Experimental method

Culture medium

strain	strain	Ingredients.	Quality.
Pseudomonas sp. QDLY500GRS	DPT	Glucose.	1g
		Tryptone.	10g
		Tyrosine.	2g
		NaCl	5g
		CaCl2	0.1g
		H₂O	1L
		Agar, used for solidifying culture media.	20g
PPICZaAPkrhdd-6xh Plasmid、Escherichia coli	Low-salt LB medium.	Tryptone.	10g
		Yeast extract.	5g
		NaCl	5g
		Agar, used for solidifying culture media.	20g
		H₂O	1L
Yeast	YPD	Tryptone.	20g
		Yeast extract.	10g
		Glucose.	20g
		Agar, used for solidifying culture media.	20g
		PH	6.5±0.2
		H₂O	1L
Microalgae(Chlorella and Gloeocapsa.)	BG11	BG11	1.7g
		H₂O	1L

Reagents.

Inducing agent (filtered by membrane filter).	Ingredients.	Quality.
20% Arabinose.	20% Arabinose.	2g
	H2O	Make up to a final volume of 10 ml.
0.5mM IPTG	IPTG	1.1865g
	H2O	Make up to a final volume of 10 ml.
100mg/ml CuSO4	CuSO4*5H2O	1g
	H2O	Make up to a final volume of 10 ml.

Antibiotics (filtered through a membrane filter):	Ingredients.	Quality.
Ampicillin (100 µg/ml)	Ampicillin	1g
	H2O	Make up to a final volume of 10 ml.
Chloramphenicol (25 µg/ml)	Chloramphenicol	250mg
	H2O	Make up to a final volume of 10 ml.
Kanamycin (10 µg/ml)	Kanamycin	10mg
	H2O	Make up to a final volume of 10 ml.
Streptomycin (50 µg/ml)	Streptomycin	50mg
	H2O	Make up to a final volume of 10 ml.

PCR

1.PCR system.

Strain	Component	Volume
Pseudomonas/Escherichia coli expressing melanin	2×Taq Master Mix	13μL
	27F	1μL
	1492R	1μL
	ddH₂O	8μL
	Template DNA	2μL
Microalgae	2×Taq Master Mix	12.5μL
	27F	1μL
	1492R	1μL
	ddH₂O	8μL
	Template DNA	2.5μL

2.PCR Program.

Temperature	Pre-denaturation at 94℃	Denaturation at 94℃	Annealing at 55℃	Extension at 72℃	Final extension at 72℃	Cycles: 30 times
time	3min	30s	30s	1min	5min

Temperature	time
Pre-denaturation at 94℃	3min
Denaturation at 94℃	30s
Annealing at 55℃	30s
Extension at 72℃	1min
Final extension at 72℃	5min
Cycles: 30 times

Isolation of Melanin-Producing Microorganisms from Soil

Objective

To isolate microorganisms that produce melanin from soil.

Materials

Equipment: Incubator, Erlenmeyer flasks, etc.

Reagents: 0.85% NaCl, DPT medium, etc.

Method

1. Sampling

Collect samples from soil that may contain melanin-producing microorganisms (riverbank mud, flower bed soil, road soil).

2. Enrichment Culture

Take 1 g of soil sample and disperse it in 9 mL of sterile 0.85% NaCl solution (in a 50 mL Erlenmeyer flask). Shake at 30 °C and 150 r·min-1 for 30 minutes in a shaking incubator. Transfer 1 mL of the soil supernatant into 50 mL of selective liquid medium (in a 250 mL Erlenmeyer flask) and shake at 28 °C and 150 r·min-1 for 24 hours in a shaking incubator.

3. Pure Culture Isolation and Screening Method

Transfer 5 mL of the enrichment culture into a flask containing 45 mL of sterile 0.85% NaCl solution. Dilute the enrichment culture to concentrations of 10^-5 and 10^-6. Spread the diluted enrichment culture on solid selective media prepared in advance and incubate at 30 °C in abiochemical incubator for 5 days. Regularly observe and record the growth of colonies on the plates during the incubation. Pick out microorganisms that produce black soluble pigments and transfer them to fresh solid selective media for streaking and separation. Perform multiple generations of subculturing on the selective media to further purify and confirm their growth and pigment production abilities. Store the purified microorganisms with good growth and high production of black soluble pigments in a 4 °C refrigerator for future use.

Ezup Column Bacteria Genomic DNA Purification Kit.

