Protocol

Introduction

A calcium chloride (CaCl₂) based method for preparing competent bacterial cells, which enhances the permeability of bacterial cell membranes to facilitate the uptake of exogenous plasmids.

Application

Plasmid transformation experiments.

Material

LB liquid medium (without antibiotics), 0.1 mol/L calcium chloride (CaCl₂) solution, 15% glycerol, pre-chilled 50 mL sterile centrifuge tubes, pre-chilled 1.5 mL sterile centrifuge tubes, constant-temperature shaking incubator, high-speed centrifuge, -80°C refrigerator.

Procedure

1. Strain activation (12-16 hours in advance)
   1. Inoculate a single colony into 5 mL of LB liquid medium (without antibiotics).
   2. Incubate in a 37°C constant-temperature shaking incubator at 180-220 rpm for 12-16 hours to obtain the seed culture.
2. Scale-up culture
   1. Inoculate the activated seed culture into 50 mL of LB liquid medium at a 1:100 ratio.
   2. Incubate at 37°C with shaking at 200 rpm for 4-6 hours until the OD₆₀₀ reaches 0.3-0.5.
3. Cell collection
   1. Transfer 50 mL of the bacterial culture into pre-chilled 50 mL sterile centrifuge tubes, and centrifuge at 4000 rpm at 4°C for 10 minutes.
   2. After centrifugation, discard the supernatant. Gently resuspend the cell pellet with 10 mL of ice-pre-chilled 0.1 mol/L CaCl₂ solution (operate gently to avoid cell rupture by pipetting).
   3. Let the mixture stand on ice for 30 minutes, then centrifuge at 4000 rpm at 4°C for 10 minutes, and discard the supernatant again.
4. Washing and resuspension
   1. Resuspend the cell pellet again with 5 mL of ice-pre-chilled 0.1 mol/L CaCl₂ solution, and let it stand on ice for 15 minutes.
   2. Centrifuge at 4000 rpm at 4°C for 10 minutes, discard the supernatant. Add 1 mL of CaCl₂ solution containing 15% glycerol, and gently pipette to resuspend the cells (the cells are now competent cells).
5. Aliquoting and storage
   1. Quickly aliquot the resuspended competent cells into pre-chilled 1.5 mL sterile centrifuge tubes, 100 μL per tube (avoid repeated freeze-thaw cycles).
   2. Immediately store the tubes in a -80°C refrigerator.

Introduction

To amplify stab cultures delivered by the company, which contain plasmids carrying the target gene. The amplified bacteria will be used for Bacterial Cell Plasmid Extraction.

Applications

Amplification of indigo synthesis gene plasmid stab cultures, blue light-controlled synthesis gene plasmid, halogenase synthesis gene plasmid, red light-controlled synthesis gene plasmid, nitrilase synthesis gene plasmid, green light-controlled plasmid.

Materials

LB medium, corresponding antibiotic for plasmid resistance, stab culture inoculated on solid medium (provided by the company).

Procedure

1. Pick the stab culture: Use a 200 µL pipette tip to collect the gel containing the stab culture.
2. Transfer the gel into a 10 mL tube containing 3 mL of LB medium. Add the corresponding antibiotic to a final concentration of 50–100 µg/mL.
3. Incubate overnight (approx. 12–16 h) at 37°C with shaking at 190 rpm.

Introduction

To extract the target plasmid from bacterial cells.

Applications

Extraction of pCDFDuet-1, pRSFDuet-1, pCDFDuet-1-PhlF, pCDFDuet-1-PhlF-YF1-fixJ, pRSFDuet-1-EcTnaA, pRSFDuet-1-EcTnaA-MaFMO,pET28a(+),pET28a(+)-fre-sttH,pCDFDuet-1-PadC4-BphO-YhjH-MrkH, pRSFDuet-1-hol-pcyA-mini_ccaS-ccaR, pCDFDuet-1-txtE-txtD-Fdr-Fdx (Extraction of plasmids and ligation products).

