Ingeneering M13

Bacteriophages play a special role in nanoscale cargo-delivery developments, because they can be regarded as naturally occurring nanomaterials. Viral nanoparticles (VNPs), in particular bacteriophages, are attractive options for cargo-delivery as they are biocompatible, biodegradable, and non-infectious to mammals.^[1]

Phage systems, like M13, have been employed in biotechnological applications, most prominently in the identification and maturation of medically-relevant binding molecules through phage display. The application of phages in materials and nanotechnology is mainly due to their nanoscale size and simple life cycles. We choose to use those application in our advantage in order to target Xylella fastidiosa and other pathogenic bacteria.

M13 is a filamentous phage that infects E. coli that carry the F-episome. Active infection with M13 does not kill the host cell. The M13 phage particle consists of a single-stranded DNA (ssDNA) genome encased in approximately 2700 copies of a major coat protein protein VIII.^[2]

The M13 life cycle begins with passage of the phage genome into a host cell in a process induced by protein III (pIII). After a while, as the concentrations of phage proteins increase, the protein V (pV) binds to the ssDNA genomes for packaging into progeny phages. pV recognise the single stranded M13 origin of replication. The pV-sequestered ssDNA is recognized by the membrane spanning phage assembly complex. ^[2]

Protein III

The molecular interactions that mediate the entry of Escherichia coli derived filamentous phages into their hosts have been studied in considerable detail. The 424-amino-acid protein III is thought to consist of a leader sequence and three domains, separated by glycine-rich regions, that serve distinct roles in phage entry and release. The first two pIII domains, D1 and D2, are required for M13 adsorption and entry, while the third domain D3 is required for the assembly and release of M13 particles from host.^[3]

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.

CLUSTAL multiple sequence alignment by MUSCLE (3.8)

pIII_CTXΦ ---------MRYFLLFLTL-------------------LFLSPSVTASAINCDPNT---- pIII_Fs2 ---------MRTVLTLFST---TLLLSISFSSFAYYQLPFWPDKTYLTPEAAAAAYLDIL pIII_VGJΦ --------MVMFMRKFFIISLVISLYFTVLPAFAAYQLPFDQTKTFSTVQAAAEYYVNLL pIII_NgoΦ6 ---------MMYSFEANAN------------------AVKISETLSVDTGQGAKVHKFVP pIII_Pf3 --------MGLHYLFGFCL--------------ALFSFSAIAAGPVSTEVAAGTTTYRVT p3_M13 ---------MKKLLFAIPL------------------VVPFYSHSAETVESCLAKPHTEN pIII_RSM1Φ ---------MATVLGFYRCAVRVGGHGRLGGWLALIVAGLLAVGTAQAQSVGVDLTVRQS pIII_RSS1Φ ---------MRVLVLLLCW---------------------WASSAWASTIPLIPPPNIVL pIII_XfasM23 -------MSIFRILVFFVI--------------------LFISRFSFACEIGEPH----- pIII_ΦLf ---------VITVRLVACI----------------LAVALWSCAFSANAAMCRDAADACD pIII_XacF1 LGRRQGVGCVRYIVVVALC----------------LAALFFSSRVTAACVQLEAPTSSHN


pIII_CTXΦ ----------------------------------------------------TTSHQLLF pIII_Fs2 GGGSCKRDTRNF-----IKASISSINTPHIAYRIDKYL--------------DDQCIVLS pIII_VGJΦ GGTSCKADGNRF-----KIYRVKSIGDPSFVIQQETYL--------------DNKCQVFS pIII_NgoΦ6 KSSNIYSSDLTK----AVDLTHIPTGAKARINAKITAS--------------VSRAGVLS pIII_Pf3 NTTVRTPPNVTLSPVRDITPYVEKIPNKGLAQAAQGRLIVAQRAASVPVTGFFNVSGAVV p3_M13 SFTNVWKDDKTL----------------DRYANYEGCL--------------WNATGVVV pIII_RSM1Φ GASVPQTPGSVMSVLVPITIGVVAVGAAAVALPATGAL--------------AITGDVIA pIII_RSS1Φ TGTGYVTTGA-------VTLSEVATATEMRAAVGAGAA--------------TIAATMTV pIII_XfasM23 -----WDPGQCL-------------------DRGEAYA---------------------- pIII_ΦLf QGQA------------------------------------------------FLAARMLA pIII_XacF1 GDWSCADQGEAF---------AKVSSFPVPADLAKCAM--------------KSVRAVAS