Objective

Extract DNA from melanin-producing microorganisms for molecular identification.

Materials

Reagents: Buffer Digestion, Proteinase K, Buffer B, Anhydrous Ethanol, Agarose, DNA Ladder (Marker), Loading Buffer

Equipment: Water Bath, Electrophoresis Apparatus, Centrifuge, Pipette, UV Spectrophotometer

Method

1. Take 0.5-1 ml of overnight cultured bacterial culture and add it to a 1.5 ml centrifuge tube. Centrifuge at 8,000 rpm at room temperature for 1 minute, discard the supernatant, and add 180 μl of Buffer Digestion. Then add 20 μl of Proteinase K solution, mix well, and incubate in a 56°C water bath for 1 hour until the cells are completely lysed.

2. Add 200 μl of Buffer BD, mix thoroughly by inversion, and incubate in a 70°C water bath for 10 minutes. (White precipitate may form after adding Buffer BD, which usually disappears after the 70°C water bath. This does not affect subsequent experiments. If the solution does not become clear, it indicates incomplete cell lysis, which may result in low DNA yield and impure DNA.)

3. Add 200 μl of anhydrous ethanol and mix thoroughly by inversion. (A translucent fibrous precipitate may form after adding anhydrous ethanol, which does not affect DNA extraction and application.)

4. Place the adsorption column in the collection tube, and use a pipette to add the solution and translucent fibrous precipitate to the column. Let it stand for 2 minutes, then centrifuge at 12,000 rpm at room temperature for 1 minute, and discard the waste liquid in the collection tube.

5. Place the adsorption column back into the collection tube, add 500 μl of PW Solution, and centrifuge at 10,000 rpm for 30 seconds, then discard the filtrate. (Before use, check if isopropanol is added to the solution in proportion.)

6. Place the adsorption column back into the collection tube, add 500 μl of Wash Solution, and centrifuge at 10,000 rpm for 30 seconds, then discard the filtrate. (Before use, check if anhydrous ethanol is added to the solution in proportion.)

7. Place the adsorption column back into the collection tube and centrifuge at 12,000 rpm at room temperature for 2 minutes to remove residual Wash Solution. (Leave the adsorption column open and place it at room temperature for a few minutes to thoroughly dry any residual Wash Solution in the adsorption material. Residual Wash Solution can affect the yield of genomic DNA and subsequent operations.)

8. Remove the adsorption column and place it in a new 1.5 ml centrifuge tube, add 50-100 μl of CE Buffer and let it stand for 3 minutes, then centrifuge at 12,000 rpm at room temperature for 2 minutes to collect the DNA solution. The extracted DNA can be used immediately for the next experiment or stored at -20°C. (To increase DNA yield, step 8 can be repeated.)

9. Add the extracted DNA to a suitable PCR system, set up the appropriate PCR program, and start the PCR machine.

10. Use the successfully amplified DNA samples for agarose gel electrophoresis (mix the samples with loading buffer before loading).

Use a High-Efficiency Plant Genome DNA Extraction Kit to extract DNA from microalgae.

Objective

After extracting the DNA from algae, perform PCR (Polymerase Chain Reaction) to prepare for subsequent detection, identification, and analysis of the algae.

Materials

Reagents: RNase A, Buffer LP2, Buffer LP3, Anhydrous Ethanol, Buffer WB2, etc.

Equipment: Centrifuge, Pipette, Centrifuge Tubes, Adsorption Columns, etc.

Method

1. Take 50-100 mg of fresh plant tissue or about 20 mg of dry weight tissue, and grind it thoroughly in liquid nitrogen. (Centrifuge the algae culture overnight and set aside.)

2. Collect the ground powder into a centrifuge tube (provided by the user), add 400 μl of Buffer LP1 and 6 μl of RNase A (20 mg/ml), vortex for 1 minute, and let it sit at room temperature for 10 minutes.

3. Add 130 μl of Buffer LP2, mix thoroughly, and vortex for 1 minute.

4. Centrifuge at 12,000 rpm for 5 minutes, transfer the supernatant to a new centrifuge tube. (Note: Only take the supernatant, do not pipette the sedimented tissue.)

5. Add 1.5 times the volume of Buffer LP3 (for example, add 600 μl of Buffer LP3 to 400 μl of supernatant), mix thoroughly for 15 seconds, at which point a flocculent precipitate may form.

   (Caution: Buffer LP3 must be mixed with anhydrous ethanol as required, add 27 ml of anhydrous ethanol to 21 ml of Buffer LP3.)