Materials

TIANprep Mini Plasmid Kit (Tiangen: DP103-02)

Procedure

1. Place the CP3 column into the collection tube. Add 500 µL of BL buffer to the column. Centrifuge at 12,000 rpm for 1 min. Discard the flow-through and reassemble the column with the collection tube.
2. Take 3–12 mL of overnight bacterial culture and transfer to a centrifuge tube. Centrifuge at 12,000 rpm for 1 min and discard the supernatant.
3. Add 250 µL of P1 buffer (with RNase A) to the pellet. Resuspend the pellet completely using a pipette or vortex mixer.
4. Add 250 µL of P2 buffer to the tube. Invert the tube gently 6–8 times to lyse the cells completely.
5. Add 350 µL of P3 buffer to the tube. Invert immediately and gently 6–8 times to mix thoroughly. Centrifuge at 12,000 rpm for 10 min.
6. Transfer the supernatant to the CP3 column placed in a collection tube. Centrifuge at 12,000 rpm for 1 min. Discard the flow-through and reassemble the column.
7. For BL21 strains: Add 500 µL of PD buffer to the CP3 column. Centrifuge at 12,000 rpm for 1 min. Discard the flow-through and reassemble the column.
8. Add 600 µL of PW buffer (with absolute ethanol) to the CP3 column. Centrifuge at 12,000 rpm for 1 min.
9. Repeat step 8.
10. Place the CP3 column in the collection tube and centrifuge at 12,000 rpm for 2 min. Incubate at 55°C on a metal bath for 2 min.
11. Transfer the CP3 column to a clean centrifuge tube. Add 20 µL of EB elution buffer to the center of the membrane. Let stand at room temperature for 2 min, then centrifuge at 12,000 rpm for 2 min.
12. Add 10 µL of EB elution buffer to the center of the membrane. Let stand at room temperature for 2 min, then centrifuge at 12,000 rpm for 2 min.
13. Add 20 µL of ddH₂O to the center of the membrane. Let stand at room temperature for 2 min, then centrifuge at 12,000 rpm for 2 min.
14. If the plasmid is to be used for sequencing, replace both EB elution buffer and ddH₂O in steps 11–13 with ddH₂O.

Introduction

To detect the presence of target DNA bands.

Applications

Detection of PCR products, restriction/ligation products, digestion results, and target plasmids.

Materials

50× TAE buffer, agarose (Shanghai Yuanye Biotechnology, S14003), GoldView nucleic acid stain (Shanghai Yuanye Biotechnology, R20977), electrophoresis power supply, electrophoresis tank, gel imaging system.

Procedure

1. Dilute 16 mL of 50× TAE buffer to 800 mL with ddH₂O to prepare 1× TAE working solution.
2. Prepare PCR samples: Mix 50 µL of sample with 6 µL of 10× loading buffer.
3. Prepare agarose gel: Mix 1 g agarose with 100 mL of 1× TAE in a 250 mL conical flask. Dissolve completely by heating in a microwave.
4. After dissolution, cool slightly and add 5 µL GoldView stain. Mix gently.
5. Pour the solution into a gel casting tray and insert a comb. Use a comb with small wells for detection and large wells for product recovery.
6. After solidification, remove the comb and place the gel in the electrophoresis tank. Submerge the gel in 700 mL of 1× TAE buffer.
7. Load samples: For large wells: Load 25 µL marker and 50 µL sample per well. For small wells: Load 2 µL marker and 10 µL sample per well.
8. Run electrophoresis at 120 V for approximately 20 min. Turn off power, remove the gel, and visualize bands using a gel imaging system.

Introduction

To recover the target DNA fragment from the correct band after agarose gel electrophoresis. Used for collecting PCR products and digested vector fragments.

Applications

Recovery of products from agarose gel electrophoresis for genes such as MaFMO, EcTnaA, PhlF, YF1, fixJ, and fre-sttH after PCR amplification.

