Part:BBa_K1445002

High copy BioBricked M13 phagemid vector

This biobrick backbone has the high copy number pUC19 origin of replication and an ampicillin resistance marker.

The part also contains the M13 origin of replication which allows the phagemid to be packaged into M13 phage. Vector does not contain any expressible phage genes; therefore, a helper phagemid is required for phage coat protein expression.

Vector includes the following features:

iGEM VF2 and VR sequencing sites

T7 promoters that flank the MCS

Translational stop sequences (BBa_B0042) that flank the MCS

Transcriptional terminator BBa_B0055 upstream of the MCS

Transcriptional terminator BBa_B0054 downstream of the MCS

Usage and Biology

This vector was packaged into phage using M13K07 as the helper phagemid. The E. coli strain ER2738 was infected with the progeny phage and plated under ampicillin selection.

Figure 1: Packaging efficiency of recombinant phagemid to helper phagemid packaging. A) Sample diluted 1:1000 and grown on 100 ug/mL Ampicillin to select for BBa_K1445002. B) Sample diluted 1:1000 and grown on 50 ug/mL Kanamycin to select for M13K07 helper phagemid. BBa_K1445002 was packaged over M13K07 at a ratio of 136:1.

Significant growth (1496 colonies) was observed for samples plated on LB with ampicillin, proving the capability of vector delivery through phage. Additionally, the same sample was plated on kanamycin to determine the packaging ratio of phagemid to helper phagemid. As shown in Figure 1B), very little growth was present on kanamycin (11 colonies) confirming that BBa_K1445002 was preferentially packaged over the M13K07 helper phagemid at a ratio of 136:1.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2990
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 2996
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2990
  Illegal BglII site found at 2852
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 2990
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 2990
  Plasmid lacks a suffix.
  Illegal XbaI site found at 3005
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 1240