Team:Aix-Marseille/M13 Design

M13 Design

Engineering M13

T--Aix-Marseille--M13K07.png

M13 is a phage that target E.coli. Our goal is to create a engineered M13 phage-like particle that will be specific to Xylella fastidiosa. To do so we look into the attachment protein of M13. This protein contain three domains (D1, D2 and D3) and a signal sequence. In filamentous phages, only D1 and D2 are crucial for target attachment. The signal sequence is crucial for the excretion of p3 in the periplasm[1] and D3 is 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 and replace them with the D1 and D2 of X. fastidiosa's filamentous phage[2]. Thus we design the biobrick with the attachment domains for E.coli [http://parts.igem.org/Part:BBa_K2255008 BBa_K2255008] and X. fastidiosa [http://parts.igem.org/Part:BBa_K2255018 BBa_K2255018]. In our design we initialy wanted to keep the signal sequence and D3 of M13, because their are crucial for the formation of the phage.

T--Aix-Marseille--P3map.png

To enable D1-D2 switch we insert two restriction site (AvrII and BspI) in the p3 gene which are compatible with XbaI and AgeI. With this modification we remove D1 abd D2 from p3 gene, but we also remove the signal sequence. As we remove it with our construction, we must put another one. Thus we design [http://parts.igem.org/Part:BBa_K2255007 BBa_K2255007].

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 BBa_K2255005], which is the domain involved in the assembly and release of M13 particles.

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 [http://parts.igem.org/Part:BBa_K1491017 Super Nova toxin], 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.

To deliver our toxin, either we created a phagemid that contain the [http://parts.igem.org/Part:BBa_K1445000 M13 origin] (oriM13) 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, [http://parts.igem.org/Part:BBa_K608002 E.coli promoter] or [http://parts.igem.org/Part:BBa_K2255004 X. fastidiosa promoter] along with SuperNova gene.

T--Aix-Marseille--pbluescript-SN.jpeg
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References

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