Materials

TIANquick Midi Purification Kit (TIANGEN, Cat#: DP214-02)

Procedure

1. Column equilibration:Add 500 µL of BL buffer to the CB2 column (placed in a collection tube). Centrifuge at 12,000 rpm (~13,400 ×g) for 1 min. Discard the flow-through and reassemble the column. (Use columns processed on the same day.)
2. Excise the target DNA band from the agarose gel (remove excess gel as much as possible) and transfer it to a clean centrifuge tube. Weigh the gel slice.
3. Add a volume of PC buffer equal to the weight of the gel slice. Incubate at 50°C for about 10 min, gently inverting the tube periodically to ensure complete dissolution of the gel.
4. Transfer the resulting solution to a CB2 column (placed in a collection tube). Centrifuge at 12,000 rpm for 1 min. Discard the flow-through and reassemble the column.
5. Add 600 µL of PW buffer (ensure absolute ethanol has been added before use) to the CB2 column. Centrifuge at 12,000 rpm for 1 min. Discard the flow-through and reassemble the column.
6. Repeat step 5.
7. Place the CB2 column in the collection tube and centrifuge at 12,000 rpm (~13,400 ×g) for 2 min to remove residual wash buffer. Dry the column at 55°C on a metal bath for 2–5 min.
8. Transfer the CB2 column to a clean 1.5 mL EP tube. Add 20 µL of EB elution buffer dropwise to the center of the membrane (pre-heat EB at 55°C for 5–10 min to improve recovery efficiency). Let stand at room temperature for 2 min, then centrifuge at 12,000 rpm (~13,400 ×g) for 2 min. Then add 10 µL of EB elution buffer dropwise to the center of the membrane, let stand for 2 min, and centrifuge again at 12,000 rpm for 2 min to collect the DNA solution.
9. Measure the concentration of the recovered DNA using Nanodrop.

Introduction

To amplify target genes from plasmid DNA.

Applications

Amplification of MaFMO, TnaA, PhlF, sttH-fre, YF1, and fixJ.

Reagents

Deionized water, template DNA, DNA polymerase (Taq or high-fidelity enzyme), forward and reverse primers (Tsingke Biotechnology), dNTP (TaKaRa, Cat#: AM31089A), 10× buffer (AM41786A).

Procedure

1. Add reagents to a PCR tube in the following order, keeping the enzyme and dNTP on ice: deionized water: 36 μL, 10× buffer: 5 μL, Forward and reverse primers: 4 μL total (2 μL each), dNTP: 4 μL, Template DNA: 1 μL, DNA polymerase (Taq or high-fidelity): 0.5 μL
2. Seal the tube and place it in the PCR machine. Set the cycling parameters as follows:

Step	Temperature (°C)	Time	Cycles
Pre-denaturation	95	5 min	1
Denaturation	95	30 sec	35
Annealing	Tm–5	30 sec	35
Extension	72	60 sec/kb	35
Final extension	72	10 min	1
Hold	12	∞	1

Note: If the Tm values of the two primers differ by >2°C, perform a gradient PCR. Set the annealing temperature range from the highest Tm decreasing in 1–2°C increments to the lowest Tm. The number of cycles per temperature is 36 divided by the number of temperature points.

Introduction

To generate compatible ends in target genes and plasmids for subsequent ligation.

Applications

Digestion of pRSFDuet-1, EcTnaA, pCDFDuet-1, phlf, MaFMO, pRSFDuet-1-EcTnaA, pet28a, SttH, fre.

Materials

Restriction enzymes (Vazyme C407-01, C412-01, C402-01, C401-01, C410-01, C420-01, C409-01, C311-02), DNA substrates (plasmids and target genes), deionized water, 10× buffer (provided with enzymes).

Procedure

1. Prepare the digestion reaction as follows:

   Reagent	Volume
   ddH₂O	18 μL
   Enzyme 1	1 μL
   Enzyme 2	1 μL
   10× Buffer	5 μL
   Plasmid/Gene	25 μL

2. Incubate at 37°C in a water bath: Linear fragments: 0.5–1.5 h; Circular plasmids: 2–3 h.

Introduction

To introduce plasmids into bacterial cells for amplification or expression.

