Part:BBa_K2230000

CP29-RBS-aeBlue/pLBA169

A vector can be used to transform Lactobacillus acidophilus by chromosomal homologous recombination at the downstream location of slpA (LBA0169), which encodes a surface layer protein A. The aeBlue gene, which is driven by the wide host range and high constitutive promoter CP29, acts as a reporter. The transformed L. acidophilus will express blue color proteins.

Cloning: Firstly, the elements of upstream and downstream recombination sites were replicated from gDNA of Lactobacillus acidophilus and put onto pSB1C3 as a L. acidophilus recombination vector, named pLBA169, at EcoRI and PstI locations. Secondly, to generate as a standard BioBrick part and assembly vector, the part of Promoter CP29-RBS-aeBlue (BBa_K1033280) was cut by EcoRI and PstI and assembled with the vector. Finally, because there’s a SpeI recognition sequence within the 169dn element, site-directed mutagenesis was performed to change a nucleotide to leave a single SpeI site in the BioBrick suffix region. (See the flow chart and data below).

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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 3356
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 3362
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3356
  Illegal XhoI site found at 1642
  Illegal XhoI site found at 2534
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 3356
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 3356
  Plasmid lacks a suffix.
  Illegal XbaI site found at 3371
  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.