iGEM 2024 — Team tupls-japan

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### Engineering

[Overview](#Engineering-nav1)

[Producing melanin in *S.oneidensis*](#Engineering-nav2)

[Rapidly producing melanin](#Engineering-nav3)

[Reference](#Engineering-nav4)

## 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.

![aaaaa](https://static.igem.wiki/teams/5140/engineering-1.png)

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.

![aaaaa](https://static.igem.wiki/teams/5140/engineering-2.png)

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 *Eco*RI-HF and *Xma*I, 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 *Eco*RI-HF and *Xma*I. 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 (pHSG*melA*) was used to transform *E. coli*via the heat shock method. We used blue-white screening to select the pHSG*melA* 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 *melA*insert in the plasmid was confirmed, we performed sequence analysis to ensure that there were no errors in the sequence.

![aaaaa](https://static.igem.wiki/teams/5140/engineering-3new.png)

Fig.3 Cloning experiment workflow using restriction enzyme digestion

【Test】
When *S.oneidensis* was transformed with pHSG398*melA*, melanin production was confirmed. The following photos show the results of transforming *E. coli* and *S.oneidensis* with *melA*.

![aaaaa](https://static.igem.wiki/teams/5140/engineering-4.png)

Fig.4 Plate with Shewanella transformed with pHSGmelA

As can be seen in the photo of the plate with *S.oneidensis* transformed with pHSG*melA*, 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.

![aaaaa](https://static.igem.wiki/teams/5140/engineering-tyrosinase1.png)

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.

![aaaaa](https://static.igem.wiki/teams/5140/engineering-tyrosinase-melanin.png)

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.

![aaaaa](https://static.igem.wiki/teams/5140/engineering-melanin-p1.png)

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 pHSG*melA* 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 *DH5*α transformed with the pHSGmelA mutant to the *DH5*α transformed with the standard pHSG*melA* to measure the difference in melanin production rates using a spectrophotometer.

![aaaaa](https://static.igem.wiki/teams/5140/engineering-5.png)

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 *melA*mut.

On the plate, DH5α *E. coli* transformed with *melA*mut and *melA* were cultured on the same medium for comparison. In the photo taken four days after plating, pHSG*melA*mut showed visible browning, while pHSG*melA* appeared to have less pigmentation and did not show significant darkening.

![aaaaa](https://static.igem.wiki/teams/5140/engineering-6.png)

Fig.9 Left: E. coli transformed with pHSGmelA
Right: E. coli transformed with pHSGmelAmut

To compare the rates of *melA*mut 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/pHSG*melA*  absorbance (400nm) | 2.089 | 2.088 | 2.125 |
| DH5α/pHSG*melA*mut  absorbance (400nm) | 2.076 | 2.102 | 2.139 |

When comparing DH5α/pHSG*melA* and DH5α/pHSG*melA*mut, it was observed that the liquid culture transformed with pHSG*melA*mut exhibited a quicker increase in absorbance than the liquid culture transformed with pHSG*melA*. This result was represented in a graph.

![aaaaa](https://static.igem.wiki/teams/5140/engineering-comparison-of-melanin-production.webp)

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 pHSG*melA*mut 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.

## Reference

---

1. Santos CNS, Stephanopoulos G2008.Melanin-Based High-Throughput Screen for l-Tyrosine Production in Escherichia coli. Appl Environ Microbiol74: <https://doi.org/10.1128/AEM.02448-07>

![rocket](https://static.igem.wiki/teams/5140/rocket2.png)

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### Experiments

[Media preparation](#Experiments-nav1)

[Refining related](#Experiments-nav2)

[Plasmid Extraction](#Experiments-nav3)

[Others](#Experiments-nav4)

## Media preparation

---

PY medium (per 150mL)

(1) 150 mL of purified water was added to the medium powder (see figure below) and dissolved.

|  |  |
| --- | --- |
| Yeast Extract | 1g |
| bacto peptone | 2g |

(2) Autoclaved at 121°C for 15 minutes.