Applications

Transformation of pCDFDuet-1, pRSFDuet-1, pCDFDuet-1-PhlF, pCDFDuet-1-PhlF-YF1-fixJ,pRSFDuet-1-EcTnaA,pRSFDuet-1-EcTnaA-MaFMO,pET28a(+),pET28a(+)-fre-sttH,pCDFDuet-1-PadC4-BphO-YhjH-MrkH,pRSFDuet-1-hol-pcyA-mini_ccaS-ccaR,pCDFDuet-1-txtE-txtD-Fdr-Fdx (Transformation of empty vectors and ligation products).

Materials

pCDFDuet-1,pRSFDuet-1,pCDFDuet-1-PhlF,pCDFDuet-1-PhlF-YF1-fixJ,pRSFDuet-1-EcTnaA,pRSFDuet-1-EcTnaA-MaFMO,pET28a(+) (Sangon Biotech,Cat#: B540183), pET28a(+)-fre-sttH,pCDFDuet-1-PadC4-BphO-YhjH-MrkH,pRSFDuet-1-hol-pcyA-mini_ccaS-ccaR, pCDFDuet-1-txtE-txtD-Fdr-Fdx,DH5α Super Competent Cells (Beyotime Biotechnology, D1031S), DH5α Competent Cells (Sangon Biotech, B528413), BL21 Super Competent Cells (Beyotime Biotechnology, D1013S), BL21(DE3) Competent Cells (Sangon Biotech, B528414)

Procedure

1. Mix 10 μL of the restriction-ligation reaction with 100 μL of competent cells in a clean bench. Incubate on ice for 30 min outside the clean bench.
2. Heat-shock at 42°C for 90 sec.
3. Place on ice for 3 min.
4. For ligation products, add 900 μL of LB liquid medium (without antibiotics) for recovery. Incubate at 37°C with shaking at 120 rpm for 1 h.
5. Centrifuge at 5,000 rpm for 5 min. Remove most of the supernatant and resuspend the pellet in the remaining medium.
6. Spread the resuspended culture onto pre-warmed solid medium containing antibiotics. Incubate overnight.

Introduction

To verify whether the engineered bacteria contain correctly ligated expression plasmids after transformation with ligation products.

Applications

Verification of transformation results for ligation products including pCDFDuet-1-PhlF, pCDFDuet-1-PhlF-YF1-fixJ, pRSFDuet-1-EcTnaA, pRSFDuet-1-EcTnaA-MaFMO, pET28a(+)-fre-sttH, pCDFDuet-1-PadC4-BphO-YhjH-MrkH, pRSFDuet-1-hol-pcyA-mini_ccaS-ccaR, pCDFDuet-1-txtE-txtD-Fdr-Fdx.

Materials

Deionized water, 2× Rapid Taq Master Mix (Vazyme, Cat#: P222-01), forward and reverse primers (synthesized by Tsingke Biotechnology Co, Ltd.), dNTP (TaKaRa, Cat#: AM31089A)

Procedure

1. Add 80 µL of 2× Rapid Taq Master Mix, 64 µL of ddH₂O, and 8 µL each of forward and reverse primers to tube No. 8 of a PCR strip tube. The total reaction volume is 160 µL.
2. Aliquot 29 µL of the mixture into tubes No. 1–7. Use tube No. 8 as a negative control.
3. Pick a single colony, briefly dip the tip into the PCR mixture, and then transfer it to a 10 mL culture tube containing 3 mL of LB medium with the corresponding antibiotic. Incubate at 37°C with shaking at 190 rpm.
4. Perform PCR with the following program:

Step	Temperature (°C)	Time	Cycles
Pre-denaturation	95	8 min	1
Denaturation	95	30 sec	35
Annealing	Tm–5	30 sec	35
Extension	72	15 sec/kb	35
Final extension	72	10 min	1
Hold	12	∞	1

Note: If the Tm values of the two primers differ by more than 2°C, perform a gradient PCR. Set the annealing temperature range from the highest Tm decreasing by 1–2°C increments to the lowest Tm. The number of cycles per temperature is 36 divided by the number of different temperatures.

