Team:Uppsala/Results naph

Naphthalene pathway

Lifting of naphthalene pathway

Our concrete goals in the lab were to extract the naphthalene degrading pathway with genes NahA to NahF both with and without its native promoter through PCR. As is confirmed by colony PCR and electrophoresis, the pathway was successfully lifted from the Nah7 plasmid.

Assembly of promoter, pathway and backbone

The PCR product, namely the naphthalene pathway, was successfully assembled into a standard iGEM backbone through 3A assembly, confirmed through colony PCR, and two different promoters were added through standard assembly due to unwanted restriction sites in the pathway sequence.

Sequencing and proteomics

Due to financial and temporal limitations, no sequencing or proteomic studies could be performed.

Plates with naphthalene in lid

To assess differences in survivability between the cells containing the naphthalene degrading pathway and negative control cells containing an RFP-coding gene. Plates were split in two parts, one containing the naphthalene degrading bacteria and one with the negative control. Fixed amounts of naphthalene crystals ranging from 50 mg to 2 g were then placed in the lid of each plate, to determine the difference in growth rate. However, no visible difference was observed. These results are consistent with results from experiments with liquid cultures where naphthalene was also supplied in gas form.

Figure 4: shows the survivability rates of modified DH5α E.coli in comparison to the negative control at a different naphthalene concentrations. Increasing concentrations from left to right. Modified DH5α are to the left of each plate, negative control on the right.

Cultures with naphthalene

The upper naphthalene pathway was introduced into both DH5α and BL21DE3 strains of E.coli, where DH5α is a cloning strain and BL21DE3 is a strain optimized for protein expression. In the DH5α cells a medium strength promoter was used to put less strain on the cells, whereas in BL21DE3 a strong promoter could be used to increase the expression level of the desired enzymes.

Both strains were grown in liquid culture with either no naphthalene, naphthalene directly supplied to the medium, or with naphthalene supplied in gas form as shown in figure 5. The OD of the cultures were measured after 24 and 48 hours at both OD₆₀₀ (for cell growth) and for OD₃₀₃ (for the presence of salicylate). The graphs in figures 6 to 11 show the experimental values obtained by spectrometry.

Figure 5 shows liquid cultures grown under three different conditions. From left to right: without naphthalene, with 500 mg of naphthalene dissolved directly into the medium and with 500 mg of naphthalene in an eppendorf tube suspended above the culture. The last picture shows a liquid culture with naphthalene dissolved directly into the medium from below.

Figure 6 shows the average OD₆₀₀ values of two different experiments, after the cells had been grown for 24 hours. The cells were grown under three different conditions; without naphthalene, with 500 mg of naphthalene dissolved directly into the medium and with 500 mg of naphthalene in an eppendorf tube suspended above the culture. The values displayed are correspondent to constructs: DH5α-pSB3C5-Upper naphthalene pathway with promoter BBa_J23101, BL21DE3-pSB3C5-Upper naphthalene pathway with promoter BBa_J23110 and DH5α-pSB1C3-RFP insert.

Figure 7 shows the OD₆₀₀ value for one experiment, after the cells have been grown for 48 hours. The cells were grown under three different conditions; without naphthalene, with 500 mg of naphthalene dissolved directly into the medium and with 500 mg of naphthalene in an eppendorf tube suspended above the culture. The values displayed are correspondent to constructs: DH5α-pSB3C5-Upper naphthalene pathway with promoter BBa_J23101, BL21DE3-pSB3C5-Upper naphthalene pathway with promoter BBa_J23110 and DH5α-pSB1C3-RFP insert.

Figure 8 shows the average OD₃₀₃ values of two different experiments, after the cells have been grown for 24 hours. The cells were grown under three different conditions; without naphthalene, with 500 mg of naphthalene dissolved directly into the medium and with 500 mg of naphthalene in an eppendorf tube suspended above the culture. The values displayed are correspondent to constructs: DH5α-pSB3C5-Upper naphthalene pathway with promoter BBa_J23101, BL21DE3-pSB3C5-Upper naphthalene pathway with promoter BBa_J23110 and DH5α-pSB1C3-RFP insert.

Figure 9 shows the average OD₃₀₃ value of one experiment, after the cells have been grown for 48 hours. The cells were grown under three different conditions; without naphthalene, with 500 mg of naphthalene dissolved directly into the medium and with 500 mg of naphthalene in an eppendorf tube suspended above the culture. The values displayed are correspondent to constructs: DH5α-pSB3C5-Upper naphthalene pathway with promoter BBa_J23101, BL21DE3-pSB3C5-Upper naphthalene pathway with promoter BBa_J23110, and DH5α-pSB1C3-RFP insert.

Figure 10 shows the average OD₃₀₃ values of the supernatant from two different experiments, after the cells have been grown for 24 hours. The cells were grown under three different conditions; without naphthalene, with 500 mg of naphthalene dissolved directly into the medium and with 500 mg of naphthalene in an eppendorf tube suspended above the culture. The values displayed are correspondent to constructs: DH5α-pSB3C5-Upper naphthalene pathway with promoter BBa_J23101, BL21DE3-pSB3C5-Upper naphthalene pathway with promoter BBa_J23110 and DH5α-pSB1C3-RFP insert.

Figure 11 shows the average OD₃₀₃ value of one experiment, after the cells have been grown for 48 hours. The cells were grown under three different conditions; without naphthalene, with 500 mg of naphthalene dissolved directly into the medium and with 500 mg of naphthalene in an eppendorf tube suspended above the culture. The values displayed are correspondent to constructs: DH5α-pSB3C5-Upper naphthalene pathway with promoter BBa_J23101, BL21DE3-pSB3C5-Upper naphthalene pathway with promoter BBa_J23110 and DH5α-pSB1C3-RFP insert.

All the cultures containing naphthalene supplied directly to the medium showed a clear trend of significantly lower growth rates in the negative control compared to the cells containing the pathway. This is to be expected as naphthalene is toxic to the cells and the negative control is unable to degrade it. After 24 hours, the BL21DE3 had grown substantially more than both the negative control and the DH5α cells. This is not surprising as this strain is better at producing the enzymes of our pathway, and thus should be better at degrading naphthalene. However, after 48 hours the DH5α culture had reached similar levels of optical density as the stabilized BL21DE3 cultures. A plausible reason is that the strain still has the pathway, though the level of expression is lower than in BL21DE3.

The naphthalene supplied in gas form did not appear to affect the cell growth at all. However, in cultures without naphthalene the negative control grew somewhat better than cells with the construct. The reason is probably that the negative control did not have to maintain a large unnecessary plasmid.

The presence of salicylate both directly in the culture and with the cells removed, was measured at OD₃₀₃. Similar results were observed at 24 hours as in above described experiment, with higher salicylate levels in BL21DE3 compared to DH5α and the negative control. After 48 hours DH5α had approximately as high levels of salicylate as BL21DE3. Levels of salicylate, however, appear to be far higher in cultures without naphthalene or with naphthalene in gas form, disagreeing with our original hypothesis. This may be due to interfering cells or substances. Regardless, results still show a clear trend both in salicylate levels and in cell growth, indicating that our construct is indeed being expressed and is degrading naphthalene to salicylate.