Part:BBa_K2350009

Pigment double-crossover homologous gene recombination bacbone (pPIGBACK)

The vector, pPIGBACK, is used to transform into S. elongatus PCC 7942 with the inserted pigment gene in our project. pPIGBACK contains 5’- and 3’-ends of the neutral site II (NSII), replication origin of pBR322, ampicillin resistance gene, and double terminator BBa_B0015. The strategy we chose to construct the vector is to fuse B0015 and AmpR together first. Secondly, we fused 5’- and 3’-ends of the neutral site II (NSII) with PBR322 replication origin (ORI) together. At last, we ligated two parts together. The aim of constructing this vector is to finish double-crossover homologous gene recombination in S. elongatus PCC 7942. To insert BBa_J04450 with EcoR1 and Pst1 cutting sites, we use site-directed mutagenesis to remove all Pst1 cutting sites in pPIGBACK.

Transformation efficiency of pPIGBACK-CrtZ is 11.4 transformants per μg DNA, and correctness is 52% (10/19), which is quite efficient because the successful rate of gene double-crossingover homologous recombination is low. See figure 1.

Figure 1

Figure 1 is pPIGBACK-CrtZ transformants electrophoresis result. C1~C20 represents the pPIGBACK-CrtZ transformant clone 1 to clone 20, and M represnets 1 kb marker.

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found at 1
  Illegal suffix found at 3880
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 1
  Illegal SpeI site found at 3881
  Illegal PstI site found at 3895
  Illegal NotI site found at 7
  Illegal NotI site found at 1321
  Illegal NotI site found at 3888
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 1
  Illegal BglII site found at 1883
  Illegal BamHI site found at 2180
  Illegal XhoI site found at 1118
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 1
  Illegal suffix found at 3881
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 1
  Plasmid lacks a suffix.
  Illegal XbaI site found at 16
  Illegal SpeI site found at 3881
  Illegal PstI site found at 3895
  Illegal NgoMIV site found at 2101
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 972
  Illegal BsaI site found at 1783