Part:BBa_K3440017

bphR2 under bphR1 promoter

Pconst(BBa_J23100) - RBS(BBa_B0034) - bphR2 mutated(BBa_K1413021) - Myc(BBa_K823036) - DT(BBa_B0015) - Pbphr1(BBa_K1155001) - RBS(BBa_B0034) - mCherry(BBa_J06504)

Usage and Biology

bphR1 promoter is found in Pseudomonas pseudoalcaligenes KF707 and regulates the transcription of a series of biphenyl catabolic genes. In our project, we rely on this promoter for the detection of polychlorinated biphenyls, pollutants similar to dioxins and found in the Baltic Sea. In particular, in our final system, bphR1 promoter is activated by the complex bphR2:PCB, with bphR2 expressed constitutively and PCB coming from the analysed sea water. bphR2 gene from the same strain produces bphR2, which together with PCB pollutants (polychlorinated biphenyls) can activate bphR1 promoter.

This part can be used to prove that bphR1 can be activated by expression of bphR2. bphR2 is constitutively expressed and can induce bphR1 promoter, under which a reporter mCherry was placed. Induction can be enhanced in the presence of polychlorinated biphenyls (PCB), which can form an activating complex with bphR2. mCherry is a fluorescent reporter gene originally found in Anaplasma marginale (UniProtKB - X5DSL3). It gives a red color (emission at 615nm) when excited at 560nm.Here the mCherry reporter was added to monitor activation of the bphR1 promoter. A myc tag was added to monitor production of bphR2.

Characterisations

This part was assembled from BBa_K3440001 and BBa_K3440009 and inserted into a plasmid and transformed E. coli TOP 10 cells with it. However after colony picking, colony PCR and sequencing, we realised that we had switched some samples and therefore did not characterise the part we thought we were working with.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 996
  Illegal BamHI site found at 1301
  Illegal BamHI site found at 1384
  Illegal XhoI site found at 1191
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 536
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 320
  Illegal SapI.rc site found at 1284