6. Transfer the solution and flocculent precipitate from the previous step into the centrifugal adsorption column, place the column in a collection tube before use, and if it cannot be transferred in one go, add it in several portions, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid in the collection tube.

   (Caution: If the membrane of the adsorption column turns green or brown, you can add 500 μl of anhydrous ethanol to the column, wait for the color to disappear, then centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid, place the adsorption column back into the collection tube.)

7. Add 600 μl of Buffer WB2 to the adsorption column, centrifuge at 12,000 rpm at room temperature for 30 seconds, and discard the waste liquid in the collection tube.

8. Add 500 μl of Buffer WB2 to the adsorption column, centrifuge at 12,000 rpm at room temperature for 2 minutes, and discard the waste liquid in the collection tube.

   (Caution: This step should not be omitted, as residual ethanol can affect the subsequent use of genomic DNA.)

9. Place the centrifugal adsorption column in a new 1.5 ml plastic centrifuge tube (provided by the user), add 50-100 μl of Buffer EB, let it stand at room temperature for 2 minutes. Centrifuge at 12,000 rpm for 1 minute, and the solution at the bottom of the centrifuge tube is the genomic DNA.

   (Caution: To increase elution efficiency, the eluent can be preheated at 60°C. If deionized water is used for elution, its pH can be adjusted to between 7.0-8.5 with NaOH. To increase DNA recovery, the obtained solution can be re-added to the centrifuge tube, let it stand at room temperature for 2 minutes, and collect again by centrifugation.)

Precautions:

• If there is precipitation in Buffer LP1 or Buffer LP3, it can be dissolved in a 37°C water bath, mixed well, and used afterward.

• All centrifugation steps should be performed using a desktop centrifuge at room temperature.

• Anhydrous ethanol should be added to Buffer LP3 and Buffer WB2 as required.

Molecular identification of DNA extracted from microorganisms isolated from soil.

Objective

Molecular identification of microorganisms through 16S rDNA sequence analysis.

Materials

Reagents: 2×Taq Master Mix, 27F primer, 1492R primer, ddH₂O, Agarose, Loading buffer, DNA ladder (marker)

Equipment: PCR machine, Pipette, Horizontal electrophoresis apparatus, UV analyzer

Method

1. PCR Amplification

Use the extracted DNA as a template and set up a 25 μL reaction system: 2×Taq Master Mix 13 μL, 27F primer 1 μL, 1492R primer 1 μL, ddH₂O 8 μL, DNA template 2 μL. Place the reaction in a PCR machine with a pre-set program for amplification. The amplified DNA is sent to Shanghai Bioengineering for sequencing and molecular identification.

2. Agarose Gel Electrophoresis

Prepare an 8% agarose solution by weighing 0.15 g of agarose and adding it to 20 mL of TAE buffer. Insert a comb into the gel mold, pour the solution into the electrophoresis tank, and wait for the gel to solidify. Fill the horizontal electrophoresis apparatus with TAE buffer, submerging the electrophoresis tank. Load the wells of the solidified gel with the DNA ladder (marker) and the amplified samples. Stain the samples with Loading buffer, and perform electrophoresis at 100 V for about 30 minutes. Observe the DNA bands under UV light.

Transformation of competent cells on ice and expression of melanin.

Objective

Utilize Escherichia coli as a chassis organism for the transformation of the hppd gene to express melanin.

Materials

Equipment: Water bath, ice maker, laminar flow hood, constant temperature shaker, centrifuge tubes, pipettes, pipette tips, etc.

Reagents: Escherichia coli TOP10, BL21 (DE3), BL21 (DE3) pLysS, LB + amp solid medium, arabinose, IPTG, etc.

Method

1. Take the Pahdd-6xH-21b/pelB--pkhppd--6xh--21b（+）plasmid, and the Top10 and BL21(DE3) competent cells, and keep them on ice;

2. In the laminar flow hood, pipette 1 μL of the Pahdd-6xH-21b/pelB--pkhppd--6xh--21b（+）plasmid and add it to the BL21(DE3)/Top10 competent cells, gently flick to mix. Be careful not to hold the competent cells in your palm or leave them off ice for too long.

