Part:BBa_K274001
melanin pigment
Produces dark brown pigment output. The gene (melA) codes for a tyrosinase which produces the pigment from L-tyrosine. Production of the pigment requires the addition of copper and L-tyrosine supplements (the copper acts as a cofactor for the gene product) but no other precursors. The BioBrick sequence includes the native ribosome binding site.
Characterization by iGEM Concordia 2016
iGEM Concordia 2016 aimed to synthesize nanoparticles for the overall goal of equipping cells with "nano-weapons" and having them fight in a microfluidics chip, in our project titled "Combat Cells: League of Enhanced MicroGladiators." One of our methods of nanoparticle synthesis and attachment to cells is titled the recombinant method. Through the use of the protein MelA with its substrate L-DOPA, eumelanin is formed, which forms gold nanoparticles from gold ions in solution.
IGEM Concordia 2016 aimed to improve the characterization of MelA by determining MelA's ability to use tyrosine as a substrate in place of L-DOPA. We cloned MelA's sequence into a plasmid, with MelA expression under the control of a constitutive promoter. This would allow us to determine if MelA had any cytotoxic effects.
After transforming this plasmid into DH5alpha E. coli cells, we set up an experiment to test MelA's ability to use tyrosine as a substrate. We set up untransformed DH5alpha cells and just tyrosine wells as controls, alongside MelA-transformed DH5Alpha cells, all containing 2mM L-tyrosine. The wells contained 180 microlitres of cells at a starting OD600 of 0.5. We placed this plate into a Tecan-Sunrise, which measured the OD450 of the well contents at every 20 minutes for 2.5 days. The value of OD450 is directly related to the amount of melanin being formed in the wells. The following graph represents our obtained data.
Graphed for each of the wells containing DH5alpha cells (transformed with MelA and not transformed) is the average of three replicates. The error bars represent standard deviation.
Tyrosine alone with no cells does not polymerize on its own, since there was no increase over time of OD450. Interestingly, there is a statistically significant difference in the OD450s when comparing the DH5alpha cells without and with MelA transformed in them. The data indicates that, in the presence of constitutive MelA production and 2mM L-tyrosine, more melanin in produced than wild-type /E.coli. This would indicate that 1) WT E. coli can produce melanin using L-tyrosine, and 2) MelA can use L-tyrosine to produce melanin, ever moreso than WT DH5Alpha.
With this data, we intended to perform nanoparticle synthesis with L-tyrosine and MelA-transformed E. coli cells, and have these cells attach the gold nanoparticles to the surface of the E. coli cells using our FhuA-GBP fusion protein (BBa_K2045000), following the protocol created by Tsai et al [1].
[1] Tsai, Y.-J., Ouyang, C.-Y., Ma, S.-Y., Tsai, D.-Y., Tseng, H.-W., & Yeh, Y.-C. (2014). Biosynthesis and display of diverse metal nanoparticles by recombinant Escherichia coli. RSC Adv., 4, 58717-58719. Royal Society of Chemistry. Retrieved from http://xlink.rsc.org/?DOI=C4RA12805B
Characterization by iGEM NCKU_Tainan 2022
NPS-tyrP-Ptrc-melA-B0015
BBa_K4171024 (NPS-tyrP-Ptrc-melA-B0015) is for tyrosinase biosynthesis based on BBa_K274001 with production optimization. BBa_K4171024 is constructed to express MelA (BBa_K274001), the tyrosinase that is essential for melanin production. TyrP (BBa_K2997000) is a transmembrane protein functions as the importer for tyrosine (Tyr). With this composite part constructed, TyrP contributes to the final production of melanin by increasing Tyr consumption.
Fig. 1. Melanin synthesis pathway
Production Optimization
Quantification of TyrP Function
Tyr, the precursor of melanin, serves as an important ingredient in melanin production. With TyrP, the production of melanin was expected to increase due to higher importation of Tyr. To verify this hypothesis, melanin production in both BBa_K274001 and BBa_K4171024 were measured by OD400, which proved that TyrP did play a important role in melanin production optimization.
