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Methods: First, the MelA gene plasmid was placed into E. coli, via transformation by heat shock. Standard lab protocols provided in the BioE44 class were used. These cells were grown in LB + KAN plates, as kanamycin resistance was programmed into the MelA gene plasmid. Additionally, untransformed bacteria was plated on an LB + KAN plate to ensure no contamination from other plasmids had occurred. After transformation occurred on the LB + KAN plates, colonies were picked and glycerol stocks were created following standard lab protocol. An initial dynamic range experiment was performed in order to test the range of concentrations of Copper sulfate and L-tyrosine, necessary cofactors for the production of melanin. Additionally, 500µM of IPTG was used across all experiments for the promoter to be properly induced 4. EZ Rich Defined Medium was used across all experiments.

Figure 4

Figure 4 shows the plate map used for the initial dynamic range experiment. A range of concentrations of Copper sulfate was used from 10µM to 1280µM. Concentrations (in g/L) of L-tyrosine were 0.1, 1.0, and 10.0. The 12th column contained no bacteria and was intended to be used as a negative control against the experiment. A spectrophotometer set at an absorbance of 475 nm, the ideal absorbance for the detection of melanin2, was used to measure OD. The blank’s OD was subtracted from each well’s measurement to ensure accurate results. Following the initial dynamic range experiment, a second dynamic range experiment was conducted with the same concentrations of Copper sulfate, but lower concentrations of L-tyrosine and a more complete set of negative controls. Figure 5 shows the plate map for the 2nd dynamic range experiment.

Figure 5

Following the successive inconsistencies in the Dynamic Range experiments conducted, a Western Blot was performed in an attempt to find if the protein Tyrosinase was being produced in any quantity. IPTG concentrations of 0, 10, 50, 100, and 1000µM were used to detect protein production. Western Blot analysis of Tyrosinase using anti-Histidine antibodies was used with the intention of detecting large quantities of Tyrosinase in the cell. The standard Western Blot protocol adapted from Endy Lab’s protocol6 was followed.

Results: The results of our first Dynamic Range experiment were inconclusive, with insignificant levels of melanin production being found and a strange dose-response graph, shown in Figure 6, because of the high growth in low levels of both Copper sulfate and L-tyrosine, as compared with low growth in high levels of Copper sulfate and L-tyrosine.

Figure 6

As can be seen in Figure 6, we found that for the L-tyrosine concentration of 0.1 g/L, there was high growth at a low CuSO4 concentration, but as this concentration increased, the growth subsequently decreased. The results from the L-tyrosine concentration of 1.0 g/L were inconclusive, with some negative values occurring after the negative control was subtracted off, indicating some form of contamination. The L-tyrosine concentration of 10.0 g/L showed a promising correlation between Copper sulfate concentration and the OD475, but the OD values were so low and similar that no significant conclusion could be drawn. We were unsure why this result occurred, but several potential explanations could be proposed, most prominently including the L-tyrosine falling out of solution. Previous research attempting to express melanin using the MelA gene recommended immediate addition of L-tyrosine prior to experimentation and for this first experiment, we added L-tyrosine to the media the night before and maintained it at 4°C overnight. When the media was taken out to be used in the morning, the solution of the 1.0 g/L concentration was cloudy, indicating that the L-tyrosine had fallen out of solution and crystallized, thus no melanin could have been produced. Following this first experiment, we conducted another Dynamic Range experiment in an attempt to rectify the previous issues of the presence and concentration of L-tyrosine. The data and dose-response graph, shown in Figure 7, was much more informative than the initial dynamic range experiment we ran, but the results were still difficult to explain.

Figure 7

The L-tyrosine concentration of 0.01 g/L had the least amount of melanin production and production continued to decrease as the Copper sulfate concentration increased. At 0.1 g/L, production consistently fell with an increasing Copper sulfate concentration, and for 1.0 g/L, there was a steady decrease in production until 1280µM, when there was a sharp increase in melanin production. These results gave the appearance of melanin production, particularly with a low Copper sulfate concentration, but no melanin could be detected by the eye when looking at the plate. A common pattern among all concentrations of L-tyrosine was the decreased melanin production as the Copper sulfate concentration increased, suggesting that a heightened CuSO4 concentration could be toxic to E. coli.

After both of the Dynamic Range experiments gave inconclusive results, we moved on to conducting a Western Blot to see if the MelA gene was being expressed and, consequently, if the enzyme Tyrosinase was being produced. The results of our Western Blot, seen in Figure 8, revealed that our desired protein (68 kDa) was being produced in extremely limited quantities at 100 and 1000µM of IPTG, but another protein was being produced at 0, 10, and 50µM that was about 19 kDa. We are unsure what protein was being produced at these limited concentrations of IPTG, but it could have been conflicting with the expression of the MelA gene and that could be a potential reason why melanin was not being produced in significant quantities. We expected the 0µM of IPTG to be our negative control for the Western Blot, but it also expressed the 19 kDa protein, leading us to the conclusion that this comparatively small protein is not inducible by the T5 promoter we chose, and could potentially always be conflicting with MelA expression.

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UNIQ8891e00cd071da0f-partinfo-00000000-QINU UNIQ8891e00cd071da0f-partinfo-00000001-QINU

Stanford Location

Plasmid name: pBlack

Organism: E. coli

Device type: Reporter

Glycerol stock barcode: 0133011949

Box label: BioE44 SP17