Part:BBa_K3440018

PCB detection

Pconst(BBa_J23100)-RBS(BBa_B0034)-bphR2(BBa_K1413021)-Myc(BBa_K823036)-DT(BBa_B0015)-Pbphr1(BBa_K1155001)-RBS(BBa_B0034)-LuxI(BBa_C0061)

Usage and Biology

This is the final construct to detect a PCB and consequently produce a quorum sensing molecule. bphR2 is produced constitutively and can bind to a PCB. The complex PCB:bphR2 can activate bphR1 promoter, under which LuxI is placed. LuxI produces the 3OC6-HSL quorum sensing molecule.

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 insertion into a pSB1C3 plasmid and transformation of E. coli TOP10 cells using heat shock, we picked several colonies corresponding to this part and PCR-amplified them using the VR and VF2 primers contained in the plasmid backbone pSB1C3.

We then ran electrophoretic gels at 180V for 30 mins (Figure 1). Ten colonies were picked from the plate and we obtained five bands for which the band height corresponded to the expected length of the construct (2493bp).

Figure 1: Colony PCR gel for BBa_K3440018

The colonies for which the size seemed correct were sent for sequencing at Microsynth AG. The sequence obtained for colony 4 corresponded to the expected part, but it was not fully sequenced due to the limitations of the Sanger sequencing method.

This construct was further analysed by Western Blot to check whether we obtained protein expression of bphR2 thanks to the added myc-tag (Figure 2). Additionally, 10e-4 M 1,1-biphenyl was added to BBa_K3440018 to compare the differences in protein expression. No bands of the correct size (35.1 kDa) were obtained for BBa_K3440018 in uninduced nor induced conditions, therefore we could not prove that this construct can express bphR2 constitutively.

Figure 2: Western blot with p3.2 and p3.2+biphenyl as BBa_K3440018

We checked that a potential toxicity of the plasmid for E .coli was not the reason behind proteins not appearing on the Western Blot by making viability tests (see Figures 3-4 for the layout of controls and the plasmid respectively and Figures 5-6 for the results of controls and the plasmid). We made growth curves by measuring OD600 of E. coli containing the part and different concentrations of pollutants. A control E. coli TOP10 strain was used as a control (Figures 3 and 5) for comparison, as well as another pollutant that should not affect growth (PFOS). It appeared that only the highest concentration, 10e-3M, of 1,1-dibiphenyl retarded the growth of E. coli. We therefore concluded that this was not the reason why the band did not appear on the Western Blot.

Figure 3: Layout of the viability test for E. coli TOP 10 (control)

Figure 4: Layout of the viability test for BBa_K3440018 (noted Plasmid 3.2)

Figure 5: Growth curves for E. coli TOP10 (control)

Figure 6: Growth curves for E. coli containing BBa_K3440018 and different concentrations of pollutants

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 BglII site found at 2108
  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