3. Place on ice for 30 minutes;

4. Heat shock at 42℃ in a water bath for 45-60 seconds, then quickly transfer to an ice bath and let sit for 2 minutes (do not shake the sample during the rest on ice, as it may reduce the transformation efficiency);

5. In the laminar flow hood, add 700 μL of sterile LB liquid medium without antibiotics to the centrifuge tube, mix well, and recover at 37℃, 200 rpm for 60 minutes;

6. In the laminar flow hood, pipette 100 μL of the recovery liquid and spread it on LB solid medium containing ampicillin (100 μg/mL). Place it upright in the 37℃ incubator for 30 minutes, then invert the plate and incubate overnight at 37℃. The remaining 700 μL of recovery liquid is added to 10 mL of sterile LB liquid medium containing ampicillin (100 μg/mL) in a 50 mL sterile centrifuge tube, and incubate overnight at 37℃ with shaking.

The impact of copper sulfate on melanin production.

Objective

Verify the impact of copper sulfate on melanin production and find the optimal concentration.

Materials

Equipment: Biochemical incubator, laminar flow hood, etc.

Reagents: Bacterial culture, copper sulfate, etc.

Method

Add 0μL, 15μL, 30μL, 45μL, 60μL, 75μL of 100mg/mL CuSO4 to 100mL of overnight cultured bacterial culture, and observe the changes during cultivation.

Protein purification.

Objective

Obtain a high-purity, homogeneous protein sample for studying protein structure and function.

Materials

Reagents: PBS (Phosphate-Buffered Saline), Binding/Wash Buffer, EMtion Buffer, ddH₂O (double-distilled water), Ethanol, Coomassie Brilliant Blue, PAGE (Polyacrylamide Gel Electrophoresis) gels, etc.

Equipment: Ultrasonic cell disruptor, vertical electrophoresis apparatus, centrifuge, Ni-IDA pre-packed column, centrifuge tubes, etc.

Method

I. Centrifugation and washing of bacterial culture:

1. Resuspend the cells in 1×PBS (approximately 5 ml of PBS per ml of bacterial pellet) and perform ultrasonication under the following conditions: 4.0 seconds on, 6.05 seconds off, for a total of 15 minutes.

2. After lysing, centrifuge at 12,000 rpm for 10 minutes to collect the inclusion bodies, and wash the inclusion bodies several times with 1×PBS.

3. Add Binding/Wash Buffer containing a denaturant under denaturing conditions (approximately 5 ml of Binding/Wash Buffer per ml of inclusion bodies), and incubate at room temperature for 30-60 minutes; homogenization or ultrasonication may be required to completely dissolve the precipitate.

4. Centrifuge at 12,000 rpm for 30 minutes to remove the remaining insoluble materials, and carefully transfer the supernatant to a clean tube without touching the pellet.

II. Purification (gravity method):

1. Select a Ni-IDA pre-packed column of the appropriate size according to your needs, and allow the storage buffer to flow out by gravity.

2. Equilibrate the column with twice the column volume of Binding/Wash Buffer, using a flow rate of 0.5-1 ml/min to slowly drain the buffer from the resin.

3. Mix the protein extract with Binding/Wash Buffer at a 1:1 ratio to prepare the sample, ensuring the total volume of the sample is twice the column volume.

4. Apply the sample to the column, and collect the flow-through in a centrifuge tube. Reapply the remaining sample to enhance the binding of the sample to the resin.

5. Elute the His-tagged proteins from the column with twice the column volume of EMtion Buffer, repeating this step and storing each eluate separately until the absorbance at 280 nm approaches baseline.

6. Post-column treatment: Elute the column with five times the volume of Elution Buffer, then equilibrate with five times the volume of Binding/Wash Buffer. Finally, wash the column with five times the volume of ddH₂O, add a 20% ethanol preservation solution, and store at 2-8 degrees Celsius; do not freeze.

III. Sample preparation:

Take 25 μl of the sample and add 5 μl of protein loading buffer, for a total volume of 30 μl. Boil for 10-15 minutes, then spin down in a centrifuge.

IV. Purification verification:

Perform a purity check on the obtained protein sample using SDS-PAGE analysis and rapid Coomassie Brilliant Blue staining.

De-stain the PAGE gel with hot water for about 5 minutes.

Add staining solution and maintain a boiling state for about 5-10 minutes.

De-stain with hot water for about 5-10 minutes and observe the results.

Transformation with the Pahdd-6xH-21b/pelB--pkhppd--6xh--21b（+）plasmid.

Objective

Utilizing Escherichia coli as a chassis organism, the PBAD--pelbpkhdd plasmid is transformed into E. coli to express melanin.

