Part:BBa_K2230004

RBS-EmR-CP29-RBS-aeBlue/pLBA169

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

Erythromycin is a common selection marker for engineering lactic acid bacteria1. And teams [http://2013.igem.org/Team:Uppsala Uppsala] in 2013 and [http://2016.igem.org/Team:TMMU_China TMMU_China] in 2016 were using it to select the engineered Lactobacillus with erythromycin resistant strains.

Cloning

RBS-EmR which was cut and gel-eluted from RBS-EmR/pSB1C3 (BBa_K2230002) was assembled with CP29-RBS-aeBlue/pLBA169 (BBa_K2230000)

Reference

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

2. Genetic engineering techniques for lactic acid bacteria: construction of a stable shuttle vector and expression vector for β-glucuronidase. Biotechnol Lett. 2014;36(2):327-35.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 4909
  Illegal suffix found in sequence at 1554
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 4909
  Illegal SpeI site found at 1555
  Illegal PstI site found at 1569
  Illegal NotI site found at 1562
  Illegal NotI site found at 4915
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 4909
  Illegal XhoI site found at 3195
  Illegal XhoI site found at 4087
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 4909
  Illegal suffix found in sequence at 1555
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 4909
  Illegal XbaI site found at 4924
  Illegal SpeI site found at 1555
  Illegal PstI site found at 1569
  
• 1000
  COMPATIBLE WITH RFC[1000]