Part:BBa_K2350009

Pigment double-crossover homologous gene recombination bacbone (pPIGBACK)

Part description

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.

Result

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.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

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