Materials

Reagents: PBAD--pelBPkhdd glycerol stock and plasmid tube, sterile water, ampicillin (amp) antibiotic, IPTG, etc.

Equipment: Laminar flow hood, biochemical incubator, centrifuge, etc.

Method

1. Centrifuge the PBAD--pelBPkhdd glycerol stock and plasmid tube at 8000 rpm for 1 minute.

2. On the laminar flow hood, open the glycerol stock tube, streak onto an LB+AMP plate, and mark it. Open the plasmid tube on the laminar flow hood and add sterile water according to the label.

3. Transform the PBAD-pelBPkhdd plasmid into BL21(DE3) or BL21(DE3)pLysS, take 100 μl to spread on the LB+AMP plate, and the rest to inoculate into LB+AMP liquid medium.

4. Induce the expression of GFP, and observe the production of melanin. E. coli BL21(DE3) or BL21(DE3)pLysS (pET-21b(+)-eGFP-pc) is grown in LB+AMP medium with shaking at 37°C overnight. Then, 1/50 of the culture is inoculated into 200 mL of the same medium and grown with shaking at 180 rpm at 30°C until the OD600 reaches 0.6. Add 100 μg/ml IPTG and further incubate the bacterial culture under the same conditions to observe melanin production.

Identify the optimal conditions for expression by varying the concentration of arabinose.

Objective

Finding the optimal conditions for expression by varying the concentration of arabinose.

Materials

Reagents: LB medium, ampicillin, 20% L-arabinose, etc.

Equipment: Laminar flow hood, incubator, etc.

Method

1. Prepare LB medium: (Tryptone 10g, Yeast Extract 5g, NaCl 10g, Agar 20g)

2. Prepare 20% L-arabinose solution (50g L-arabinose, 200ml H₂O).

3. For each transformation sample, inoculate 2ml of E. coli in LB medium containing 50mg/ml ampicillin.

4. Grow overnight at 37°C with a shaking speed of 225-250rpm until OD600 reaches 1.2.

5. Label test tubes 1-5 and add 5ml of LB containing 100μg/ml ampicillin.

6. Inoculate each tube with 0.1ml of overnight culture.

7. Grow at 37°C with vigorous shaking until OD600 reaches 0.5 (i.e., logarithmic phase).

8. During cell growth, prepare four 10-fold serial dilutions of 20% L-arabinose with sterile water (e.g., 2%, 0.2%, 0.02%, and 0.002%).

9. Take 1μl of culture from each tube, centrifuge at maximum speed in a microcentrifuge for 30 seconds, and aspirate the supernatant.

10. Freeze the cell pellets at -20°C. This is the zero-time sample.

11. Add 0.1ml of L-arabinose to five 10ml cultures, with concentrations of 0.002%, 0.02%, 0.2%, 2%, and 20%.

12. Grow with shaking at 37°C for 4 hours.

13. After 4 hours, take a 1ml sample and repeat steps 9 and 10.

Extract the plasmid pPICZαA-PKrhdd.

Objective

Extract the plasmid pPICZαA-PKrhdd for transformation in Pichia pastoris to express melanin.

Materials

Reagents: Buffer S1, Buffer S2, Buffer S3, Buffer W1, Buffer W2, Buffer EB, etc.

Equipment: Pipettes, Adsorption column, Centrifuge, Filter membrane, etc.

Method

1. Take 1-5 ml of cultured bacterial liquid, centrifuge at 12,000 rpm for 1 min. Remove the supernatant.

2. Add 250 μl of Buffer S1 and mix well with a pipette.

3. Add 250 μl of Buffer S2, gently invert to mix well until the bacterial cells are completely lysed and the liquid becomes clear and viscous.

4. Add 350 μl of Buffer S3, immediately invert and mix 6-8 times, a white precipitate will form, let it stand at room temperature for 2 min, then centrifuge at 12,000 rpm for 5 min.

5. Carefully transfer the supernatant to the adsorption column, centrifuge at 12,000 rpm for 0.5 min and discard the filtrate in the collection tube.

6. Add 500 μl of Buffer W1 to the adsorption column, centrifuge at 12,000 rpm for 0.5 min and discard the filtrate.

7. Add 600 μl of Buffer W2, centrifuge at 12,000 rpm for 0.5 min at room temperature and discard the filtrate.

8. Add 500 μl of Buffer W2, centrifuge at 12,000 rpm for 0.5 min at room temperature, and discard the filtrate.

9. Dry: Centrifuge the adsorption column at 12,000 rpm for 2 min to spin dry.

10. Place the adsorption column into a new sterile 1.5 ml centrifuge tube, add 50-100 μl of elution buffer Buffer EB, let it stand at room temperature for 2 min, then centrifuge at 12,000 rpm for 1 min. The solution at the bottom of the centrifuge tube is the plasmid.

