Difference between revisions of "Team:Aix-Marseille/M13 Design"

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{{Aix-Marseille|title=M13 Design|toc=__TOC__}}
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{{Aix-Marseille|title=M13 Design|toc=__noTOC__}}
  
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==Engineering M13==
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==Engineering M13 helper phage==
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[[File:T--Aix-Marseille--M13K07.png|350px|right]]
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[[File:T--Aix-Marseille--M13K07-2.png|400px|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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[[Team:Aix-Marseille/M13|M13]] is a phage that targets ''E.coli''. Our goal is to create an engineered M13 phage-like particle that will be specific to [[Team:Aix-Marseille/Xylella_fastidiosa|''Xylella fastidiosa'']]. To do so, we look into the attachment protein of [[Team:Aix-Marseille/M13|M13]]. This protein contains three domains (D1, D2, and D3) and a signal sequence. In filamentous phages, only D1 and D2 are crucial for target attachment while 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> and D3 is important for phage formation.
  
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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 chose 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 [[Team:Aix-Marseille/M13|M13]] origin of replication.
  
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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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In order to engineer multiple phages to infect various pathogens, we used two strategies.  
  
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[[File:T--Aix-Marseille--P3map.png|700px|center]]
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The first one is to remove D1 and D2 and replace them with the D1 and D2 of [[Team:Aix-Marseille/Xylella_fastidiosa|''X. fastidiosa'']]'s filamentous phage<ref>Chen, J. & Civerolo, E. L. Morphological evidence for phages in ''Xylella fastidiosa''. Virology Journal 5, 75 (2008).</ref>. Thus we designed the biobrick with the attachment domains for ''E.coli'' [http://parts.igem.org/Part:BBa_K2255008 BBa_K2255008] and [[Team:Aix-Marseille/Xylella_fastidiosa|''X. fastidiosa'']] [http://parts.igem.org/Part:BBa_K2255018 BBa_K2255018]. In our design, we initially wanted to keep the signal sequence and D3 of M13, because there are crucial for the formation of the phage.
  
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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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[[File:T--Aix-Marseille--P3map-3.png|1000px|center]]
  
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===Attachment protein===
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[[File:T--Aix-Marseille--mutation-2.png|400px|right|thumb|A) Agarose gel in which we can see PCR amplification of M13KO7 before (WT) and after (mut) deletion of p3. As you can see, the mutant has a loss the p3 gene. B) Agarose gel in which we can see the digestion by AvrII and BspI of M13KO7 WT and M13KO7 after mutation (M13KO7AB). As you can see, only the mutant has been digested.]]
  
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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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To enable the D1-D2 switch, we inserted two restriction sites (AvrII and BspI) in the p3 gene which are compatible with XbaI and AgeI. With this modification, we could remove D1 and D2 from the p3 gene, but we also removed the signal sequence. As we removed it with our construction, we had to put another one. We therefore designed [http://parts.igem.org/Part:BBa_K2255007 BBa_K2255007]. We couldn't swap only D1 and D2 because of the need to substitute only 3 nucleotides to create the new restriction site.
  
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{|
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The second strategy to engineer [[Team:Aix-Marseille/M13|M13]] is to entirely remove the protein III from the phage genome and to reconstruct it in another plasmid. Using this method, we created another part, [http://parts.igem.org/Part:BBa_K2255005 BBa_K2255005], which is the domain involved in the assembly and release of M13 particles.
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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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|-
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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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|-
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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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|-
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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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We have succeeded to engineered both design, with de-insertion of AvrII and BspI(M13KO7AB) and the deletion of the whole p3 (M13KO7Δp3). Verification has been done with the sequencing of both designs.
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|-
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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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|-
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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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|-
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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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==Phagemid==
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|-
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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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|-
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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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|-
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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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As the M13KO7 genome is only poorly assembled into phage, we decided to use a phagemid to carry a toxin gene and be assembled into our engineered phage-like particles.  
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|-
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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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We first thought about using the SuperNova toxin ([http://parts.igem.org/Part:BBa_K1491017 BBa_K1491017]), made by the iGEM team Carnegie Mellon in 2014. This toxin has multiple benefits: because of the production of ROS occurring only in a yellow-orange light, we could produce phages-like particles carrying this toxin without killing ''E.coli''. However, when this toxin would be produced in [[Team:Aix-Marseille/Xylella_fastidiosa|''X. fastidiosa'']] with the light coming from the sun, the bacterium will be harmed, even if it is in the Xylem vessels.
 +
To optimize the production of this toxin in [[Team:Aix-Marseille/Xylella_fastidiosa|''X. fastidiosa'']] we tried to find a strong and constitutive promoter in this bacterium.
  
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===Signal sequence===
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To deliver our toxin, we either created a phagemid that contains the [http://parts.igem.org/Part:BBa_K1445000 M13 origin] (oriM13) which gives it the opportunity to be used in the phage construction. As the part [http://parts.igem.org/Part:BBa_K1445000 BBa_K1445000] didn't work for us, we choose to follow a new strategy to creates our PLPs.
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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 also thought about the used of pBluescript II KS(+) phagemid. Both of those phagemid contain a M13 origin of replication and a gene for antibiotic resistance. We inserted it in ''E.coli'' ([http://parts.igem.org/Part:BBa_K608002 BBa_K608002]) or ''X. fastidiosa'' ([http://parts.igem.org/Part:BBa_K2255004  BBa_K2255004]) promoter along with the SuperNova gene.
 +
[[File:T--Aix-Marseille--pSB1C3-SN-1.png|400px|left]]
 +
[[File:T--Aix-Marseille--pbluescript-SN-1.png|400px|centre]]
  
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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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[[File:T--Aix-Marseille--testSN-1.png|400px|right]]
  
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[[File:T--Aix-Marseille--pbluescript-SN.jpeg|400px|left]]
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The verification of the concept has been conducted in ''E. coli'', with the Super Nova toxin gene. The Toxin + plate are ''E. coli'' illuminated with a 16,5 Candela 595nm Diode (lumex ssl-lx5093syc/g) for 2 hours and the Toxin - plate are the same bacteria but put in the dark for two hours before incubation.
  