pIII_CTXΦ GFGSPIVQSVLFDGCMLDIEKD--DYGFVWSCLSNENGDYCKGLYKPRFSQGVSP----- pIII_Fs2 STGNASVTLNVVDNCPDGTSPD--LSTGMCKPKPDTPQYCGTS---AMFEDVANL----- pIII_VGJΦ SKGDISVTLIVVDDPTTCEASK--GQTGKVGWNSYFWGSATPSRYICSSAYGGCV----- pIII_NgoΦ6 GVGKLVRQGAKFGTRAVPYVGT--ALLAHDVYETFKEDIQARG---CRYDPETDK----- pIII_Pf3 KSGAKSFLRSAGRASGIGLGLA--ALLEAADWVFDEEGEIVKP-----LPGGGSPVLMPR p3_M13 CTGDETQCYGTWVPIGLAIPEN--EGGGSEGGGSEGGGSEGGG---TKPPEYGDT----- pIII_RSM1Φ SAGMAAIRKGAIQGAALAGVIA--LIGAPSGIALDLNGNVVAP---AVSANAGDIGFNGF pIII_RSS1Φ GEGAAAVALAALRATPAIATATSLAYLAQLGIQKCLDGTWCTS---KRSPAAGDT----- pIII_XfasM23 ---IASASYQMWRSNQLKDSNI--PGLQVVDCPMTDDGHIIGFGGYTTAPGYPSS----- pIII_ΦLf DQRGVDLCKLVGGSNSLYKGPD--VVAESAGIYNAQATCSIGG-----PAGAGST----- pIII_XacF1 GPGFTQRMTYPGNTCGIGYELD--IGTGS-----------------AQFPEAS-------


pIII_CTXΦ ---NWPM------------CDLSGASAERCIYPYCPEGE-----------ECVPLPPSPP pIII_Fs2 ---------------------YNACYEQNGILSYTCDEST----------QALDAKCDLT pIII_VGJΦ ALTGDHLCINIDPEALKNDPSLYDCDALYIVQDTPCSPSGDYP-------FCTDENCSSF pIII_NgoΦ6 FVKGYEYANCLWYEDERRINRTYGCYGVDSSIMRLMPDRSRFPEVKQLMESQMYRLARPF pIII_Pf3 PVILNEYT-------------VTGSAGQWSISKEYEPDP----------------RSVPG p3_M13 PIPGYTY--------------INPLDGTY------PPGT-----------EQNPANPNPS pIII_RSM1Φ ---GWLW--------------IDGSGAHYAV----SPGA-----------ACAAKAATSG pIII_RSS1Φ GFNGWGWTYGYN---------TSATGGNIANGVAASPGA-----------ACSAMLAADA pIII_XfasM23 ----------------------DSCSNSLVYFQRVYPEGK----------TCLTRSPKSL pIII_ΦLf ---------------------FYSK-------------------------TCAQRPP--- pIII_XacF1 --------------------------------------------------TCAKRPAQSG


