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| − | {{Aix-Marseille|title=Design|toc=__TOC__}}
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| − | ===Engineering M13===
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| − | [[File:T--Aix-Marseille--M13K07.png|350px|right]]
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| − | 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.
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| − | 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.
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| − | 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).
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| − | [[File:T--Aix-Marseille--P3map.png|700px|center]]
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| − | 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.
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| − | ===Attachment protein===
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| − | 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.
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| − | {|
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| − | ! scope="col" |Pathogene
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| − | ! scope="col" |Filamentous phage
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| − | ! scope="col" |gene ID
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| − | |''Escherichia coli''
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| − | |M13 (fd,ff)<ref name=Smeal>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).</ref>
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| − | |927334
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| − | |''Neisseria gonorrheae''
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| − | |NgoΦ6<ref>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).</ref>
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| − | |1260906
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| − | |''Pseudomonas aeruginosa''
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| − | |Pf3<ref>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).</ref>
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| − | |1260906
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| − | | rowspan="2" | ''Ralstonia solanacearum''
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| − | |RSM1Φ<ref name="T,K">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).</ref>
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| − | |5179368
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| − | |RSS1Φ<ref name="T,K">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).</ref>
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| − | |4525385
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| − | | rowspan="3" | ''Vibrio Cholerea''
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| − | |CTXΦ<ref name="Heilpern">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).</ref>
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| − | |26673076
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| − | |-
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| − | |VFJΦ(fs2)<ref>Ikema, M. & Honma, Y. A novel filamentous phage, fs-2, of Vibrio cholerae O139. Microbiology 144, 1901–1906 (1998).</ref>
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| − | |1261866
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| − | |VGJΦ<ref>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).</ref>
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| − | |1260523
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| − | |''Xanthomonas campestris''
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| − | |ΦLf<ref>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).</ref>
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| − | |3730653
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| − | |''Xanthomonas fucans''
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| − | |XacF1<ref>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).</ref>
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| − | |17150318
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| − | |''Xylella fastidiosa''
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| − | |XfasM23<ref>Chen, J. & Civerolo, E. L. Morphological evidence for phages in Xylella fastidiosa. Virology Journal 5, 75 (2008).</ref>
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| − | |6203562
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| − | |}
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| − | Table showing the attachment proteins from various filamentous phages.
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| − | ===Signal sequence===
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| − | The signal sequence is crucial for the excretion of p3 in the periplasm.<ref name="Heilpern">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).</ref> 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.
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| − | [[File:T--Aix-Marseille--M13pIII-Sequencesignal.jpeg|400px|center]]
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| − | 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.
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| − | 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.
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| − | ===Phagemid===
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| − | 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.
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| − | 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.
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| − | To optimise the production of this toxin in ''X. fastidiosa'' we tried to find a strong and constitutive promoter of this bacterium.
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| − | We thought about multiple ways to engineer our phagemid.
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| − | [[Team:Aix-Marseille/phagemid|Read more…]]
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