(3) While autoclaving, the substances in the figure below were filter-sterilized.

|  |  |
| --- | --- |
| CaCl2 (0.7M) | 2ml |

(4) When the beaker after autoclaving reached about 70°C, the reagents sterilized in (3) were mixed.

(5) The material was poured into petri dishes and allowed to dry until it cooled and solidified.

LB medium(per liter)

(1)Medium powder (see figure below) was dissolved with 1 L of purified water.)

|  |  |
| --- | --- |
| tripton | 10g |
| Yeast Extract | 5g |
| NaCl | 10g |

(2) Autoclave at 121°C for 15 minutes and cool to about 70°C.

(3) The material was poured into petri dishes and allowed to dry until it cooled and hardened.

M9YE medium (per 150mL)

(1) Medium powder (see figure below) was dissolved with 150 mL of purified water.

|  |  |
| --- | --- |
| KH2PO4 | 0.15g |
| Na2HPO4 | 0.54g |
| NH4Cl | 0.15g |
| NaCl | 0.075g |
| L-Tyrosin | 0.075g |
| Yeast Extract | 0.75g |
| Agar(Agar medium only) | 2.25g |

(2) Autoclaved at 121°C for 15 minutes.

(3)While autoclaving, the substances in the figure below were filter-sterilized.

|  |  |
| --- | --- |
| CaCl2 (0.7M) | 20µL |
| MgSO4 (1M) | 300µL |
| CuSO4 (0.5M) | 3µL |
| Glucose (1M) | 3mL |
| 50mg/mL Cm | 60µL |
| IPTG (1M) | 75µL |

(4)When the beaker after autoclaving reached about 70°C, the reagents sterilized in (3) were mixed.

(5)The material was poured into petri dishes and allowed to dry until it cooled and solidified.

## Refining related

---

Protocol for total DNA purification using DNAzol(invitrogen)

(1) Centrifuge and remove the bacteria.

(2) Add 200 μL of DNAzol to 200 μL of bacteria, and suspend well by pipetting. Incubate for 2~5 min.

(3) Add 250 µL of 99.5% EtOH, and mix well by invert.

(4) After 3 min of incubation, centrifuge at 15,000 rpm for 2 min at 4°C, and remove the supernatant by decantation.

(5) The pellet was rinsed with 400 μL of 70% EtOH and centrifuged at 15,000 rpm at 4°C for 2 min.

(6) The supernatant was removed by decantation and centrifuged again at 15,000 rpm at 4°C for 1 min.

(7) The supernatant was completely removed by pipetting, and the lid was opened and allowed to dry for 1 min.

(8) The pellet was dissolved in 200 μL of 8 mMNaOH.

Protocol for DNA purification using NucleoSpin gDNA Clean-up

Sample Preparation

150 μl of DNA solution was prepared. If the volume is less than 150 μl, add water to make it 150 μl.

①Addition of Buffer

Add 450 μl of Binding Buffer DB to 150 μl of DNA solution, and mix by stirring strongly for 5 seconds.

②Adsorption on column

A NucleoSpin gDNA Clean-up Column (green ring) was set in a 2 ml collection tube.
The solution of ① was added to the column and centrifuged at 11,000 × g for 30 seconds.
The filtrate was discarded and the column was reassembled in the same tube.

③Membrane washing

First washing

Add 700 μl of Buffer DW to the column, agitate strongly for 2 seconds, and centrifuge at 11,000 × g for 30 seconds.
The filtrate was discarded and the column was re-set in the same tube.

Second washing

Add 700 μl of Buffer DW to the column, agitate strongly for 2 seconds, and centrifuge at 11,000 × g for 30 seconds.
The filtrate was discarded and the column was reassembled in the same tube.

④Membrane drying

The column was centrifuged at 11,000 × g for 1 minute.
The column was placed in a microtube (1.5 ml) (prepared by each person).

⑤Elution of DNA

50 μl of Buffer DE was added to the column and allowed to stand for 1 minute without closing the lid, then the lid was closed and centrifuged at 11,000 × g for 30 seconds.

Protocol for Gel purification

(1) Buffer QX1 was added in 3x volume (930 μL).

(2) Vortexed for 30s, and added QX2 (30 μL).

