Results

• Enzymatic degradation
• Naphthalene pathway
• Biosurfactants

Enzymatic degradation

Naphthalene pathway

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

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.

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.

The PCR product 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.

To assess differences in survivability between 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.

The upper naphthalene pathway was introduced into both DH5α and BL21 strains of E.coli, where DH5α is a cloning strain and BL21 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 BL21 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.

All cultures containing naphthalene supplied directly to the medium show 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 BL21 have 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 BL21 cultures. A plausible reason is that the strain still has the pathway, though the level of expression is lower than in BL21.

The naphthalene supplied in gas form does 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 does not have to maintain a large unnecessary plasmid.

The presence of salicylate both directly in culture and with cells removed was measured at OD₃₀₃. Similar results were observed at 24 hours as in above described experiment, with higher salicylate levels in BL21 compared to DH5α and the negative control. Once again, after 48 hours DH5α had approximately as high levels of salicylate as BL21. 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.

Biosurfactants

Figures 3 and 4 shows bands for each construct approximately as expected according to table 1. All biobrick constructs were verified by Sanger sequencing.

Red fluorescent color expression of cells from figure 5 indicates that the mono-rhamnolipid gene construct is working, in effect the genes rhlA and rhlB are transcribed.

Table 2 and figure 6 displays data of drop expansion test with standard mono-rhamnolipids (0, 0.2, 0.4, 0.6 1 and 1.6 mg/ml). Table 3 and figure 7 displays the data of drop expansion test of LB medium, supernatant extracted from E.coli BL21DE3 with BBa_K1688000 respectively untransformed and supernatant extracted from E.coli DH5α with BBa_K1688000 respectively untransformed.

Table 2 shows that a higher concentration of mono-rhamnolipids causes the drop to expand more and collapse faster. This verifies that presence of rhamnolipids can be indicated from drop collapse tests. The drop from sample BBa_K1688000 in BL21 from table 3 collapsed after 30 seconds and expansion of drop diameter was 120% within 5 minutes from 1 cm to 2.2 cm which indicate presence of biosurfactant. The drop from sample BBa_K1688000 in DH5α collapsed and diameter expansion of drop was 90% after 20 minutes. This indicates some presence of biosurfactants. As expected the test indicate that BBa_K1688000 was more expressed and rhamnolipid production was higher in BL21DE3 than in DH5α as BL21DE3 is good for protein expression. The negative controls, LB medium and un-transformed BL21DE3 and DH5α showed very little expansion or no expansion, which is expected as they do not produce biosurfactants.

The appearance of halos around the colonies on CTAB plates, figure 8 indicates the expression of rhamnolipids.

Clear spots were detected in lane 2, 4, 5 and 6 in figure 9 corresponding to the sample extracted from BL21DE3 cells with biobrick BBa_K1688000 and standard mono-rhamnolipid 10, 30 respectively 50 μg. The detection spot of BBa_K1688000 had a retention factor 0,82, the same or similar retention factor as the detection spots for standard mono-rhamnolipids (table 4), which confirms mono-rhamnolipid synthesis by BBa_K1688000 in BL21DE3 cells.

Negative control; BL21DE3 un-transformed in lane 1 (figure 9) showed no spot which is expected as BL21DE3 do not produce biosurfactants naturally. P. putida as a positive control showed no spot. This might be because of too low concentration of rhamnolipids in sample, problems with extraction of rhamnolipids or sample contamination. Low concentration of rhamnolipids in supernatant might be because of used medium and growth conditions.

Although our tests, drop collapse test, CTAB test and TLC, indicated the presence of biosurfactants, rhamnolipids, and mono-rhamnolipids respectively, we still have to perform quantitative assays such as mass spectrometry, HPLC, etc. to identify the concentration of rhamnolipids produced. The data that could be obtained from these assays will help in maintaining the bacterial growth in the degradation reactor. Our future plan is that biosurfactant strains will be used together with the strains that expresses the PAH degrading enzymes. The biosurfactants will break down the clustered PAHs and make them available to degrading enzymes for an efficient degradation.