Overview
Overview
The ultimate goal of our project is to develop a mechanism that converts "radiation energy," which is harmful to living organisms, into useful energy. To achieve this, we focused on melanin, which has radiation resistance and the ability to transfer electrons in response to radiation, as well as S. oneidensis, which releases electrons extracellularly, to realize this project.
Fig.1 Here are three key points in the project
Melanin is produced by the binding of tyrosinase expressed from the melA gene with tyrosine. To apply this to Shewanella, we plan to synthesize melanin in the periplasm of Shewanella, allowing it not only to acquire radiation resistance but also to enhance the production of electrical energy by utilizing the electrons generated from melanin.
Fig.2 Schematic Diagram for Producing Melanin in Shewanella
The first step in wet lab experiments to realize this mechanism is to produce melanin in S.onendensis. Since attempts to produce melanin in S.oneidensis have not been made before, this wiki page introduces the DBTL cycle we conducted to express melanin in S.oneidensis.
Producing melanin in S.oneidensis
【Design】
We first decided to conduct an experiment to transform Shewanella with melA. The part used is (BBa_K193600). This part was created by iGEM09_Tokyo_Tech (2009-10-14). Additionally, this part was introduced into the pHSG398 plasmid through restriction enzyme digestion, resulting in the creation of pHSG398melA.
【Build】
We performed cloning through restriction enzyme digestion. First, we amplified melA, which is needed for transforming Shewanella, using PCR. Then, we confirmed the amplification of melA by gel electrophoresis. After confirming the amplification of melA, we performed column purification using the PCR product. The purified DNA was treated with restriction enzymes EcoRI-HF and XmaI, and the solution containing the digested product was subjected to column purification again, followed by ethanol precipitation to extract melA.
Meanwhile, to prepare the vector for cloning, we prepared LB + Cm plates and transformed E. coli with pHSG398. We extracted the plasmid from the cultured E. coli containing pHSG398 and performed restriction enzyme digestion using EcoRI-HF and XmaI. To verify successful digestion, we conducted gel electrophoresis and column purification.
With the desired fragments obtained from both the insert and the vector, we performed ligation. The resulting plasmid (pHSGmelA) was used to transform E. coli via the heat shock method. We used blue-white screening to select the pHSGmelA containing the insert. A white colony, which was presumed to have the correct insert, was cultured in liquid medium, and plasmid extraction was performed. To verify whether the extracted plasmid contained the correct sequence, we conducted an insert check by restriction enzyme digestion and gel electrophoresis. Once the presence of the melAinsert in the plasmid was confirmed, we performed sequence analysis to ensure that there were no errors in the sequence.
Fig.3 Cloning experiment workflow using restriction enzyme digestion
【Test】
When S.oneidensis was transformed with pHSG398melA, melanin production was confirmed. The following photos show the results of transforming E. coli and S.oneidensis with melA.
Fig.4 Plate with Shewanella transformed with pHSGmelA
As can be seen in the photo of the plate with S.oneidensis transformed with pHSGmelA, the colonies cultured on the plate have turned black.
【Learn】
It was found that transformation of S.oneidensis with melA is possible. However, it remains unclear whether exposure to radiation enhances electricity production through S.oneisensis's electron transport mechanism and how electrons are transferred to the EET pathway when S.oneidensis produces melanin. In the future, it will be necessary to create a part with a signal peptide attached to melA to determine whether radiation resistance can be conferred and whether electricity production capacity increases when exposed to radiation.
Rapidly producing melanin
1 Tyrosinase production.
【Design】
The results of the wet experiments showed that melanin synthesis takes a significant amount of time. This means that the waiting time during experiments is long, which may lead to various limitations. Moreover, this could also result in weakened radiation resistance after cell division during implementation.
Therefore, in the dry experiments, we decided to investigate the dynamics of melanin production.
【Build】
To verify melanin production, a model of tyrosinase production required for melanin synthesis was constructed based on the lactose operon model.
【Test】
A model was constructed using MATLAB, and a simulation was performed.
Fig.5 This represents the time course of tyrosinase expression. The horizontal axis indicates time (minutes), while the vertical axis shows concentration (nM).
It was determined that the tyrosinase concentration at steady state is approximately 2500 nM.
【Learn】
From this model, it was determined that the tyrosinase concentration is approximately 2500 nM. Using this result, execute the model for melanin production.