Chemical transformation

Objective

Transform the yeast Pichia pastoris with the plasmid pPICZαA-PKrhdd using a chemical method to express melanin.

Materials

Reagents: Buffer A, Buffer A, etc.

Equipment: Water bath, pipette, etc.

Method

1. When the fresh culture reaches an OD600 of 0.6-0.8, centrifuge at 3000×g for 3 minutes at room temperature.

2. Add 5 ml of Buffer A to resuspend the bacterial liquid, centrifuge at 3000×g for 3 minutes at room temperature, discard the supernatant, and retain the bacterial pellet.

3. Add 400 μl of Buffer A to resuspend the bacterial pellet, aliquot 100 μl per tube into 1.5 ml sterile centrifuge tubes, add 5.5 μl of DMSO to each tube and mix well. Directly transform and store at -80°C.

4. Combine 3 μg of linearized plasmid DNA with 10 μl of heat-denatured Carrier DNA, vortex to mix, and then add to 100 μl of competent cells, ensuring the total volume of the DNA sample does not exceed 20 μl.

5. Incubate in a 37°C water bath for 5 minutes, mix the sample up and down 1-2 times during the incubation to ensure complete melting of the frozen competent cells (this step can be omitted for freshly prepared cells).

6. After removing from the 37°C water bath, add 750 μl of Buffer B solution and mix by inverting.

7. Incubate in a 30°C water bath for 1 hour, mixing the sample up and down every 15 minutes to improve transformation efficiency.

8. Centrifuge at 2000×g for 10 minutes at room temperature, discard the supernatant, and resuspend the bacterial pellet in 750 μl of Buffer C.

9. Centrifuge at 2000×g for 10 minutes, discard the supernatant, and gently resuspend the bacterial liquid in 200 μl of Buffer C.

10. Culture the resuspended bacteria in gradients of 50 μl, 100 μl, and 200 μl on YPD + seocin plates.

Enzymatic digestion (pPLCZαA-PKrhdd)

Objective

In order to construct the recombinant plasmid pPLCZαA-PKrhdd in molecular cloning, restriction enzymes (restriction endonucleases) are used to cut the plasmid vector and the target DNA fragment at specific sequences, producing complementary sticky ends or smooth ends. The target DNA fragment is then inserted into the plasmid vector to create a recombinant DNA molecule. This recombinant molecule can subsequently be used to transform host cells, enabling the cloning and expression of the target gene.

Materials

Reagents: 10xCutEZTM Buffer, pPLCZαA-PKrhdd plasmid, Beyo Tast TM Pmel, ultrapure water, etc.

Equipment: Ice maker, pipette, centrifuge, etc.

Method

1. The reaction system for single enzyme digestion is as follows: perform the operation on ice.

Total reaction system	10xCutEZTMBuffer	extracted plasmid DNA	Beyo Tast TM Pmel	ultrapure water
20ul	2ul	0.5ul	1ul	16.5ul

Incubate at 37°C for 15 minutes.

Add the reagents from the table above, mix well with a pipette, and then briefly centrifuge to pellet the liquid at the bottom of the tube.

2. Incubate in a 37°C water bath for 5 minutes.

Electroporation transformation

Objective

Perform electroporation transformation of the plasmid pPICZαA-PKrhdd in Pichia pastoris to express melanin.

Materials

Reagents: Pichia pastoris strain, sterile water, IM sorbitol, linearized plasmid pPICZαA-PKrhdd, Zeocin+YPD plates, etc.

Equipment: Incubator, centrifuge, electroporation cuvettes, electroporator, ice maker, etc.

Method

1. Cultivate 5 ml of Pichia pastoris strain in a 50 ml conical flask overnight at 30°C.

2. Inoculate 500 ml of fresh medium with 0.1-0.5 ml of overnight culture in a 2 L flask and grow overnight to an OD600 of 1.3-1.5.

