[1]. Thus we design [http://parts.igem.org/Part:BBa_K2255008 BBa_K2255008] biobrick.

Signal sequence

The signal sequence is crucial for the excretion of p3 in the periplasm.^[2] As we remove it with our construction, we must put another one. Thus we design [http://parts.igem.org/Part:BBa_K2255007 BBa_K2255007].

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.

References

1. ↑ Chen, J. & Civerolo, E. L. Morphological evidence for phages in Xylella fastidiosa. Virology Journal 5, 75 (2008).
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).