Part:BBa_K2230000

CP29-RBS-aeBlue/pLBA169

The 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.

Design

To engineer Lactobacillus acidophilus by chromosome integration through homologous recombination, the selection of integration site is very important for successful recombination and not disturbing bacterial internal metabolism. Based on the method created by Grace L. Douglas and Todd R. Klaenhammer (Appl Environ Microbiol. 2011), the region between slpA gene (LBA0169) stop codon and the terminator was chosen as the intergenic insertion location. The gene of slpA encodes a surface-layer protein with a strong constitutive promoter activity, which can also drive the expression of the inserted gene.

Cloning

Firstly, the elements of upstream and downstream recombination sites were amplified 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).

Reference

Directed chromosomal integration and expression of the reporter gene gusA3 in Lactobacillus acidophilus NCFM. Appl Environ Microbiol. 2011;77(20):7365-71.

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.