2 Melanin production
【Design】
Verify the time required to reach a steady state and the amount of melanin produced.
【Build】
Melanin is synthesized through the oxidation of tyrosine. The enzyme tyrosinase catalyzes the conversion of tyrosine to dopaquinone, which then undergoes spontaneous oxidation to form melanin. A simplified schematic diagram of this reaction is shown below.
Fig.6 A schematic diagram illustrating the reaction of melanin synthesis from tyrosine.
A model was constructed based on this reaction.
【Test】
A model was constructed using MATLAB, and a simulation was performed.
Fig.7 The changes in concentration of melanin, tyrosine, and dopachrome over time are represented. The horizontal axis indicates time (hours), while the vertical axis shows concentration (nM).
It was found that it takes approximately 160 hours or more for melanin to reach a stable state. Additionally, melanin was produced at the same concentration as the tyrosine concentration in the medium (0.6 g/L).
【Learn】
When a cell divides, its contents are halved. Even if the concentration of tyrosine in the medium is maintained at a constant level, it is expected to take a long time for melanin synthesis to produce the same level of melanin as the original parent cell. This biological system is intended for use in radiation environments, but the lack of melanin after cell division may weaken radiation resistance. Additionally, the long waiting time for melanin synthesis during experiments could pose various limitations. Therefore, we considered the need to synthesize melanin more quickly.
3 Rapidly producing melanin using melA mutants
【Design】
Based on the content of the dry experiments, we need to implement the expression of melA rapidly. Therefore, we have planned to conduct experiments using melanin mutants. The melA mutant is one in which the 1000th nucleotide of the melA gene is mutated from C to T, resulting in the change of the 334th amino acid from proline to serine, which has been shown to increase the speed of melanin production (1). Thus, we will create the melA mutant and conduct experiments to demonstrate that melanin can be produced more rapidly.
【Build】
Using the already constructed pHSGmelA as a template, we will perform PCR with primers that introduce mutations in the overlap region. Once we confirm the amplification of the desired fragment through gel electrophoresis, we will purify the DNA fragment and conduct the transformation. We will compare the DH5α transformed with the pHSGmelA mutant to the DH5α transformed with the standard pHSGmelA to measure the difference in melanin production rates using a spectrophotometer.
Fig.8 p">How to create pHSGmelAmut based on pHSGmelA.
The principle of PCR for introducing mutations is that when mutations are incorporated into the overlap region of the primers, the annealing occurs at regions other than the mutated site, followed by elongation in the 5' to 3' direction. The elongation is performed by KOD One, extending up to just before the overlap region. After transforming the microorganisms with the generated fragment, the overlap region will anneal within the microorganism, resulting in the formation of a circular plasmid.
【Test】
Plate and liquid cultures were performed to compare melA and melAmut.
On the plate, DH5α E. coli transformed with melAmut and melA were cultured on the same medium for comparison. In the photo taken four days after plating, pHSGmelAmut showed visible browning, while pHSGmelA appeared to have less pigmentation and did not show significant darkening.
Fig.9 Left: E. coli transformed with pHSGmelA
Right: E. coli transformed with pHSGmelAmut
To compare the rates of melAmut and melA production in liquid medium, transformed DH5α E. coli were cultured and absorbance was measured every 24 hours, respectively. The results are shown in the following table.
| cultivation time (h) | 24 | 48 | 72 |
| DH5α/pHSGmelA absorbance (400nm) | 2.089 | 2.088 | 2.125 |
| DH5α/pHSGmelAmut absorbance (400nm) | 2.076 | 2.102 | 2.139 |
When comparing DH5α/pHSGmelA and DH5α/pHSGmelAmut, it was observed that the liquid culture transformed with pHSGmelAmut exhibited a quicker increase in absorbance than the liquid culture transformed with pHSGmelA. This result was represented in a graph.
Fig.10 A graph showing the absorbance (400 nm) measured over time.
【Learn】
The results from both the plate culture and the liquid culture suggest that pHSGmelAmut may have facilitated faster melanin production. However, the limited number of samples and the fact that observations were only conducted for part of the duration mean that the data obtained is insufficient for a more accurate assessment.
If these results are correct, using the melA mutant could contribute to the simplicity of implementing the project, allowing for rapid melanin production during cell division after implementation, thereby maintaining radiation resistance.