3. Centrifuge: 1500 rpm for 5 minutes, resuspend the pellet with 500 ml of 0°C sterile water.

4. Repeat the centrifugation step 3, resuspend the pellet with 250 ml of 0°C sterile water.

5. Repeat the centrifugation step 3, resuspend the pellet with 20 ml of 0°C IM sorbitol.

6. Repeat the centrifugation step 3, resuspend the pellet in 1 ml of 0°C IM sorbitol. The final volume should be 1.5 ml, keep it on ice and use it on the same day.

7. Mix 80 μl of the cells from step 6 with 5-10 μl of the enzymatically digested linearized plasmid pPICZαA-PKrhdd in 8-10 μl of sterile water and transfer to a 0.2 cm electroporation cuvette kept at 0°C, place on ice for 5 minutes.

8. Electroporate the cells (Set the electroporator to program Sc2).

9. Immediately add 1 ml of sorbitol to the cuvette, transfer the contents to a 1.5 ml tube, and incubate at 30°C for 1-2 hours without shaking.

10. Culture in gradients of 50, 100, and 200 μl on Zeocin+YPD plates.

11. Incubate at 30°C for 2-3 days.

12. Pick colonies for purification.

Building an algal-bacterial symbiosis system.

Objective

Microalgae provide oxygen to bacteria, while bacteria supply microalgae with carbon dioxide and other nutrients required by the algae. Meanwhile, the melanin secreted by the bacteria forms a protective shell against radiation for the microalgae.

Materials

Reagents: Microalgae (Chlorella, Gloeocapsa), bacterial strains (Pseudomonas, Escherichia coli expressing melanin), DPT medium, BG11 medium, etc.

Equipment: Algal culture chamber, laminar flow hood, etc.

Method

Cultivate microalgae to the logarithmic growth phase (for four days) using BG11 medium, and use a suitable overnight cultured bacterial liquid; mix the bacterial liquid in a certain ratio, and observe cell growth using an optical microscope.

Preparation of sodium alginate hydrogel

Objective

Add sodium alginate solution to a calcium chloride solution to prepare a hydrogel, which is further used for bacterial culture.

Materials

Reagents: Sodium alginate, anhydrous calcium chloride, pure water, etc.

Equipment: Water bath, plastic boxes, plastic shallow dishes, beakers, stirring rods, etc.

Method

1. Take 5.4g of sodium alginate and add it to 200ml of pure water.

2. Heat in a 55°C water bath while stirring until fully dissolved.

3. Prepare a 0.9% concentration calcium chloride solution (for this procedure, use 12.88g of calcium chloride and 1400ml of pure water).

4. Pour the sodium alginate solution into a shallow dish and slowly immerse it in the calcium chloride solution, then let it sit until it is completely solidified (this can be observed by gently shaking the dish).

5. Remove the sheet-like gel (discard any residue of sodium alginate in the dish), and place it in the calcium chloride solution for soaking.

Preparation of alginate hydrogel for bacterial culture experiment.

Objective

Replace the water inside the alginate hydrogel with LB liquid medium containing engineered E. coli, and then soak it in the bacterial liquid to culture E. coli within the hydrogel, verifying whether E. coli can survive within the hydrogel.

Materials

Reagents: Overnight cultured E. coli, ampicillin (amp) + LB liquid medium, hydrogel, etc.

Equipment: Incubator, laminar flow hood, etc.

Method

1. Remove the prepared hydrogel stored in 4°C PBS and soak it in ampicillin-containing LB liquid medium.

2. Place it at 4°C overnight.

3. Take out the hydrogel and place it in the liquid medium containing E. coli for incubation at room temperature overnight.

Optimization of calcium ion concentration in the crosslinking of sodium alginate hydrogel.

Objective

Verify the optimal concentration of calcium chloride for crosslinking sodium alginate.

Materials

Reagents: Sodium alginate, sterile water, calcium chloride, etc.

Equipment: Water bath, stirrer, etc.

Method

1. Prepare a high-viscosity sodium alginate solution of 100 ml (stir until completely dissolved in a 60°C water bath).

2. Prepare calcium chloride solutions of 100 ml at gradient concentrations: 2%, 1%, 0.5%, 0.25%, and 0.125%.

3. Use a 6-well plate as a template to record the gelation time.

Preparation of double-layer hydrogel.

Objective

Encapsulate the E. coli expressing GFP (Gloeocapsa) with sodium alginate and LB liquid medium, and then coat it with a polyacrylamide shell in a nitrogen atmosphere to verify whether the bacteria can grow within the hydrogel.

Materials

Reagents: Alginate, MilliQ water, ampicillin (amp) and other antibiotics, etc.

