Part:BBa_K3440002

mCherry under prma promoter

Pprma (BBa_K2911000) - RBS(BBa_B0034) - mCherry (BBa_J06504) - DT(BBa_B0015)

Usage and Biology

This part produces mCherry under prma promoter, which can be activated by PFAS/PFOA. mCherry is a fluorescent reporter gene originally found in Anaplasma marginale [1]. It gives a red color (emission at 615nm) when excited at 560nm. prma promoter is found in Rhodococcus jostii RHA1 [2] and regulates the transcription of prma, a gene that produces propane monoxygenase. In our project, we rely on this promoter for the detection of polyfluoroalkyl substances, a pollutant found in the Baltic Sea. Literature [3] shows that perfuloroalkyl substances activate the prma promoter. In particular, in our final system, prma promoter is activated by perfluorooctanoic acid.

In our project, we use the BBa_K344002 part in order to test the leakiness of prma 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 of the pSB1C3 plasmid containing the BBa_K3440002, we picked colonies from plates (Figure 1) and PCR amplified them with primers VF and VR2. We ran gels of the product at 180V and for 30 mins (Figure 2). We obtained the expected size for the bands (1361bp) for C1,C2,C3 and C5.

We therefore prepared plasmid preparations and glycerol stocks of those and sent them for sequencing to Microsynth AG. The sequence obtained corresponded to the expected part for C3.

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  Figure 1: Transformation plate of BBa_K3440002 (noted C)
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  Figure 2: colony PCR Gel for BBa_K34440002 (noted C on the gel) and BBa_K3440003 (D)

We then proceeded to test the leakiness of prma promoter thanks to the mCherry reporter added in the part. We measured fluorescence intensity (excitation 560nm, emission 615nm) of mCherry for C3 with (1.56 μM) and without PFOA and calibrated the values with OD600 measurements. The obtained results (Figure 3) show, as expected, very low intensity for the uninduced promoter. However, the induced promoter did not seem to give fluorescence. Literature suggests that concentrations of 2mg/L (which is much higher than what the Baltic sea contains) can activate the promoter, which might expalin our results.

Figure 3: Fluorescence measurements for BBa_K34440001 (B) and BBa_K3440002 (C)

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 137
  Illegal NgoMIV site found at 148
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

[1] Uniprot entry X5DSL3

[2] Uniprot entry Q0SJK9

[3] https://mountainscholar.org/bitstream/handle/11124/166686/Weathers_mines_0052E_10892.pdf?sequence=1