(3) Incubated at 50°C for 10 min (vortexed every 2 min) using a heat block (large).

(4) Centrifugation (13,500 rpm, rt, 1min) was used to remove the supernatant.

(5) 500 μL of BufferQX1 was added, followed by vortexing.

(6) Centrifugation (13,000, 1min, re) followed by removal of the supernatant.

(7) After adding 500 μL of BufferPE, vortexed.

(8) The pellet was air-dried for 40 min.

(9) 20 μL of MiliQ was added and vortexed or pipetted, followed by incubation at 50°C for 5 min.

(10) Centrifugation (13,000, 1min, re) was performed and the supernatant was transferred to a new tube.

Protocol for column purification

(1) All amounts were appliqued to the column.

(2) Centrifugation (13,500 rpm, rt, 1min) was performed.

(3) Discard the flow-through and add 750 μL of PEbuffer.

(4) Centrifugation (13,500 rpm, rt, 1 min) was performed, and after discarding the flow-through, further centrifugation (13,500 rpm, rt, 1 min) was performed.

(5) The column was transferred to a new 1.5-mL tube and 10 μL of dH2O was added.

(6) Incubated for 1min and centrifuged (13,500 rpm, rt, 1min).

(7) The amount of DNA was measured using Nano drop.

Cleanup of PCR products (using QIA quick PCR Purification Kit)

(1) A 5x volume of Buffer PB was added to the PCR product and pipetted.

(2) The column was incubated (RT. 1 min).

(3) Incubated (RT. 1min).

(4) Centrifuged (13000 rpm, 1min, RT) and discarded the filtrate.

(5) Add 700 µL of Buffer PE.

(6) Centrifuge (13000 rpm, 1min RT) and discard the filtrate.

(7) Further centrifugation (13,000 rpm, 1 min, RT) was performed.

(8) Place the column in a new 1.5-mL tube.

(9) Add 30 µL of Buffer EB.

(10) Incubate at 50°C for 5 min.

(11) Centrifugation (13000 rpm, 1min, RT).

## Plasmid Extraction

---

Miniprep method

(1) About 2~5ml of E. coli bacteria solution containing plasmid was collected.
※Collection of about 2~3ml of bacteria was performed 2~3 times in a centrifuge (15000rpm, 1min.rt).

(2) The supernatant was discarded and centrifuged (15,000 rpm, 1 min, rt).

(3) The supernatant was completely removed using P200.

(4) 250 μl of Butter P1 (stored at 4°C) was added and subjected to a vortex mixer.

(5) Buffer P2 250 μl was added and mixed by vortex mixer 4~6 times.

(6) Add 350 μl of Buffer N3 and mix immediately by inverting 4~6 times.

(7) After centrifugation (13000 rpm, 10 min, rt), the supernatant was transferred to the attached column.
※⑤ to ⑦ were generally performed within 20 min.

(8) After centrifugation (13000rpm,1min,rt), the filtrate was discarded.

(9) Add 500 μl of Buffer PB and centrifuge (13000 rpm, 1min, rt).

(10) Discard the filtrate and add 750 μl of Buffer PE.

(11) After centrifugation (13000 rpm, 1min, room temperature), the filtrate was discarded.

(12) Centrifuge again (13,000 rpm, 1 min, room temperature).

(13) The column was placed in a new 1.5-ml tube, and 50 μl of Buffer EB was added so as not to contact the wall.

(14) The column was allowed to stand for 1 min.

(15) Centrifuged (13000 rpm, 1min., room temperature) and collected the concentrated liquid.

(16) The amount of extracted DNA was measured using Nanodrop.

Alkali SDS

(1) Six colonies were inoculated into LB + Km liquid medium and incubated overnight.

(2) 2 mL of the culture solution was transferred to a 2 mL tube and centrifuged at 15,000 rpm for 1 min at room temperature.

(3) The supernatant was removed by decantation and centrifuged at 15,000 rpm for 1 min at room temperature.

(4) The supernatant was completely removed by pipetting.

(5) 100 μL of TEG buffer was added to the bacterial pellet, and the pellet was completely suspended by vortexing.