pIII_CTXΦ SDSPVDGLSSSFKSAFNQVYKNQSEMASTLNHVSGQVSHSQDMVQLNTKFHADRVLESV- pIII_Fs2 TSD-----QCVIGRPTWPDCLDK-------------PHQPNDPTNPLPPVGGFNPSPVN- pIII_VGJΦ LPE-----PNPNPNPEPEPEPEP---------QPDPEHNPSDPTAPLPGSGGEVINPTVP pIII_NgoΦ6 WNWRKEE-LNKLSSLDWNNFVLN-RCTFDWNGGGCAVNKGDD-----FRAGASFSLGRN- pIII_Pf3 W-------YSYNGNPVWVSAVEDVGFTWRYWYFADVLMDGQGRPNYLVAYSDSGPNEYWQ p3_M13 LEE-----SQPLNTFMFQNNRFR-NRQGALTVYTGTVTQGTDPVK---TYYQYTPVSSK- pIII_RSM1Φ --------ATIVSVQVGSNPQQY-NCVARAADGSE-FGIGTSQGD--ACIGGYVLKSGS- pIII_RSS1Φ YLAGQK--AKFAGMKATGNGTSY-ECHYT-NDGGDNFYAGTSQAS--SCVDGYVVSGSA- pIII_XfasM23 LGL-----TLPSGVRVPSTACYD-GCSYDLDRSHGIIGVGQDDGKVRYVLPGMTPNGNL- pIII_ΦLf --------LIGASSSDGSGFSCDDGCFYN-------FTVGASGGS------GMYPSGAT- pIII_XacF1 W-------TNP-TAPTPSDVCND-GCYYTY---------AVDPGN--PKGYSYTPSGAT-

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pIII_CTXΦ LFR--------------- pIII_Fs2 LFTGVTPVAGTVPYFSRR pIII_VGJΦ IFGG-------------- pIII_NgoΦ6 VFGSLGGE---------- pIII_Pf3 TLGVVR------------ p3_M13 FANILRNKES-------- pIII_RSM1Φ FF---------------- pIII_RSS1Φ VIGVGRKGEE-------- pIII_XfasM23 TLR--------------- pIII_ΦLf VT---------------- pIII_XacF1 VT----------------

Phages-like particules

Bacteriophages are capable of expressing their genomes, and generating new copies of themselves. We choose to limit the phage ability to reproduce itself in order to contain it. As it is possible to produce recombinant viruses that express foreign proteins, it is possible to restrain their capacity to reproduce themself. ^[12] Virus-like particles (VLPs) are multiprotein structures that mimic the organization and conformation of authentic native viruses but lack the viral genome. They have been applied not only as prophylactic and therapeutic vaccines but also as vehicles in drug and gene delivery and, more recently, as tools in nanobiotechnology. ^[12]

Genomic modification of M13

In order to engineered multiple phages to infect various pathogenes we 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 manage 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).

Signal sequence

The signal sequence is crucial for the excretion of p3 in the periplasm.^[3] 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.

Construction of our Phagemids

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. This phagemid , will contain the oriM13 which will gives it the opportunity to be used in the phage construction.

We thought about using the KillerRed toxin (Bba_K1184000), made by iGEM13_Carnegie_Mellon. 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.

Promoter for production of the toxins in X. fastidiosa

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.^[13]

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 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.

References

1. ↑ Czapar, A. E. & Steinmetz, N. F. Plant viruses and bacteriophages for drug delivery in medicine and biotechnology. Current Opinion in Chemical Biology 38, 108–116 (2017).
2. ↑ ^{2.0 2.1 2.2} 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).
3. ↑ ^{3.0 3.1 3.2} 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).
4. ↑ 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).
5. ↑ 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).
6. ↑ ^{6.0 6.1} 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).
7. ↑ Ikema, M. & Honma, Y. A novel filamentous phage, fs-2, of Vibrio cholerae O139. Microbiology 144, 1901–1906 (1998).
8. ↑ 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).
9. ↑ 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).
10. ↑ 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).
11. ↑ Chen, J. & Civerolo, E. L. Morphological evidence for phages in Xylella fastidiosa. Virology Journal 5, 75 (2008).
12. ↑ ^{12.0 12.1} Roldão, A., Silva, A. C., Mellado, M. C. M., Alves, P. M. & Carrondo, M. J. T. Viruses and Virus-Like Particles in Biotechnology: Fundamentals and Applications. in Reference Module in Life Sciences (Elsevier, 2017).
13. ↑ 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).