Part:BBa_K390000

pSB1A3_Int (BamHI, XhoI, NdeI, NheI added)

This part is based on pSB1A3, with BamHI, XhoI, NdeI and NheI sites added through site-directed mutagenesis. This plasmid backbone retains compatibility with BioBricks (except with RFC 21 - BamBgl), its antibiotic resistance, and its high copy number in E. coli.

The additional restriction sites allow the addition of DNA sequences homologous to genomic sequences in Synechocystis sp. PCC 6803 and other species, enabling the plasmid to be used to integrate the BioBrick construct and an antibiotic resistance gene into the chromosome at a specific location through homologous recombination. Integration sequences can be designed so that the BioBrick construct is added in the middle of a gene knocking the gene out in the process, or between two genes without interfering with native expression.

The BioBrick construct can be assembled in E. coli cells without changes to the standard procedure, and when completely assembled, the plasmid can be introduced into the target species.

Synechocystis sp. PCC 6803 naturally takes up DNA from its environment, and if sequences are homologous to regions of the genome, will naturally undergo homologous recombination. Other species may lack one or both of these abilities, and the addition of another plasmid carrying a recombinase gene may be necessary for genomic integration.

Recombination region RS2 can be added with a NdeI and NheI digestion and ligation. To add RS1, you must first make a fusion of the resistance gene you want to add to the RSI region (using a HindIII site), and then insert that complex with a BamHI and XhoI digestion and ligation (see figure below for final plasmid appearance).

Presence of restriction sites was verified by gel electrophoresis, and sequencing verification is pending.

Figure 1. Plasmid backbone BBa_K390000 before addition of recombination regions and resistance gene (left) and after (right). Final plasmid appearance example shown is part BBa_K390300.

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 2136
  Illegal NheI site found at 230
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 2142
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2136
  Illegal BamHI site found at 1992
  Illegal XhoI site found at 2009
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 2136
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 2136
  Plasmid lacks a suffix.
  Illegal XbaI site found at 2151
  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.rc site found at 1175