Experiment 1: Melanin Expression in Escherichia coli

We have isolated melanin-producing Pseudomonas strains from environmental samples. Next, we aim to express the genes responsible for melanin production in E. coli and use engineered E. coli to produce melanin.

Design

We plan to express the relevant melanin production genes in E. coli. The genes we selected are based on the melanin-producing strains we isolated from environmental samples. We reviewed the literature and designed the melanin synthesis genes from the Pseudomonas genus [1-2].

Build

We constructed an E. coli expression vector containing the Pseudomonas 4-hydroxyphenylpyruvate dioxygenase gene. This gene was placed under the control of a T7 or arabinose promoter, and expression was induced using IPTG or arabinose.

Figure 1  The construction of 4-hydroxyphenylpyruvate dioxygenase gene

Test

We tested the expression of 4-hydroxyphenylpyruvate dioxygenase in E. coli. On solid LB plates and in liquid LB medium, melanin production was visibly observed.

Figure 2  Melanin production

Learn

We successfully expressed the 4-hydroxyphenylpyruvate dioxygenase gene in E. coli, which produced melanin in LB medium. This result provides a basis for the next step of our experiments.

Experiment 2: Interaction Between Green Algae and Melanin-Producing Bacteria

Our goal is to examine the radioprotective effects of melanin on photosynthetic organisms and planktonic algae. We are focusing on two aspects: the protective effect of melanin-producing microorganisms on photosynthetic organisms (radiation protection, light/dark response), and the influence of photosynthetic organisms on melanin-producing bacteria (oxygen supply, oxygen sensing).

Design

We designed co-culture systems between algae and bacteria, including in liquid medium, on plates, and within hydrogels or other transparent porous systems. These setups will allow us to study algal-bacterial symbiosis and their interactions[3].

Figure 3  A simple schematic diagram of the co-cultivation system between microalgae and Pseudomonas or E. coli expressing melanin.

Test

Oxygen plays a crucial role in melanin production, which involves multi-step reactions (both enzymatic and purely chemical) that are oxidative in nature. Typically, in laboratory settings, microbial cultures are oxygenated through shaking or aeration.

We tested the changes in melanin production in stationary cultures of melanin-producing Pseudomonas after adding green algae.

Figure 4  Culture system of green algae and Melanin-Producing Bacteria

Our results indicate that adding Chlorella (500 μL of Chlorella culture to 3 liters of Pseudomonas culture) in a stationary led to rapid melanin production within a short period (less than 6 hours).

Learn

The algal culture medium contains a significant amount of dissolved oxygen, which can provide a temporary oxygen boost to the stationary bacterial culture. Further research is needed to explore the detailed interactions between algae and bacteria.

Experiment 3: The melanin’s protective effects of X-ray ion radiation, and microalgal-bacterial co-culture model

Design

The radiation protection model is designed to place melanin producing bacteria and green algae in the same space. Green algae (Chlorella vulgaris/Chlorella vulgaris) and black pigment producing pseudomonas co cultured in a greenhouse with artificial bubble protection film.

Green algae use urea, urine, carbon dioxide, and other substances for photosynthesis to release oxygen and produce sugars. The melanin produced by Pseudomonas aeruginosa helps to protect against X-rays and ultraviolet radiation from the sun. Synthetic biology regulates the relationship between two types of algae and bacteria to adapt them.

Test

We had coated Chlorella sp. in sodium alginate hydrogel. After microalgea growing, we coated the sodium alginate sphere of chlorella in agar with melanin and metal ions in the culture dish, as well as agar without melanin and metal ions. Then the culture dish received 1 Gray’s X-ray ion radiation, to test the protective effect of hydrogel spheres and melanin on ion radiation of Chlorella.

Result

Exposure to 1Gy of radiation, photographed under white light. On the left; On the right, melanin plus copper ions.	Photograph taken under white light without radiation exposure. On the right, melanin plus copper ions.

Figure 5  The melanin’s protective effects of microalgea against X-ray ion radiation.

The green algae embedded in hydrogel, which againwas embedded in the agar environment of melanin+metal ions, which is easier to receive X-ray radiation and die. A possible principle was the surrounding melanin+metal ions may transmit the radiation energy received to the green algae embedded in the water gel, causing death. And an environment without melanin and metal ions is less likely to cause radiation death.

Learning

It is possible that melanin and metal ions can absorb more radiation. If they are not spatially separated from the protected organism, cell and organelle, they are likely to transfer the energy or ions generated after radiation to the protected part, which may actually kill the protected part and the protected organism, cell, organelle and molecule.

When we use melanin for radiation protection, we must consider the temporal, spatial, and sequential relationships between melanin and the protected object.

Therefore, we revised our model for algal bacterial symbiosis and melanin radiation protection, distinguishing melanin from protected objects in time, space, and sequence. One of the simple diagrams is shown below.

Figure 6  The revised model will be planned for the next phase, for the purpose of radiation protection, melanin, the part that receives radiation, is seperate melanin from protected objects in time, space, and sequence.

This DBTL cycle has been instrumental in advancing our understanding and engineering capabilities, bringing us closer to achieving our project goals.

References

[1] ChiaraScanferla EnricoCaruso Viviana TeresaOrlandi FabrizioBolognese. Bacterial melanin production by heterologous expression of 4hydroxyphenylpyruvate dioxygenase from Pseudomonas aeruginosa[J]. International Journal of Biological Macromolecules, 2019, 133: 1072-1080.

[2] S.eskandari Z.Etemadifar. Melanin biopolymers from newly isolated Pseudomonas koreensis strain UIS 19 with potential for cosmetics application, and optimization on molasses waste medium[J]. Journal of Applied Microbiology, 2021.

[3] ManoranjanNayak AmitGhosh AyusmitaRay. A review on co-culturing of microalgae: A greener strategy towards sustainable biofuels production[J]. Science of the Total Environment, 2022, 802: 149765.