Results

Creation of pHSGmelA

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Using the melA gene from Rhizobium etli as a template, we performed PCR to amplify the melA necessary for transforming S.oneidensis. We used Extaq for the final PCR. After amplification, we confirmed the melA amplification through gel electrophoresis and subsequently carried out column purification.

The results of the electrophoresis are shown below.

After purification, the Nanodrop measurement result was 100.8 ng/µL.

Once the amplification of melA was confirmed, we performed column purification using the PCR product. We then treated the purified DNA with EcoRI-HF and XmaI for restriction enzyme digestion. After the digestion, we conducted another column purification using the digested solution and extracted melA through ethanol precipitation.

To create the cloning vector, we prepared LB + Cm plates and transformed E. coli with pHSG398. We then extracted the proliferated pHSG398 plasmid and treated it with EcoRI-HF and XmaI for restriction enzyme digestion. To confirm whether the digestion was successful, we performed electrophoresis. The composition of the restriction enzyme treatment solution are as follows:

[Restriction enzyme processing composition]

dH2O	6µL
plasmid	2µL
rCutsmart	1µL
EcoRI-HF	0.5µL
XmaI	0.5µL
total	10µL

[PCR conditions]

CYCLE STEP	CYCLES	TEMP	TIME
Initial Denaturation	1cycle	98℃	1min
Denaturation	30cycles	98℃	10sec
Annealing		71℃	5sec
Extension		68℃	18sec
Final Extension	1cycle	72℃	30sec
Hold	1cycle	4℃	∞

The results of the electrophoresis at this stage are shown below.

The DH5α/pHSG398 that was cultured in liquid medium was recovered, and a mini-prep was performed to extract the plasmid. A total of 10 µL of the extracted plasmid was subjected to restriction enzyme treatment, confirming successful cleavage. The concentrations of the extracted plasmids were as follows:
Sample 1: 237.9 ng/µL
Sample 2: 256.1 ng/µL
Sample 3: 230.8 ng/µL
Sample 4: 228.6 ng/µL

With the desired fragments obtained from both the insert and the vector, ligation was performed. The resulting plasmid, named pHSGmelA, was used to transform E. coli via the heat shock method. During this process, selection was carried out using blue-white screening to identify colonies containing the pHSGmelA with the insert successfully integrated.

From the white colonies that were believed to have the insert correctly integrated, a liquid culture was grown overnight, and the plasmid was extracted from this culture. To confirm the presence of the melA insert, an insert check was performed.

The results of the insert check yielded the following electrophoresis results:

Using glycerol stocks prepared in advance from the liquid culture, the cells were spread onto M9YE medium to produce melanin. For comparison, E. coli transformed with a control plasmid that did not contain melA was also spread onto the same medium. The cultured plates appeared as shown in the following photograph.

When spreading on the plate, there was no change in the color of the colonies transformed with pHSGmelA compared to the control colonies transformed with pHSG398. Normally, if melA is successfully integrated, tyrosinase would be produced, leading to melanin formation through the binding of tyrosine. However, since the colonies remained white and no melanin was produced, it was considered that mutations might have occurred in the nucleotide sequence during the experimental process. Therefore, plasmids were extracted from the liquid culture, and sequencing analysis was conducted.

The results of the sequencing analysis are as follows.

From the photos and the results of the sequencing analysis, it was confirmed that several mutations had occurred within the melA gene. To resolve this issue, an approach was taken to use a DNA polymerase with higher accuracy in order to address this problem.

This time, in order to perform PCR using the highly accurate KOD one, we confirmed whether melA could be amplified with KOD one. The composition of the PCR solution and the reaction times were conducted as follows.

The results of the electrophoresis when melA was amplified using KOD one are shown below.

Column purification was performed, and the results measured by NanoDrop are as follows.

The amplified melA obtained from KOD one was restriction digested and ligated with the restriction-digested pHSG398. The blue-white screening plate from this process is shown below.

After again spreading the white colonies into liquid medium and allowing them to grow overnight, I spread both the cultures transformed with pHSGmelA and those transformed with pHSG398 onto M9YE agar plates. After about two weeks, the results were observed, as shown in the following photos.

Additionally, after extracting the plasmid from the liquid culture and performing sequence analysis, no mutations were found within melA. The mutations observed on the vector side were located in non-coding regions, so they are considered to have no impact.

Having confirmed the production of melanin after transforming E. coli, we proceeded to transform S.oneisensis via electroporation.

When comparing S.oneidensis transformed with pHSG398 and S.oneidensis transformed with pHSGmelA, it was observed that the S.oneidensis transformed with pHSGmelA was darker in color.

Evaluation of melAmut

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First, the previously constructed pHSGmelA was used as a template, and PCR was performed using primers with mutations introduced in the overlap region. The amplification of the desired fragment was then confirmed by electrophoresis.

The fragment's amplification was confirmed, so I selected one PCR product for purification. After purification, I checked the DNA concentration using a NanoDrop.

The results from the NanoDrop are shown below.

The concentration of the purified DNA was 270.1 ng/µL.

Next, the purified DNA fragment was used to transform DH5α. After incubating overnight during the transformation, four colonies were selected for liquid culture. Following overnight liquid culture, plasmid extraction was performed from the liquid medium, and the concentration was measured using NanoDrop.

