Part:BBa_K2491030

RK2 Broad Host Range Vector with Kanamycin Resistance

The goal of this improvement was to replace p15a origin of replication in pSB3K3 (https://parts.igem.org/Part:pSB3K3) with the RK2 ori to generate a shuttle vector as a broad host range vector (i.e. can replicate in E.coli and other microorganisms).

The RK2 origin of replication is a broad-host-range plasmid belonging to the incP incompatibility group that can be maintained in a large number of bacteria. The minimal region for replication and maintenance consists of an origin of replication, oriV, and the plasmid replication initiator protein TrfA protein, that activates oriV. The copy number of RK2 is about 4-7 per cell in E. coli, 3 in Pseudomonas aeruginosa, and 4-7 in Agrobacterium

Proof Of Concept

Clone verification. The following primers annealing in the TRFA region of the RK2 backbone vector and the kanamycin region were used to verify the construct by amplification and sequencing of the backbone: TRFA_F: 5’-CCGACGGATGTTCGACTATTT-3’, TRFA_R: 5’-AGTAAAGCGCTGGCTGAA-3’, KM_F: 5’-AAAGCCGTTTCTGTAATGAAGG-3’, and KM_R: 5’-CCTGTTGAACAAGTCTGGAAAG-3’. The data showed that the 2 regions were present. In addition, cultures containing the RK2 backbone harboring the RFP gene [biobrick part BBa_K2491030 containing biobrick part BBa_J04450] showed a red pellet (right vial) compared to a control (left vial) without the RFP gene.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found at 1508
  Illegal EcoRI site found at 3141
  Illegal XbaI site found at 3156
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 1508
  Illegal EcoRI site found at 3141
  Illegal NheI site found at 1384
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 1514
  Illegal NotI site found at 3147
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 1508
  Illegal EcoRI site found at 3141
  Illegal XhoI site found at 177
  Illegal XhoI site found at 1020
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 1508
  Illegal suffix found at 2
  Illegal EcoRI site found at 3141
  Illegal XbaI site found at 3156
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 1508
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3141
  Illegal XbaI site found at 1523
  Illegal XbaI site found at 3156
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NgoMIV site found at 1552
  Illegal NgoMIV site found at 2143
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.