(6) 200 µL NaOH-SDS was added, and after gentle invert, the pellets were kept on ice for 5 min.

(7) 150 µL 5M KOAc was quickly added, and after invertting, the suspension was kept on ice for at least 5 min.

(8) Phenol/chloroform was added, and after invertting, the suspension was kept on ice for at least 5 min.

(9) Centrifuge at 15,000 rpm for 5 min at room temperature.

(10) Transfer 400 μL of the upper layer to a new 1.5-mL tube.

(11) 1mL of 99.5% EtOH was added and vortexed.

(12) Centrifuge at 15,000 rpm for 5 min at room temperature, and remove the supernatant.

(13) 400 μL of 70% EtOH was added, and the tube was gently rotated to wash the pellet.

(14) Centrifuge at 15,000 rpm for 3 min at room temperature, and remove the supernatant completely.

(15) Open the lid of the tube and let it dry for 5~10 min, and dissolve the pellet in 50 μL of TE + RNase.

(16) Incubated at 37°C for 1h and stored at -20°C.

## Others

---

Sequence Analysis

|  |  |  |
| --- | --- | --- |
| Plasmid | | 300ng-600ng |
| PCR product | 100-200bp | 2-6ng |
| 200-500bp | 6-20ng |
| 500-1000bp | 10-40ng |
| 1000-2000bp | 20-80ng |
| >2000bp | 40-100ng |
| Primer | | 6.4pmol |

(1) After adjusting the template DNA, agarose gel electrophoresis was always performed and the concentration was measured by comparison with a size marker of known concentration.

(2) After mixing the template DNA and analysis primers as follows, the mixture was adjusted to 14 μl.

(Ex) In case of plasmid DNA concentration of 100ng/ml and primer concentration of 10pmol/μl

Dispense 4 μl of DNA (400 ng) + 0.64 μl of primer (6.4 pmoles) + 9.36 μl of sterile water into 1 tube (total 14 μl)

Autoclave

(1) Tap water was filled to the level that the Sunoko (a disk with holes laid on the bottom) was slightly submerged.

(2) The apparatus and reagents in the basket were placed on top of it.

(3) The lid was set in the correct position and fixed by tightening the handle.

(4) The weight of the safety valve was checked to make sure that it was placed correctly, the exhaust cock was closed, the timer was set, and heating was started.

(5) As the temperature inside the equipment gradually increased, steam came out of the exhaust hose, but after a while the valve automatically closed and the internal pressure increased.

(6) After the set time passed, the temperature and internal pressure gradually decreased, and the exhaust valve opened at nearly 100°C.

(7) When the pressure became normal and the temperature dropped to below 100°C, the lid was opened and the instruments and reagents were removed after they cooled down enough to be held.

Transformation
**heat shock method**

(1) To a DH5α competent cell (100µL), 2.0µL of plasmid solution (270.1ng/µL) was added.

(2) Incubated (on ice, 20 min).

(3) Incubate (42°C, 45sec).

(4) Incubated on ice for 2 min.

(5) 1mL of LB medium was added and suspended.

(6) Incubated (37°C, 1h).

(7) Centrifuge (13,000 rpm, 4°C, 1 min) and remove the supernatant.

(8) Suspended in 100 µL of LB medium.

(9) Two 10-fold dilution series were made.

(10) Spread onto LB+Cm (20 µM) plates.

(11) Incubated (37°C, overnight).

**electroporation**

(1) Cells were harvested and counted.

(2) Cells were centrifuged and resuspended in electroporation reagent.

(3) Nucleic acid was added, and the mixture of nucleic acid and cells was transferred to a cuvette.

(4) The cells were transferred to a cuvette immediately after electroporation.

(5) The cells were incubated for 12 to 72 hours.

(6) Assay was performed.

Restriction endonuclease

(1) Preparation of reaction solutionThe composition of the solution is shown below.

| Component | 50 µl Reaction |
| --- | --- |
| DNA | 1 µg |
| 10X rCutSmart Buffer | 5 µl (1X) |
| restriction endonuclease | 1.0 µl (20 units)† |
| Nuclease-free Water | to 50 µl |

(2) ncubated at 37°C for 5-15 min.