Introduction

This program uses recombinase to directionally clone the PCR product with the same sequence at both ends into the linearized plasmid DNA to generate the recombinant plasmid carrying the target gene.

Application

Changing the promoter of pRSFDuet-1-EcTnaA-MaFMO plasmid.

Materials

Linearized vector of pRSFDuet-1-EcTnaA-MaFMO plasmid after the first inverse PCR, PCR product of promoter pPphlF, ddH₂O, DH5α competent cells, ClonExpress MultiS One Step Cloning Kit-C113 from Vazyme (C113-02).

Procedure

1. Calculate the volume that the recombination reaction needs
   The formula of calculating the suitable volume of target genes and linearized vector is [0.02 X the number of bases]ng.
2. Formulate the recombination reaction system
   1. Add YμL the Nth insert (n≤5) and XμL linearized vector to a 200μL PCR tube.
   2. Add 4μL 5xCE Multis Buffer to the tube.
   3. Add 2μL Exnase Multis to the tube.
   4. Add ddH₂O to the tube to make the total volume of the mixing system reach 20μL.
   5. Mix the mixture system and briefly centrifuge.
3. Start reacting
   Incubate the tubes in a PCR instrument at 37°C for 30 minutes, and cool the tubes at 4°C after completion.
4. Transformation
   1. Defrost the stored DH5α competent cells on ice.
   2. Add 10μL recombinant plasmid to 100μL competent cells, flick the tube wall to mix well, and let it stand for 30min on ice.
   3. After heat shocking the competent cells 90s in a water bath at 42°C, cool it on ice immediately for 3min.
   4. Add 900μL LB liquid medium to the bacterial solution, and shake the bacteria solution at 200rpm for 1h at 37°C.
   5. Preheat the corresponding resistant LB solid medium in an incubator at 37°C.
   6. Centrifuge the tube at 5000 rpm for 5 min and pour off 900μL of supernatant.
   7. Resuspend the cells with the remaining liquid medium and use glass beads to evenly spread the bacterial suspension on prewarmed LB solid mediums.
   8. Incubate the cells in an incubator at 37°C for 12-16 hours.

Introduction

To use the lactose analog IPTG to induce transcription of the target gene under the control of a T7 promoter in the expression vector, resulting in production of the target protein.

Applications

Expression of MaFMO, EcTnaA, PhlF, YF1, FixJ, and SttH-Fre proteins.

Materials

LB liquid medium, appropriate antibiotic, glucose fermentation medium, 1 M IPTG stock solution, successfully transformed BL21(DE3) strain.

Procedure

1. Inoculate successfully transformed BL21(DE3) into a 10 mL culture tube and incubate overnight for 12–16 h.
2. Perform a 1:100 dilution for expansion: Transfer 200 µL of overnight culture to 20 mL of liquid medium containing the appropriate antibiotic at working concentration.
3. Incubate at 37°C with shaking at 200 rpm for approximately 3 h, until OD₆₀₀ ≈ 1 (measured by UV spectrophotometer).
4. Add IPTG to a final concentration of 0.5–1 mM. Induce expression at 16°C for 16–20 h.

Introduction

To rapidly separate and identify the expression of insoluble proteins (in pellet) and soluble proteins (in supernatant) based on molecular weight, providing preliminary identification of the target protein.

Applications

Detection of MaFMO, EcTnaA, PhlF, YF1, FixJ, SttH-Fre, hol, PcyA, mini_CcaS, CcaR, TxtE, TxtD, Fdr, and Fdx proteins.

Materials

SDS-PAGE precast gel (Beyotime Biotechnology, P0052A), 1× PBS (Yongjin Biology, YJ0014), 5× SDS loading buffer (Wanlei Biotechnology, 22A071), Color Mix Protein Marker (ABclonal, RM19001), 5× running buffer.

