Part:BBa_K4727003

M13 standard phagemid

This part, is a new backbone intended to be useful in the production of M13 bacteriophages carrying a genome of desire. It has been developed starting from a commercially available phagemide (pTZ19R) that has been deleted of all the prohibited restriction sites. Following this, BioBrick RFC[10] standard prefix and suffix were added. Any part of interest can now be easily cloned between the prefix and suffx in order to trasduce the cell if interest. It has been developed with the aim of being used together with part BBa_K4727002 or any other M13 helper phage. The phagemid and the helper plasmid must be co-transformed in E. coli, thus allowing the production of phage particles. Its inability to propagate within host cells due to the absence of phage protein encoding genes, ensures the prevention of unintended dissemination of engineered constructs. Whereas, its adaptability is a remarkable feature - it can be easily tailored to the specifics of individual cases, thus presenting a versatile and readily adjustable platform tailored to different purposes.

Usage and Biology

This newly introduced phagemid has been used to produce recombinant phage particles bearing an RFP expression cassette (BBa_I13521), by using the helper plasmid assembled by Chasteen et al.[1]. This first attempt was made to assess the ability of the phagemid to be encapsidated inside the phage particle. After producing and purifying the phages infection of E. coli TOP10 F’ cells has been attempted. Following the infection the bacteria have been plated on selective LB agar plates and incubated overnight at 37 C. The following day colonies could be seen on the plate with a weak expression of RFP.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found at 623
  Illegal suffix found at 645
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 623
  Illegal SpeI site found at 646
  Illegal PstI site found at 660
  Illegal NotI site found at 629
  Illegal NotI site found at 653
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 623
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 623
  Illegal suffix found at 646
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 623
  Plasmid lacks a suffix.
  Illegal XbaI site found at 638
  Illegal SpeI site found at 646
  Illegal PstI site found at 660
  Illegal NgoMIV site found at 127
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI.rc site found at 2026
  Illegal SapI site found at 943
  

References

[1] Chasteen, L., Ayriss, J., Pavlik, P. & Bradbury, A. R. M. Eliminating helper phage from phage display. Nucleic Acids Res. 34, e145–e145 (2006).