PCR

Extaq

(1) Keep all reagents on ice until use.

(2) Prepare the reaction mixture on ice.

|  |  |
| --- | --- |
| TaKaRa Ex Taq (5 U/μl) | 0.25 μl |
| 10X Ex Taq Buffer (Mg2＋ plus) (20 mM) | 5 μl |
| dNTP Mixture (2.5 mM each) | 4 μl |
| Template | < 500 ng |
| Primer 1 | 0.2 - 1.0 μM (final conc.) |
| Primer 2 | 0.2 - 1.0 μM (final conc.) |
| Sterile purified water | up to 50 μl |

(3) Program a thermal cycler with the desired cycling conditions.

**3stepPCR**

| CYCLE STEP | CYCLES | TEMP | TIME |
| --- | --- | --- | --- |
| Initial Denaturation | 1cycle | 98℃ | 30 sec |
| Denaturation | 30 cycles | 98℃ | 10 sec |
| Annealing | 55℃ | 30 sec |
| Extension | 72℃ | 1 min |
| Final Extension | 1cycle | 72℃ | 7 min |
| Hold | 1cycle | 4℃ | ∞ |

**2stepPCR**

| CYCLE STEP | CYCLES | TEMP | TIME |
| --- | --- | --- | --- |
| Initial Denaturation | 1cycle | 98℃ | 30 sec |
| Denaturation | 30 cycles | 98℃ | 10 sec |
| Extension | 68℃ | 1 min |
| Final Extension | 1cycle | 68℃ | 7 min |
| Hold | 1cycle | 4℃ | ∞ |

Denaturation conditions vary depending on the thermal cycler and tubes used for PCR. We recommend denaturing for 5 - 10 secat 98℃ or 20 - 30 sec at 94℃.

(4) Set the tubes in a thermal cycler and start thermal cycling immediately.

KOD one

(1) Keep all reagents on ice until use.

(2) Prepare the reaction mixture on ice.

|  |  |
| --- | --- |
| Autockaved,distilled water | XμL |
| KOD one PCR Master Mix | 25μL(1×) |
| Primer | 1.5μL(0.3μM each) |
| template | Genomic DNA～200ng/50μL |
| Plasmid DNA～50ng/50μL |
| cDNA(Equivalent amount of RNA)～750ng/50μL |
| Biological samples or crude extracts～5μL/50μL |

(3) Program a thermal cycler with the desired cycling conditions.

**3stepPCR**

| CYCLE STEP | CYCLES | TEMP | TIME |
| --- | --- | --- | --- |
| Initial Denaturation | 1cycle | 98℃ | 30sec |
| Denaturation | 25~45 cycles | 98℃ | 10 sec |
| Annealing | (Tm-5)℃ | 5 sec |
| Extension | 68℃ | 1~10 sec/kb |
| Final Extension | 1cycle | 68℃ | 30 sec |
| Hold | 1cycle | 4℃ | ∞ |

**2stepPCR**

| CYCLE STEP | CYCLES | TEMP | TIME |
| --- | --- | --- | --- |
| Initial Denaturation | 1cycle | 98℃ | 30 sec |
| Denaturation | 25~45 cycles | 98℃ | 10 sec |
| Extension | 68℃ | 5~10 sec/kb |
| Final Extension | 1cycle | 68℃ | 30 sec |
| Hold | 1cycle | 4℃ | ∞ |

**Step-down PCR**

| CYCLE STEP | CYCLES | TEMP | TIME |
| --- | --- | --- | --- |
| Initial Denaturation | 1cycle | 98℃ | 30 sec |
| Denaturation | 5cycles | 98℃ | 10 sec |
| Extension | 74℃ | 5～10 sec/kb |
| Denaturation | 5cycles | 98℃ | 10 sec |
| Extension | 74℃ | 5～10 sec/kb |
| Denaturation | 5cycles | 98℃ | 10 sec |
| Extension | 70℃ | 5～10 sec/kb |
| Denaturation | 15~30cycles | 98℃ | 10 sec |
| Extension | 68℃ | 5～10 sec/kb |
| Final Extension | 1cycle | 68℃ | 30 sec |
| Hold | 1cycle | 4℃ | ∞ |

(4) Set the tubes in a thermal cycler and start thermal cycling immediately.

