Part:BBa_K3142002

Acid-induced promoter and repressor lacR of lac operon

The rcfB promoter, a member of the CRP-FNR positive regulation family, was amplified from chromosomal DNA lactis subsp. lactis IL1403. It is an important part of the P170 promoter regulated by acid. Lac repressor (LacR) and operator sequences (O1 and O2) is part of the lactose operon derived from Lactobacillus delbrueckii. O1 and O2 may serve as operators for the binding of the lac repressor (LacR) to prevent repressing the expression of downstream genes.When the external pH value declines to 5.5, rcfB promoter will be highly upregulated. Along with that, the LacR gene, which is downstream of rcfB will be highly transcribed. The large translation of LacR, the repressor of lac operon in Lactobacillus delbrueckii, will inhibit the use of lactose in the bacteria.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 9
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 9
  Illegal NotI site found at 15
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 9
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 9
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 9
  Illegal XbaI site found at 24
  Illegal NgoMIV site found at 149
  Illegal NgoMIV site found at 1052
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 989
  Illegal SapI.rc site found at 718
  

SZPT-China

Verification by A11Sg-AHPMprotein expression

First, we constructed a vector that regulates the expression of a protein of interest through the PrcfB promoter. The vector has the characteristic that when the pH is less than 5.5, the PrcfB promoter strongly expresses the LacR, and the LacR binds to the O1/O2 site to repress the production of the target protein. When the pH is greater than 5.5, the PrcfB promoter is weakly expressed, and the protein of interest can be expressed without being repressed. Fig 1 shows the regulation mechanism.

Reference:

• Madsen S M, Arnau J, Vrang A, et al. Molecular characterization of the pH-inducible and growth phase-dependent promoter P170 of Lactococcus lactis. Molecular Microbiology, 1999, 32(1):75-87.
• Søren M. Madsen, Thomas Hindré, Jean-Paul Le Pennec, et al. Two acid-inducible promoters from Lactococcus lactis require the cis-acting ACiD-box and the transcription regulator RcfB. Molecular Microbiology, 2005, 56(3):735-746.
• Ismail AKYOL, Ugur COMLEKCIOGLU, Asuman KARAKAS, Kalbiye SERDAROGLU, Mehmet Sait EKINCI, Emin OZKOSE. Regulation of the acid inducible rcfB promoter in Lactococcus lactis subsp. Lactis. Annals of Microbiology, 2008, 58 (2) 269-273.