The measurement results for each colony were as follows: Colony 1 had a concentration of 75.2 ng/µL, Colony 2 had 78.3 ng/µL, Colony 3 had 76.0 ng/µL, and Colony 4 had 89.6 ng/µL. Based on these concentrations, a solution for sequencing analysis was prepared, and sequencing was performed. The results of the sequencing analysis are as follows:

Sequencing analysis confirmed that all plasmids (pHSGmelAmut) contained the correct mutations. Therefore, Colony 1 was chosen for cultivation. Initially, to visually compare pHSGmelAmut and pHSGmelA, E. coli was transformed using each plasmid on M9YE agar plates.

To compare the differences in melanin production rates between E. coli transformed with the pHSGmelA mutant and the standard pHSGmelA, absorbance measurements were conducted.

In liquid culture, DH5α E. coli transformed with either melAmut or melA was cultivated, and absorbance was measured every 24 hours. The results are shown in the table below.

	24h	48h	72h
DH5α/pHSGmelA	2.089	2.088	2.125
DH5α/pHSGmelAmut	2.076	2.102	2.139

From the table, a comparison between DH5α/pHSGmelA and DH5α/pHSGmelAmut showed that the absorbance in the liquid culture transformed with pHSGmelAmut increased more rapidly than that in the culture transformed with pHSGmelA. This result is illustrated in the graph below.

UV Irradiation

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Colonies transformed with melA were diluted in PBS, and a solution of colonies transformed with the control plasmid was also diluted in PBS. Both solutions were subjected to ultraviolet irradiation to measure the differences in resistance. The irradiation times were set at 0 minutes, 5 minutes, 10 minutes, and 15 minutes. The diluted solutions were then cultured on LB + Cm agar plates. This allowed us to evaluate how the presence of melanin affects resistance to ultraviolet radiation. The results showed that there was no difference between the colonies transformed with melA and those with the control plasmid, as all E. coli, except for the 0-minute group, were killed.

To examine the irradiation times, an experiment was conducted using only E. coli transformed with melA. This time, DH5α/pHSGmelA was grown in liquid culture using M9YE medium, and the production of melanin was confirmed. The OD600 was adjusted using PBS before the bacterial solution was placed on plates for ultraviolet irradiation. The irradiation times were set at 0 minutes, 3 minutes, 5 minutes, and 7 minutes. After irradiation, the bacteria were diluted to concentrations of 1,000, 100, and 10 cells, and cultured on LB + Cm agar plates. The culture results and colony counts are shown in the following photos and tables.

When measuring the number of colonies, the following data was obtained:

Time (min)	0	3	5	7
Number of Cells (1,000)	105	0	0	0
Number of Cells (100)	11	0	0	0
Number of Cells (10)	2	0	0	0

↑Measurement of CFU

The ultraviolet irradiation time was further shortened, and an experiment similar to the first trial was conducted. The conditions included irradiation for 0 seconds, 5 seconds, 10 seconds, 20 seconds, and 30 seconds in liquid medium, followed by diluting the samples and spreading them on agar plates to measure CFU (colony-forming units). The culture results and colony counts are shown in the following photos and tables.

When measuring the number of colonies, the following data was obtained:

Time (seconds)	0	5	10	20	30
Number of Cells (10,000)	133	113	101	42	11
Number of Cells (1,000)	23	8	6	5	1
Number of Cells (100)	1	2	2	0	0

↑Measurement of CFU

Discussion and Outlook

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The transformation of S.oneidensis with melA to produce melanin is our first attempt, and it has shown that it is indeed possible for S.oneidensis to generate melanin. However, it remains unclear how much radiation resistance is conferred by the presence of melanin within the intracellular membrane. Therefore, we believe it is necessary to test the radiation resistance of S.oneidensis that produces melanin. We anticipate that generating melanin within the inner membrane of S.oneidensis may not confer resistance to radiation; hence, it should be produced in the periplasm. This could lead to the creation of a state referred to as "melanin ghost," which may acquire resistance through Compton scattering (refer to HP page). To generate melanin in the periplasm, we would like to conduct experiments by introducing signal peptides. If we can confirm an increase in the electricity production capacity of S.oneidensis, it will likely mark a significant step toward the project's implementation.

We successfully created melA mutants based on pHSGmelA. Comparative experiments between pHSGmelA and pHSGmelAmut suggest that the production of melanin may have progressed faster with pHSGmelAmut, based on the culture results from transformed E. coli plates and liquid media. However, due to the small sample size and the observation period being cut short, the data collected is insufficient for drawing accurate conclusions. Therefore, it is necessary to conduct more precise experiments to enhance the accuracy of our results.

If this result is confirmed, utilizing melA mutants could simplify the implementation of the project, ensuring that melanin is rapidly purified during subsequent cell division, thereby maintaining radiation resistance.

Unfortunately, we could not conduct comparative experiments between the control plasmid and the melA mutant due to time constraints. However, we were able to determine the duration for which the E. coli transformed with melA exhibits resistance. By applying UV radiation to the control plasmid for the same duration, we can evaluate how much UV resistance is gained by the microorganisms transformed with melA.