Phusion HF

(1) Keep all reagents on ice until use.

(2) Prepare the reaction mixture on ice.

|  | 20 µl REACTION | 50 µl REACTION | FINAL CONCENTRATION |
| --- | --- | --- | --- |
| Nuclease-Free Water | to 25 µl | to 50 µl |  |
| 5X Phusion HF or GC Buffer | 4 µl | 10 µl | 1X |
| 10 mM dNTPs | 0.4 µl | 1 µl | 200 µM |
| 10 µM Forward Primer | 1 µl | 2.5 µl | 0.5 µM |
| 10 µM Reverse Primer | 1 µl | 2.5 µl | 0.5 µM |
| Template DNA | variable | variable | < 250 ng |
| DMSO (optional) | (0.6 µl) | (1.5 µl) | 3% |
| Phusion DNA Polymerase | 0.2 µl | 0.5 µl | 1.0 units/50 µl PCR |

(3) Program a thermal cycler with the desired cycling conditions.

**3stepPCR**

| CYCLE STEP | CYCLES | TEMP | TIME |
| --- | --- | --- | --- |
| Initial Denaturation | 1cycle | 98℃ | 30 sec |
| Denaturation | 30 cycles | 98℃ | 5~10 sec |
| Annealing | 45~72℃ | 10~30 sec |
| Extension | 72℃ | 15~30 sec/kb |
| Final Extension | 1cycle | 72℃ | 7 min |
| Hold | 1cycle | 4℃ | ∞ |

(4) Set the tubes in a thermal cycler and start thermal cycling immediately.

Electrophoresis

(1) Gel preparation: The concentration of the gel to be used was determined according to the length of the target DNA.

(2) Sample preparation: DNA samples and other samples to be investigated were diluted with appropriate solvents. For dilution of samples, appropriate solvents were selected according to the desired concentration and reaction conditions. The choice of solvent was carefully determined based on the purpose of the experiment and the detection method to be used.

(3) Loading samples onto gels: Samples prepared on gel cast trays were injected into the holes. Samples were loaded precisely using a micropipette or micropipette. Care should be taken during sample loading to prevent cross contamination. Point-like loading and accurate positioning of samples are also important.

(4) Performing electrophoresis: The gel cast tray was set into the electrophoresis bath and TAE buffer was poured into the bath. Electrophoresis was conducted at the appropriate power and time using a power supply device with electrodes connected to the electrophoresis tank. The conditions of electrophoresis (voltage and time) should be optimized according to the size and purpose of the sample.

(5) Visualization of bands: Once electrophoresis was complete, the gels were stained with MIDORIGREEN. The staining allowed DNA fragments to be visualized as bands. After staining, the gel was photographed and the position and intensity of the bands were recorded.

(6) Analysis of results: The position and size of the stained bands were analyzed to obtain the desired information. Gel image analysis software or dedicated analysis tools can be used for the analysis. The analysis results were accurately recorded and necessary statistical analysis and graphing were performed.

Nanodrop

(1) Measured in milli-Q water.

(2) Blank was measured in buffer.

(3) Concentration of the solution with plasmid was measured.

Glycerol stock
**(1) Prepare glycerol solution:.**

A 50% glycerol solution was prepared by diluting 100% glycerol and MiliQ in a 1:1 ratio. The solution was then autoclaved.

**(2) Bacterial culture:.**

Bacteria were cultured overnight in appropriate culture medium (e.g., LB medium).

**(3) Glycerol stock preparation:.**

500 µL of the cultured bacterial stock was taken and mixed with 500 µL of 50% glycerol solution.

This mixture was placed in a sterile 2 ml tube.

**(4) Cryopreservation:**

Freeze the tubes at -80°C.

The tubes were frozen at -80°C.

**Notes.**

Glycerol stock can be stored stably at -80°C for a long period of time, but repeated cycles of freezing and thawing will degrade it, so avoid as much as possible.

