Lux I with myc tag under Promoter prmA activated in the presence of PFOS
Pprma(BBa_K2911000)- RBS(BBa_B0034) - LuxI(BBa_C0061) - Myc(BBa_K823036)
Usage and Biology
This part was built to show that LuxI can be expressed under prma promoter, and to compare its expression under prma promoter with/without PFOS. prma promoter is found in Rhodococcus jostii RHA1 (UniProtKB - Q0SJK9) and regulates the transcription of prma, a gene producing a propane monoxygenase. In our project, we rely on this promoter for the detection of polyfluoroalkyl substances, a pollutant found in the Baltic Sea. LuxI is found in Vibrio fischeri (UniProtKB - P12747) and can produce 3OC6-HSL, a lactone that can be used as a signalling molecule for which the sender is LuxI and the receiver is LuxR. In our project LuxI is in E. coli, in the detector module, while LuxR is in Shewanella oneidensis, in the electricity output module.
In our project, part BBa_K3440005 is the final construct to insert in E. coli in order to detect the pollutant perfluorooctanoic acid (PFOS) in the sensing module. We added a myc-tag to the part in order to be able to characterize it by Western Blotting.
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_K3440005, 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 (1159bp) for F5 and F7.
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 F5, but not for F7, which contained a mismatch affecting the aminoacid sequence.
We then proceeded to characterize by Western blotting F5 thanks to the added myc-tag (Figure 3) in order to prove that PFOS could induce production of LuxI (and there signalling molecule 3OC6-HSL). The results did not show any expression of F5 either with our without the pollutants. This was not very surprising given the results obtained with BBa_K344002 in which mCherry could not be produced under induced prma. Our hypothesis is that mcuh bigger quantities of pollutant are needed to create a visible signal, thus this detection part should be improved and made more sensitive in the future.
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 2-3 for the layout of controls and the plasmid respectively and Figures 4-5 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 2 and 4) for comparison, as well as another pollutant that should not affect growth (1,1-dibiphenyl). It appeared that only the highest concentration, 10e-3M, of 1,1-dibiphenyl retarded the growth of E. coli. PFOS did not affect the growth of the culture, although the other pollutant did. We therefore concluded that this was not the reason why the band did not appear on the Western Blot.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal BglII site found at 837
Illegal BglII site found at 875
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 137
Illegal NgoMIV site found at 148
- 1000COMPATIBLE WITH RFC
|origin||prma promoter from Rhodococcus jostii RHA1 and LuxI from Vibrio fischeri|