Equipment: Autoclave, etc.

Method

I. Preparation of Alginate Cores

1. Dissolve medium-viscosity sodium alginate (Sigma-Aldrich A2033, sodium alginate) in MilliQ water (with a resistivity of 18.2 MΩ·cm) to make a 5% alginate solution.

2. Then sterilize at 120°C in an autoclave for 20 minutes to ensure sterility.

3. Mix fresh bacterial culture (~109 cells/ml in LB broth with antibiotics) with the alginate solution at a 1:1 volume ratio, resulting in a final alginate concentration of 2.5 wt%.

4. Load the bacteria-alginate premix into a syringe and place it on parafilm to form droplets.

5. Immerse the droplets in a 5% calcium chloride (ionic crosslinking agent, Sigma-Aldrich 223506) solution for 15 minutes to solidify.

II. Coating with a Tough Hydrogel

1. Prepare a precursor solution consisting of 2 wt% sodium alginate, 30 wt% acrylamide, 0.046 wt% ammonium persulfate, and 0.015 wt% N,N-methylenebisacrylamide.

2. Degas the precursor solution thoroughly.

3. Before the coating process, mix the viscous precursor solution with the accelerator N,N,N′,N′-tetramethylethylenediamine (0.1% of the precursor solution by volume) to form a rapidly curing pre-gel solution.

4. Immerse the alginate gel cores made in the previous step in the pre-gel solution to form an adjustable thin shell layer of about 100 - 1000 μm around the core under a nitrogen atmosphere.

5. To stabilize the shell, immerse the hydrogel in MES buffer solution (0.1 M MES and 0.5 M NaCl, pH 6.0) containing crosslinking agents and catalysts for 3 hours to form the shell. The crosslinking agents and catalysts include 0.00125 wt% 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 0.000375 wt% N-hydroxysuccinimide, and 0.00075 wt% adipic acid dihydrazide.

Irradiation of ADE2-gal1-egfp-CYC1+ yeast strain.

Objective

Exposing yeast to radiation for extended periods can lead to the death of the yeast. By irradiating the yeast with X-rays of the same intensity for different durations and observing their survival rates, one can assess the yeast’s radioresistance.

Materials

Reagents: YPDA medium, SD-20 solid medium, ADE2-gal1-egfp-CYC1+

Equipment: Irradiation device, incubator, pipette, tips, solid medium

Method

1. Remove the solid culture medium from the refrigerator and place it in a 29°C incubator to thaw.

2. Thaw the bacterial culture.

3. First, aspirate 500 μl of YPDA medium, then 0.5 μl of bacterial culture, and mix well in a small test tube.

4. Aspirate 100 μl of the diluted culture and evenly drop it onto the surface of the solid medium, spreading it evenly with a spreader.

5. Place the solid culture medium in the incubator to allow the liquid to be absorbed, and then it can be put into the irradiation device for exposure.

Measure the OD value.

Objective

By measuring the absorbance at a specific wavelength, the quantity of microorganisms in a cell suspension can be estimated. The OD value is proportional to the cell concentration, thus it can be used to monitor cell growth; by measuring the OD value at regular intervals, a growth curve of the cells can be plotted, providing insights into the growth rate and phase of the cells.

Materials

Reagents: Bacterial culture, PBS, etc.

Equipment: Microplate, microplate reader, pipette, etc.

Method

1. Sample preparation: First, prepare microbial cultures, such as suspensions of bacteria or yeast.

2. Select the appropriate wavelength and measurement range of 280-700 nm.

3. Using a microplate reader, with PBS as a control, add 200 μl of the culture to each well of the microplate, and then measure its OD value.

4. Record the measured OD values; these data can be used to plot growth curves or assess cell density.

5. Sample dilution: If the OD value is too high (typically greater than 1.0), it may be necessary to dilute the sample and measure again to ensure the accuracy of the measurement results.

UV irradiation experiment

Objective

Through the UV irradiation experiment, it is determined whether our bacterial strains have radioresistance capabilities.

Materials

Reagents: Bacterial culture, saline solution, antibiotics

Equipment: Dark chamber UV analysis, thin-layer transmission and reflection spectrophotometer, fluorescence photometer, laminar flow hood, etc.

Method

Dilute the overnight cultured bacterial culture with saline solution in a gradient, spread it onto agar plates, and expose them to UV light for 0h, 2h, 4h, and 8h; cultivate under the same conditions for the same duration and count the number of colonies.