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[[File:T--Aix-Marseille--pSB1C3-SN.jpeg|400px|centre]]
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This test was conducted with PLPs that contain a pBluescript II KS(+) phagemid. This experiment shows us that PLPs can be used as vehicles in toxin gene delivery. With this in mind, we can imagine that we could use a more powerful toxin gene in order to increase the efficiency of our PLPs.
  
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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(+).
 
  
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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.
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We made a [[Team:Aix-Marseille/SN|Supernova activity test]] to count the remaining colonies after illumination.
  
 +
An [[Team:Aix-Marseille/Amelioration_part:BBa_K1445000|amelioration]] of the [http://parts.igem.org/Part:BBa_K1445000 BBa_K1445000]part was done : we used the pBlueScript II instead of the BBa_K1445000 because the phagemid encapsidation didn't work.
  
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===References===
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==References==

Latest revision as of 02:20, 2 November 2017

M13 Design

Engineering M13 helper phage

T--Aix-Marseille--M13K07-2.png

M13 is a phage that targets E.coli. Our goal is to create an 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 contains three domains (D1, D2, and D3) and a signal sequence. In filamentous phages, only D1 and D2 are crucial for target attachment while 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 chose 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 engineer multiple phages to infect various pathogens, we used two strategies.

The first one is to remove D1 and D2 and replace them with the D1 and D2 of X. fastidiosa's filamentous phage[2]. Thus we designed 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 initially wanted to keep the signal sequence and D3 of M13, because there are crucial for the formation of the phage.

T--Aix-Marseille--P3map-3.png
A) Agarose gel in which we can see PCR amplification of M13KO7 before (WT) and after (mut) deletion of p3. As you can see, the mutant has a loss the p3 gene. B) Agarose gel in which we can see the digestion by AvrII and BspI of M13KO7 WT and M13KO7 after mutation (M13KO7AB). As you can see, only the mutant has been digested.

To enable the D1-D2 switch, we inserted two restriction sites (AvrII and BspI) in the p3 gene which are compatible with XbaI and AgeI. With this modification, we could remove D1 and D2 from the p3 gene, but we also removed the signal sequence. As we removed it with our construction, we had to put another one. We therefore designed [http://parts.igem.org/Part:BBa_K2255007 BBa_K2255007]. We couldn't swap only D1 and D2 because of the need to substitute only 3 nucleotides to create the new restriction site.

The second strategy to engineer M13 is to entirely remove the protein III from the phage genome and to reconstruct it in another plasmid. Using this method, we created another part, [http://parts.igem.org/Part:BBa_K2255005 BBa_K2255005], which is the domain involved in the assembly and release of M13 particles.

We have succeeded to engineered both design, with de-insertion of AvrII and BspI(M13KO7AB) and the deletion of the whole p3 (M13KO7Δp3). Verification has been done with the sequencing of both designs.

Phagemid

As the M13KO7 genome is only poorly assembled into phage, we decided to use a phagemid to carry a toxin gene and be assembled into our engineered phage-like particles.

We first thought about using the SuperNova toxin ([http://parts.igem.org/Part:BBa_K1491017 BBa_K1491017]), made by the iGEM team Carnegie Mellon in 2014. This toxin has multiple benefits: because of the production of ROS occurring only in a yellow-orange light, we could produce phages-like particles carrying this toxin without killing E.coli. However, when this toxin would 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 optimize the production of this toxin in X. fastidiosa we tried to find a strong and constitutive promoter in this bacterium.

To deliver our toxin, we either created a phagemid that contains the [http://parts.igem.org/Part:BBa_K1445000 M13 origin] (oriM13) which gives it the opportunity to be used in the phage construction. As the part [http://parts.igem.org/Part:BBa_K1445000 BBa_K1445000] didn't work for us, we choose to follow a new strategy to creates our PLPs.

We also thought about the used of pBluescript II KS(+) phagemid. Both of those phagemid contain a M13 origin of replication and a gene for antibiotic resistance. We inserted it in E.coli ([http://parts.igem.org/Part:BBa_K608002 BBa_K608002]) or X. fastidiosa ([http://parts.igem.org/Part:BBa_K2255004 BBa_K2255004]) promoter along with the SuperNova gene.

T--Aix-Marseille--pSB1C3-SN-1.png
T--Aix-Marseille--pbluescript-SN-1.png


T--Aix-Marseille--testSN-1.png

The verification of the concept has been conducted in E. coli, with the Super Nova toxin gene. The Toxin + plate are E. coli illuminated with a 16,5 Candela 595nm Diode (lumex ssl-lx5093syc/g) for 2 hours and the Toxin - plate are the same bacteria but put in the dark for two hours before incubation.

This test was conducted with PLPs that contain a pBluescript II KS(+) phagemid. This experiment shows us that PLPs can be used as vehicles in toxin gene delivery. With this in mind, we can imagine that we could use a more powerful toxin gene in order to increase the efficiency of our PLPs.


We made a Supernova activity test to count the remaining colonies after illumination.

An amelioration of the [http://parts.igem.org/Part:BBa_K1445000 BBa_K1445000]part was done : we used the pBlueScript II instead of the BBa_K1445000 because the phagemid encapsidation didn't work.

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).