Scribe the contents with an oil-based pen so that the contents can be identified.

Ultraviolet irradiation experiment
**DH5α/pHSG398 plate**
**DH5α/pHSGmelA plate**
**(-)PBS**
**LB+Cm(20) plate**

methods

The following operations are performed inside the safety cabinet.

(1) Add 5 mL of PBS to 2 mL tubes.

(2) Remove E.coli from the plate and collect. (3 samples each)

(3) Measure OD600 and align the OD value.

(4) Add 5 mL of bacteria solution to the empty plate.

(5) Irradiate with UV light (15 W).

(6) Collect the bacteria solution in 1.5 mL tubes.

(7) Dilute the solution with 900µL of PBS and 100µL of bacteria solution.

|  | 1 | 10-1 | 10-2 | 10-3 | 10-4 |
| --- | --- | --- | --- | --- | --- |
| PBS | 0 | 900 | 900 | 900 | 900 |
| BS | 1000 | 100 | 100 | 100 | 100 |

(8) Add 100µL of the bacteria solution to the LB + Cm(20) plate and spread.

![rocket](https://static.igem.wiki/teams/5140/rocket2.png)

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### Results

[Creation of pHSG*melA*](#Results-nav1)

[Evaluation of *melA*mut](#Results-nav2)

[UV Irradiation](#Results-nav3)

[Discussion and Outlook](#Results-nav4)

In our Wet experiments, we conducted studies to produce melanin in *Shewanella oneidensis*, evaluate the *melA* mutants, and assess resistance to ultraviolet (UV) radiation.

## Creation of pHSG*melA*

---

### Amplification and Restriction Enzyme Treatment of *melA* Gene

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.

![aaaaa](https://static.igem.wiki/teams/5140/results-1.png)

Fig.1 From left to right: Marker (50), PCR sample 1, PCR sample 2.

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 *Eco*RI-HF and *Xma*I for restriction enzyme digestion. After the digestion, we conducted another column purification using the digested solution and extracted *melA* through ethanol precipitation.

### Amplification and Restriction Enzyme Treatment of pHSG398

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 |
| *Eco*RI-HF | 0.5µL |
| *Xma*I | 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.

![aaaaa](https://static.igem.wiki/teams/5140/results-2.png)

Fig.2 Electrophoresis results after restriction enzyme treatment of pHSG398 and melA (from left to right: Lane 1: Marker 6, Lanes 2-4: melA, Lanes 5-8: pHSG398, Lane 9: Marker 6).

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

### Creation of pHSG*melA* via Ligation

With the desired fragments obtained from both the insert and the vector, ligation was performed. The resulting plasmid, named pHSG*melA*, 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 pHSG*melA* with the insert successfully integrated.

![aaaaa](https://static.igem.wiki/teams/5140/results-3.png)

Fig.3 Blue-white selection after ligation

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:

![aaaaa](https://static.igem.wiki/teams/5140/results-4.png)

Fig.4 Electrophoresis (from left to right: Lane 1: Marker 6, Lanes 2-4: pHSGmelA, Lane 5: Marker 6)

### Confirmation of Melanin Production

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.

![aaaaa](https://static.igem.wiki/teams/5140/results-5.png)

Fig.5 Results of transformation with pHSGmelA

When spreading on the plate, there was no change in the color of the colonies transformed with pHSG*melA* 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.

![aaaaa](https://static.igem.wiki/teams/5140/results-6.png)

![aaaaa](https://static.igem.wiki/teams/5140/results-7.png)

![aaaaa](https://static.igem.wiki/teams/5140/results-8.png)

Fig.6 Mutation location of melA

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.

### Reevaluation of PCR for *melA* Gene Amplification

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.

| Solution composition | |
| --- | --- |
| dH2O | 34µL |
| 2x KOD One Mix | 50µL |
| Primer-F | 3µL |
| Primer-R | 3µL |
| Template | 10µL |
| Total | 100µL |

| Reaction temp | time |
| --- | --- |
| 98℃ | 1min |
| [ |  |
| 98℃ | 10s↓ |
| 71℃ | 5s↓ |
| 68℃ | 16s↓ |
| [ |  |
| 68℃ | 30s |

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

![aaaaa](https://static.igem.wiki/teams/5140/results-9.png)

Fig.7 Lane 1: Marker 6, Lane 2: PCR product.

