Part:BBa_K3440001

mCherry under bphR1 promoter

PbphR1(BBa_K1155001) - RBS(BBa_B0034) - mCherry(BBa_J06504) - DT(BBa_B0015)

Usage and Biology

Produces mCherry under bphR1 promoter, which can be activated by bphR2:Polychlorinated biphenyl.

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. 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.

This construct was used by our tram to determine the leakiness of bphR1 promoter.

Characterization

Due to the pandemics, we haven’t been able to use biobricks to create the iGEM Stockholm 2020 parts. Those parts were ordered as gene blocks from Integrated DNA Technologies Inc.. As a result, the sequences of the biobricks used are the same, but the scars between biobricks might differ, as well as the final size of the part.

After heat shock transformation into E. coli TOP10 cells and incubation (plate on figure 1), we picked colonies of BBa_K3440001, PCR amplified them with the VF and VR2 primers found in the plasmid backbone pSB1C3 and ran electrophoresis gels of the products at 180V for 30 mins (Figure 2). The gel showed two bands corresponding to the expected size of BBa_K3440001 (1492bp), and they are denoted B7 and B8. Thus, we prepared glycerol stocks and plasmid preparations of B7 and B8, and we send them both for sequencing at Microsynth AG. The sequence obtained by Sanger sequencing corresponded to the expected part.

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  Figure 1: Transformation plate of BBa_K3440001 (noted B)
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  Figure 2: Colony PCR gel for BBa_K3440001 (noted B)

We then proceeded to characterise this part by fluorescence intensity measurements, since it contains a reporter mCherry. Our aim was to obtian the leakiness of bphR1 promoter and see if it could be induced by PCB pollutants (1,1-dibiphenyl in DMSO at 10e-5M)(Figure 3).The measurements were calibrated with OD600 absorbance measurements performed on a plate reader so that the intensity would only reflect expression of GFP not be affected by the number of plasmids. As expected, fluorescence was very low for both uninduced bphR1 promoter and bphR1 induced with only 1,1-dibiphenyl, since induction should only appear for biphenyls complexed with bphR2 protein.

Figure 3: Fluorescence measurements for BBa_K34440001 (B) and BBa_K3440002 (C), uninduced and with pollutants

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 156
  Illegal BamHI site found at 239
  Illegal XhoI site found at 46
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 139