Procedure

1. Cell Harvesting: Collect bacterial culture by centrifugation at 6,000 rpm, 4°C for 10 min. Wash the pellet with 1× PBS, resuspend, and repeat centrifugation 3–4 times.
2. Cell Lysis: Perform all steps on ice. Sonicate the resuspended cells (3 sec ON, 7 sec OFF, 35% amplitude) for 5 min. Centrifuge at 14,000 rpm, 4°C for 10 min. Collect the pellet and supernatant.
3. Pellet Treatment: Resuspend a small amount of pellet in 160 μL PBS. Centrifuge at 14,000 rpm, 4°C for 10 min. Collect the supernatant as the "pellet fraction".
4. Sample Preparation: Mix 160 μL of supernatant or pellet fraction with 40 μL of 5× loading buffer. Heat at 95°C for 5 min (vent tubes at 2 min). Cool to room temperature before loading.
5. Buffer Dilution: Dilute 5× running buffer to 1× with ddH₂O.
6. Gel Assembly: Remove the precast gel from packaging and install it in the electrophoresis tank.
7. Buffer Addition: Fill the inner chamber with 1× running buffer until it overflows. Add 1× running buffer to the outer chamber to cover the electrodes.
8. Comb Removal: Slowly remove the comb vertically. Ensure the inner chamber is completely filled with buffer.
9. Loading: Load 5 μL protein marker and 20 μL samples into wells according to experimental groups.
10. Electrophoresis: Run at 80 V for ~20 min (until bromophenol blue enters the separation gel). Switch to 120 V until the dye front reaches the bottom. Monitor and replenish the inner chamber buffer as needed.
11. Gel Removal: Carefully disassemble the plates using a gel releaser. Discard the stacking gel and unnecessary portions.
12. Staining: Incubate the gel in Coomassie Brilliant Blue R-250 staining solution. Shake at 150 rpm for 30 min or microwave until boiling, then shake for 5 min.
13. Destaining: Replace with destaining solution and shake at 150 rpm. Destain for 10–30 min, replace solution, repeat twice, and destain overnight or microwave until boiling, then shake for another 5 min.

Introduction

To perform immunoblot analysis of SDS-PAGE-separated proteins using commercial antibodies targeting fusion tags, enabling detection of target protein presence, molecular weight, and expression levels.

Applications

Detection of MaFMO, EcTnaA, PhlF, YF1, FixJ, SttH-Fre, hol, PcyA, mini_CcaS, CcaR, TxtE, TxtD, Fdr, and Fdx proteins.

Materials

Cellulose acetate/NC membrane, transfer filter paper, transfer apparatus, TBST, commercial primary antibodies: S-tag Mouse Monoclonal Antibody (Huabio, EM50105), His-tag Mouse McAb (San Ying, 66005-1-Ig); commercial secondary antibody: HRP-conjugated Goat anti-Mouse IgG (H+L) (ABclonal, AS003); 5% skim milk; Enhanced ECL Kit (Solution I) (ABclonal, RM00021); ECL Enhanced Kit (Solution II) (ABclonal, RM00021); 1× transfer buffer.

Procedure

1. Wet Transfer: Place the transfer cassette in a tray and add 1× transfer buffer to submerge the sandwich. Sequentially place sponge, filter paper, PVDF/NC membrane, SDS-PAGE gel, filter paper, and sponge. Avoid bubbles. Ensure the sandwich is fully immersed.
2. Insert the sandwich into the transfer holder and place it in the electrophoresis tank. Fill the tank with 1× transfer buffer until the sandwich is completely covered. Perform all steps on ice.
3. Run transfer at 300 mA constant current for 1.5 h.
4. Membrane Retrieval: Remove the membrane and place it in a water-filled container to keep it wet.
5. Blocking: Discard water, add blocking buffer (5% skim milk), and incubate at room temperature for 1 h.
6. Washing: Discard milk, wash with 1× TBST on a shaker 3 times (5 min each).
7. Primary Antibody Incubation: Prepare diluted primary antibody in 5% BSA according to the manufacturer’s instructions. Add 5 mL diluted primary antibody, and incubate overnight at 4°C on a shaker.
8. Washing: Wash with 1× TBST 3 times (5 min each) to remove residual primary antibody.
9. Secondary Antibody Incubation Discard TBST, add secondary antibody, and incubate at room temperature for 1 h.
10. Washing: Wash with 1× TBST 3 times (5 min each) to remove residual secondary antibody.
11. ECL Solution Preparation: Mix 1 mL Solution I and 1 mL Solution II. Add to the membrane, ensuring full coverage.
12. Detection: Develop and visualize results.