Fig. 2. Relative melanin production of E. coli with and without TyrP.
As the result shows, melanin production increased significantly with the addition of TyrP.
Culturing Conditions Adjustments
Besides this improvement, other experiments were also conducted to maximize the production of melanin. First, the bacteria were cultured in various growing conditions to find the best condition for culturing.
Tyrosinase requires the cofactor Cu2+ to function properly [1]. However, the concentration of cofactor in previous studies for incubation varied. To determine the best concentration of the cofactor, we added Cu2+ to the bacteria with MelA overexpression when OD600 reached 0.6. The result is shown in Fig. 3 (A).
Furthermore, Since studies have shown that melanin production prefers to occur under 32°C, we compared the efficiency of melanin polymerization under 30°C and 37°C. The result is shown in Fig. 3 (B).
Fig. 3. With the optimization of the Cu2+ concentration (A) and temperature (B) for in vivo melanin production in E. coli DH5α.
As Fig. 3 shows, melanin production was better when cultured in 0.5 mM Cu2+ and 37°C. WIth the optimization of each parameter, melanin production had been promoted.
Selenomelanin Synthesis and Function Test
Once melanin was prepared, Sec was added and they together polymerized into selenomelanin, which showed the best ability to protect microorganisms from radiation than melanin.
Fig. 4. Selenoelanin synthesis pathway
The function of selenomelanin was verified. Selenomelanized bacteria were placed under UV irradiation with the comparison of melanized and non-melanized bacteria as controls to determine if Se coli, the engineered kind of E. coli which produced selenomelanin automatically, was capable to survive under radiation exposure for a longer period.
As Fig. 5 shows, 17.9% of Se coli survived, while only 5% of melanized bacteria and 3.2% of non-melanized bacteria survived respectively. Selenomelanized bacteria (Se coli) is 3.6 times more tolerant to radiation than melanized-bacteria, and 6 times than non-melanized ones.
Fig. 5. Bacteria under exposure to UV-C (A) Survival rate (B) CFU comparison
Characterization by iGEM Leiden 2022
Binanox aimed to synthesize bimetallic nanoparticles with a silver core and golden spikes by overexpressing certain genes in E. coli by using a cell-free system. We chose melA for its ability to form gold nanoparticles.
We obtained the melA gene from the ASKA collection, where the gene is in the plasmid pCA24N. This plasmid was then transformed into E. coli BL21. The strain was induced with IPTG to express MelA, this was shown on an SDS gel. The protein size for melA is 51kDA.
Fig. 1. SDS gel comparing non-induced MelA to induced MelA. 5. protein ladder 8. MelA non-induced. 9. MelA induced with IPTG.
We set up an experiment to test MelA’s ability to form bimetallic nanoparticles in a cell-free system. In this set up we grew the strains in MH broth and either lysed the cells or spun them down and used the supernatant for the production of nanoparticles. The melA strains were compared to the control of E. coli BL21 strains (WT). We also compared the nanoparticle formation in the presence or absence of L-DOPA. The silver and gold was added under the form of AgNO3 and HAuCl4 salts. The absorbance was measured after 24 hours.
Fig. 2. Absorbance graph obtained at 800 nm after addition of gold and silver ions and/or L-DOPA to MH broth media, BL21 supernatant, MelA lysate and a combination of MelA lysate and BL21 supernatant. These readings were taken at 24h after the addition of gold and silver salts, at a ratio of 1.85:1 M.
The graph shows absorbance obtained for MelA at 800 nm. As discussed earlier, MelA uses L-DOPA for metal ion reduction. Thus, L-DOPA was used to test the effect on nanoparticle production in the presence of MelA. The addition of L-DOPA to these exact reactions causes an increase in absorbance at an A800 nm. The wells containing L-DOPA developed an orange color within a few minutes of addition, which may explain the higher absorbance values.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1332
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1184
Illegal BsaI.rc site found at 489
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