Overview
In order to test the protection efficacy of melanin, colonies from both melanized E. coli and non-melanized E. coli were exposed to UV radiation and their survival rates were compared. After colonies of engineered bacteria with dual plasmids containing melA (BBa_K4171024) and gadB (BBa_K4171023) respectively turned dark, they would be exposed to UV radiation and receive the same radiation dosage. With the definition of survival rate deriving from the colony forming unit (CFU) ratio between bacteria with and without UV exposure, we can obtain the survival rate by re-culturing. By comparing the follow-up survival rate of bacteria with melanin and without melanin, protection efficacy of melanin could be determined. The same process was conducted to test the protective effect of selenomelanin.
Procedure
Survival Rate Test
Fig. 1. Function test design for melanin and selenomelanin
[click the picture to watch animation]
Dual plasmids containing melA and gadB sequences were transformed into E. coli DH5α, and treated with and without Tyr to yield melanized and non-melanized colonies respectively. After the exposure under UV-B (24 W, 10 min), the medium containing bacteria cells was collected. The medium with bacteria was adjusted to fixed OD600 value to control the total amount of bacteria pigment, followed by serial dilution from 102 to 106. Each diluted ratio sample was dropped on agar plates and incubated overnight. The CFU of different conditions were calculated to quantify the survival rate.
GABA Production
Bacteria supernatant was reacted with 0.4 M boric acid, 6% phenol, 6% sodium nitrate for 5 minutes, and heated at 100℃ for 10 minutes to turn it into a blue liquid. A standard curve was generated by measuring the different concentrations of standards in OD630. GABA production was calculated using this standard curve.
Result 1: Melanin
The graph below shows after 10 minutes of UV exposure, 60% of the melanized cells survived, while only 18% of the non-melanized cells survived, which was less than one-third of melanized cells. This proved melanin has a significant resistance against UV, and it also indicated that the measuring method was feasible.
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Fig. 2. Survival rate of bacteria without melanin and with melanin (A) Survival rate (B) CFU comparison
Result 2: Selenomelanin
The success of the above measuring method built us an available measuring platform. The effect of Se coli was verified through the same process. Se coli function was examined by testing its radiation resistance with the comparison to non-melanized cells as well as melanized cells, and it was expected to have the best survival tolerance under the pressure of radiation among the three groups.
The result suggested that Se coli had the best radiation tolerance towards UV irradiation exposure for an hour (12 W, UV-C) among the three groups. 17.9% of Se coli survived, while only 5% of melanized bacteria and 3.2% of non-melanized bacteria survived respectively. In general, selenomelanized bacteria (Se coli) is 3.6 times more tolerant to radiation than melanized-bacteria, and 6 times than non-melanized ones. This indicates the success of selenomelanin biosynthesis in Se coli.
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Fig. 3. Survival rate of bacteria without melanin, with melanin and with selenomelanin (A) Survival rate (B) CFU comparison
Result 3: GABA Production in the Dual-Plasmid Bacteria
As the result below indicates (Fig. 4), Se coli contains the satisfying efficacy of GABA biosynthesis, in which the concentration of produced GABA surpasses 2.3 g/L, though being inevitably less than the control group. By easy-conducting measurements, it is furthermore proved that Se coli is able to bear the burden of two different sets of plasmids, synthesizing both selenomelanin and GABA in vivo.
Fig. 4. The yield of GABA while producing melanin simultaneously