Introduction

A fermentation method for enhancing the production of indigo and its derivatives through a fed-batch strategy.

Application

Pigment synthesis.

Material

LB liquid medium, antibiotic, IPTG solution, glucose, L-tryptophan, 0.22 μm filter-sterilized.

Procedure

1. Add 5 mL of LB liquid medium and 5 μL of Kanamycin (Kan) stock solution (50 mg/mL) to a 10 mL culture tube. Pick an appropriate single colony from the agar plate using a pipette tip, immerse the tip in the culture medium, and incubate in a shaking incubator at 37°C with 190 rpm for 12-14 hours.
2. In a 250 mL Erlenmeyer flask, add 25 mL of LB liquid medium, 20 μL of Kan stock solution (50 mg/mL), and 5 mL of the overnight culture. Incubate in a shaking incubator at 37°C with 190 rpm until the OD₆₀₀ reaches 0.8-1.0.
3. Add 5 μL of IPTG stock solution (1 M) to achieve a working concentration of 0.2 mM. Incubate in a shaking incubator at 16°C with 190 rpm for 16 hours.
4. Add 3.71 mL of filter-sterilized glucose concentrate (44 g/L, passed through a 0.22 μm syringe filter) and 7.15 mL of filter-sterilized L-tryptophan solution (40 mM, passed through a 0.22 μm syringe filter).
5. Incubate in a shaking incubator at 30°C with 200 rpm for 24 hours.
6. Add another 3.71 mL of glucose concentrate and 7.15 mL of L-tryptophan solution to the culture. Continue incubation in a shaking incubator at 30°C with 200 rpm for an additional 24 hours.

Introduction

A method for cultivating fre-sttH engineering bacteria, inducing enzyme expression via red light, and achieving the synthesis of 6-bromoindole and its derivatives through subsequent transformation reactions.

Material

LB liquid medium, streptomycin, kanamycin, L-tryptophan (Trp), potassium bromide (KBr), glucose, 50 mM phosphate buffer (pH 7.8), E. coli BL21(DE3)ΔtnaA (host strain), tnaA/MaFMO bacterial strain, LED strip (emission peak at 710 nm, total luminous flux of 240 lm).

Procedure

1. Cultivation and enzyme induction of fre-sttH engineering bacteria
   1. Seed culture: Pick a positive single colony, inoculate into 5 mL of LB liquid medium containing streptomycin (100 μg/mL) + kanamycin (50 μg/mL), and incubate at 37°C with 200 rpm for 12 hours (OD₆₀₀≈2.0).
   2. Scale-up culture: Transfer 500 μL of the seed culture to 50 mL of LB medium with the same double antibiotics, incubate at 37°C with 200 rpm until OD₆₀₀ reaches 0.6-0.8.
   3. Cool down to 16°C, continue incubation with 200 rpm for 16 hours. Meanwhile, irradiate with an LED strip (710 nm) for 12 hours, followed by 4 hours in the dark.
2. Cell collection and washing
   1. Cell collection: Transfer 50 mL of the induced bacterial culture to a centrifuge tube, centrifuge at 3500×g at 4°C for 10 minutes, and discard the supernatant.
   2. Cell washing: Resuspend the cell pellet with 50 mM phosphate buffer, gently pipette to mix, then centrifuge again at 3500×g at 4°C for 10 minutes, discard the supernatant. Repeat twice.
   3. Cell resuspension: Resuspend the washed cells with 50 mM phosphate buffer to an OD₆₀₀ of 20.
3. Whole-cell bromination reaction
   Configure reaction system:

