Team:Paris/Transduction overview fusion

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Overview

• Introduction
• A. Fusion
  • A.1 jun/fos
  • A.2 G3P
  • A.3 Snares
• B. Our strategy
• C. Construction

A. Fusion

A.1 Jun/Fos

A.2 G3P

What is the G3P and how could it be a key part in the vesicles-bacteria fusion ?

The viral protein known as G3P is naturally exposed at the surface of the filamentous bacteriophage which enable it to get in the bacteria. The M13 phage has a high affinity for E.coli, and if we could place its G3p on the surface of the vesicles it could activate the fusion with the Outer membrane of the targeted bacteria.

To be sure to target the receiving bacteria we separe the donnor from the receiver with the criterium of the presence or not of pilli, because the G3P need a pillus to start its incorporation process. So the donnor would be pillus negative and the receiver pillus positive.

OmpA-Linker is the second protein required because it is a protein that target any protein that is fuse to it to the surface of the Outer membrane, consequently we fuse G3P with OmpA-Linker

• Infection of Escherichia coli by filamentous bacteriophages as M13, fd, f1, is mediated by the phage gene 3 protein (g3p or pIII). This protein of 406 amino acid residues, has a signal peptide, two N-terminal domains and one C-terminal domain, separated by two flexible glycin-rich linkers. All three domains are indispensable for phage infectivity.
  
• The signal peptide (1-18aa) address the protein to the cell membrane before being cleaved. (We deleted it).
  
• The first N-terminal domain (N1) binds to the bacterial periplasmatic domain of TolA (TolAII - see http://biocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG11007 ), receptor presumably at the inner face of the outer membrane.
  
• The second N-terminal domain (N2) gives recognition of the host cell by binding the F-pilus on the surface of E. coli. F-pilus is encode by the F episome of male E. coli, and is the primary receptor of the host cell.
  
• In fact, N1 and N2 interact with each other to form a blocked di-domain (N1G1N2). The binding of N2 to the tip of the bacterial F-pilus releases N1, which becomes free to interact with its receptor TolA (TolAIII).
  
• The C terminus (CT) of g3p anchors the g3p in the phage coat by interacting with phage coat protein 6, at the tip of the phage. Its seem that phages are released from the bacterial membrane by a two-step mechanism involving a short C-terminal fragment of g3p.
  
• N1, N2 and N3 domain are linked by flexible glycin-rich domains (G1 and G2). G1 is composed of four tandem copies of the sequence Glu-Gly-Gly-Gly-Ser. In a recent study it has been showed that it may have an active role in F-pilus-dependent infection.
  
• Fusion of peptides or proteins to the N-terminus of intact g3p does not compromise infectivity of the phage, but insertion of polypeptides between N2 and N3 appear to reduce the infectivity.
  

Design Notes

• In our project we use g3p as a fusion to OmpA-Linker (BBa_K103996) which need SacI restriction site for inframe fusion.
  
• So we design g3p with SacI site at the N-terminal. SacI (GAGCT^C) site is shared with XbaI (T^CTAGA) in order to have SacI site for fusion and standard sites.
  
• Moreover we decide to suppres the signal peptide (18 first amino acids) which is cleaved in order to conserve the N-ter fusion.
  

• g3p could be found in filamentous bacteriophages like M13, fd, f1, etc... or in phage helper like M13KO7, etc...
  

References

• The Mechanism of Bacterial Infection by Filamentous Phages Involves Molecular Interactions between TolA and Phage Protein 3 Domains. Fredrik Karlsson, Carl A. K. Borrebaeck, Nina Nilsson, and Ann-Christin Malmborg-Hager
  
• Interdomain interactions within the gene 3 protein of philamentous phage. Jean Chatellier, Oliver Hartley, Andrew D. Grifths, Alan R. Fershta, Greg Wintera, Lutz Riechmannb
  
• A prokaryotic membrane anchor sequence: Carboxyl terminus of bacteriophage fl gene III protein retains it in the membrane. Jef D. Boeke AND Peter Model
  

Direct link to our part :

end of SacI site for fusion 1 3
N1 domain 5 205
G1 (Gly-rich linker, EGGGS motif) 206 262
N2 domain 263 655
G2 (Gly-rich linker) 656
CT domain 788 1237
Double stop codon 1238

References

A.3 Snares