Team:Aix-Marseille/Part Collection

Part Collection

Contents


Our collection of parts is designed to allow the creation of proteins, or phage-like particles, that target a wide range of different Gram-negative bacteria. Each part corresponds to the domains 1 (D1) and 2 (D2) from the p3 protein of filamentous phages targeting the different organisms. The parts in the collection are expected to be used as fusion proteins and so conform to the [http://parts.igem.org/Assembly_standard_25 Rfc25] standard. To know more about the design of these parts, and how we used them to make phage-like particles in our project, you can check out our design page.

This part collection contains the following biobricks all in [http://parts.igem.org/Assembly_standard_25 Rfc25] format:

  • p3_E.coli (RFC25) - [http://parts.igem.org/Part:BBa_K2255008 BBa_K2255008]
  • p3_ N.gonorrhoeae (RFC25) - [http://parts.igem.org/Part:BBa_K2255009 BBa_K2255009]
  • p3_P.aeruginosa (RFC25) - [http://parts.igem.org/Part:BBa_K2255010 BBa_K2255010]
  • p3_R.solanacearum_RSM1 (RFC25) - [http://parts.igem.org/Part:BBa_K2255011 BBa_K2255011]
  • p3_R.solanacearum_RSS1 (RFC25) - [http://parts.igem.org/Part:BBa_K2255012 BBa_K2255012]
  • p3_V.Cholerae_CTXΦ (RFC25) - [http://parts.igem.org/Part:BBa_K2255013 BBa_K2255013]
  • p3_V.Cholerae_fs2 (RFC25) - [http://parts.igem.org/Part:BBa_K2255014 BBa_K2255014]
  • p3_V.Cholerea_VGJΦ (RFC25) - [http://parts.igem.org/Part:BBa_K2255015 BBa_K2255015]
  • p3_X.campestris (RFC25) - [http://parts.igem.org/Part:BBa_K2255016 BBa_K2255016]
  • p3_X.fastidiosa (RFC25) - [http://parts.igem.org/Part:BBa_K2255017 BBa_K2255017]
  • p3_X.fuscans (RFC25) - [http://parts.igem.org/Part:BBa_K2255018 BBa_K2255018]

Design

Design

The domain 3 (D3) and the signal sequence are both the best conserved part from the attachment protein. Using a global protein alignment (Needleman-Wunsch and MUSCLE alignments), using two or three sequence at one time, we were eventually able to determinate domain 1 (D1) and domain 2 (D2) from the attachment protein of each phages.

We were able to found these domains because each of them is separated by a flexible sequence composed of Glycine and Serine [1]. Then we retrotranslate this sequence in a nucleotidic sequence and we used iDT to optimise this sequence for E.coli production.

Pathogene Filamentous phage GI Part ID
Escherichia coli M13 (fd,ff)[2] 927334 BBa K2255008
Neisseria gonorrheae NgoΦ6[3] 1260906 [http://parts.igem.org/Part:BBa_K2255009 BBa_K2255009]
Pseudomonas aeruginosa Pf3[4] 215374 [http://parts.igem.org/Part:BBa_K2255010 BBa_K2255010]
Ralstonia solanacearum RSM1Φ[5] 5179368 [http://parts.igem.org/Part:BBa_K2255011 BBa_K2255011]
RSS1Φ[5] 4525385 [http://parts.igem.org/Part:BBa_K2255012 BBa_K2255012]
Vibrio Cholerea CTXΦ[6] 26673076 [http://parts.igem.org/Part:BBa_K2255013 BBa_K2255013]
VFJΦ(fs2)[7] 1261866 [http://parts.igem.org/Part:BBa_K2255014 BBa_K2255014]
VGJΦ[8] 1260523 [http://parts.igem.org/Part:BBa_K2255015 BBa_K2255015]
Xanthomonas campestris ΦLf[9] 3730653 [http://parts.igem.org/Part:BBa_K2255016 BBa_K2255016]
Xylella fastidiosa XfasM23[10] 6203562 [http://parts.igem.org/Part:BBa_K2255017 BBa_K2255017]
Xanthomonas fucans XacF1[11] 17150318 [http://parts.igem.org/Part:BBa_K2255018 BBa_K2255018]
Table showing the attachment proteins from various filamentous phages.
  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. ↑ 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. ↑ 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).
  4. ↑ 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).
  5. ↑ 5.0 5.1 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).
  6. ↑ 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).
  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. ↑ Chen, J. & Civerolo, E. L. Morphological evidence for phages in Xylella fastidiosa. Virology Journal 5, 75 (2008).
  11. ↑ 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).