   Component	Amount
   Resuspended fre-sttH bacterial cells	~5 mL
   L-tryptophan (Trp, 40 mM/L)	37.5 mL
   KBr	1.785 g
   Glucose (44 g/L)	7 mL
   50 mM phosphate buffer (pH 7.8)	Make up to 50 mL total volume

   
   Incubate at 30°C with 200 rpm for 24 hours. Then centrifuge at 3500×g at 4°C for 10 minutes, discard the pellet, and collect the supernatant containing 6-Br-Trp.
4. Transformation reaction between supernatant and tnaA/MaFMO bacteria

   Configure reaction system:

   Component	Amount
   Supernatant containing 6-Br-Trp	35 mL
   Resuspended tnaA/MaFMO bacterial cells	~5 mL
   Glucose (44 g/L)	3.5 mL
   50 mM phosphate buffer (pH 7.8)	Make up to 50 mL total volume

   Incubate at 30°C with 200 rpm for 8-12 hours.

Introduction

Using organic solvents to extract pigment from engineered bacterial strains.

Material

DMSO

Procedure Ⅰ

1. Add 500 μL of fermentation broth to a 1.5mL EP tube and collect the pellet by high-speed centrifugation (12,000 g, 5 min).
2. Add absolute methanol to rinse the pellet in order to remove impurities.
3. Redissolve the indigo with 2 mL of dimethyl sulfoxide (DMSO).

Procedure Ⅱ

1. Add all fermentation broth to a 50mL centrifuge tube. Centrifuge the cell cultures at 5000 rpm for 10 min to collect the cell pellet.
2. Add 2mL DMSO to resuspend the pellet. The mixture was sonicated on ice for 30 cycles; each cycle comprised 1-s sonication and 6-s break.
3. Centrifuge the sonicated mixture at 10,000 rpm for 5 minutes, collect the supernatant for indigo analysis. Repeat the extraction process until no blue color is detected in the supernatant.

Component	Manufacturer	Amount
NaCl	AOBOXING Biotech	10.0g/L
Tryptone	AOBOXING Biotech	10.0g/L
Yeast Extract	Sangon Biotech	5.0g/L
Agar*	AOBOXING Biotech	20.0g/L
Corresponding antibiotics	Corresponding manufacturer	Corresponding amount

*When preparing solid culture media, add Agar.

Component	Manufacturer	Amount
Na₂HPO₄	Sinopharm Chemical Reagent	6.8g/L
KH₂PO₄	Sinopharm Chemical Reagent	3g/L
NaCl	AOBOXING Biotech	0.5g/L
NH₄Cl	Aladdin Biochemical Technology	1g/L
MgSO₄·7H₂O	Sinopharm Chemical Reagent	0.49g/L
CaCl₂	Aladdin Biochemical Technology	0.02g/L
Glucose	Fuchen (Tianjin) Chemical Reagent	4g/L
Tryptophan	Aladdin Biochemical Technology	2.86g/L
Yeast Extract	Sangon Biotech	5g/L
Corresponding antibiotics	Corresponding manufacturer	Corresponding amount

Antibiotic drugs are prepared according to the concentration of the mother liquor. After the solid drugs are dissolved, they are filtered through a 0.22μm filter membrane (from Biosharp) in a laminar flow hood to remove microorganisms, and then stored in a -20°C refrigerator.

After the culture medium is sterilized and cooled, antibiotics should be added according to the corresponding working concentration of antibiotics.

 

Name	Component	Manufacturer	Mother liquor concentration	Working concentration
Kanamycin	Kanamycin sulfate	yuanye Bio-Technology	50mg/mL	50μg/mL
Ampicillin	Ampicillin sodium	yuanye Bio-Technology	100mg/mL	100μg/mL
Streptomycin	Streptomycin sulfate	yuanye Bio-Technology	50mg/mL	50μg/mL

Component	Manufacturer	Amount
Na₂HPO₄	Sinopharm Chemical Reagent	40mM
KH₂PO₄	Sinopharm Chemical Reagent	10mM