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

![aaaaa](https://static.igem.wiki/teams/5140/results-10.png)

Fig.8 Measurement of Concentration After Purification of PCR Products Amplified from melA Using KOD One

### Reconstruction of pHSG*melA* via Ligation

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.

![aaaaa](https://static.igem.wiki/teams/5140/results-11.png)

Fig.9 Appearance of the Plate Used for Blue-White Screening

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

![aaaaa](https://static.igem.wiki/teams/5140/results-12.png)

Fig.10 On the left is the E. coli transformed with the control plasmid, and on the right is the E. coli transformed with melA.

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.

![aaaaa](https://static.igem.wiki/teams/5140/results-13.png)

Fig.11 Schewanella transformed with melA on a plate

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

## Evaluation of *melA*mut

---

### Creation of pHSG*melA* Mutant

First, the previously constructed pHSG*melA* 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.

![aaaaa](https://static.igem.wiki/teams/5140/results-14.png)

Fig.12 From left to right, Lane 1: Marker 6, Lanes 2–7: PCR Product 2, Lane 8: Marker 6.

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.

![aaaaa](https://static.igem.wiki/teams/5140/results-15.png)

Fig.13 Results of Nano Drop measurement after generating PCR products

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.

![aaaaa](https://static.igem.wiki/teams/5140/results-16.png)

Fig.14 Nano drop measurement results①

![aaaaa](https://static.igem.wiki/teams/5140/results-17.png)

Fig.15 Nano drop measurement results②

![aaaaa](https://static.igem.wiki/teams/5140/results-18.png)

Fig.16 Nano drop measurement results③

![aaaaa](https://static.igem.wiki/teams/5140/results-19.png)

Fig.17 Nano drop measurement results④

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:

![aaaaa](https://static.igem.wiki/teams/5140/results-20.png)

Fig.18 Sequencing results of the created pHSGmelAmut

### Comparison of pHSG*melA*mut and pHSG*melA*

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

![aaaaa](https://static.igem.wiki/teams/5140/results-21.png)

Fig.19 Left: E. coli transformed with melA, Right: E. coli transformed with melAmut

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

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

|  | 24h | 48h | 72h |
| --- | --- | --- | --- |
| DH5α/pHSG*melA* | 2.089 | 2.088 | 2.125 |
| DH5α/pHSG*melA*mut | 2.076 | 2.102 | 2.139 |

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

![aaaaa](https://static.igem.wiki/teams/5140/engineering-comparison-of-melanin-production.webp)

Fig.20 Comparison of absorbance of E. coli transformed with pHSGmelA and pHSGmelAmut

## UV Irradiation

---

### Ultraviolet Irradiation Experiment (1st Trial)

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.

### Ultraviolet Irradiation Experiment (2nd Trial)

To examine the irradiation times, an experiment was conducted using only *E. coli* transformed with *melA*. This time, DH5α/pHSG*melA* 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.

![aaaaa](https://static.igem.wiki/teams/5140/results-23.png)

Fig.21 Results of CFU measurement in UV irradiation experiments (2nd trial)

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

### Ultraviolet Irradiation Experiment (3rd Trial)

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.

![aaaaa](https://static.igem.wiki/teams/5140/results-24.png)

Fig.22 Results of CFU measurement in UV irradiation experiments (3rd trial)

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

---

### Transformation of *S.oneidensis* with *melA*

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](https://2024.igem.wiki/tupls-japan/human-practices)). 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.

### Creation of *melA* Mutants and Comparison of Production Rates

We successfully created *melA* mutants based on pHSG*melA*. Comparative experiments between pHSG*melA* and pHSG*melA*mut suggest that the production of melanin may have progressed faster with pHSG*melA*mut, 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.

### Ultraviolet Irradiation Experiments

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*.

![rocket](https://static.igem.wiki/teams/5140/rocket2.png)

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