Results

Plasmid Construction

We constructed the plasmid pRSFDuet-1-Fre-L3-SttH-rrnB T1 terminator. Gene expression on this plasmid are regulated by the T7 promoter.

Figure38 :Plasmid map of pRSFDuet-1-Fre-L3-SttH-rrnB T1 terminator

Successful transformation verification

A long DNA fragment, Fre-L3-SttH-rrnB T1 terminator, was amplified by high-fidelity PCR from the cloning vector. This fragment was then digested and ligated into the Multiple Cloning Site 1 (MCS1) region of the pRSFDuet-1 vector, as illustrated in the plasmid map(Figure 38).

Figure39: PCR Amplification of the Fre-L3-SttH-rrnB T1 terminator Gene Fragment.

The observed band aligns with the expected product size of 2494 bp, confirming the successful amplification of the target fragment.

Figure40: Colony PCR of E. coli DH5α Clones Transformed with pRSFDuet-1-Fre-L3-SttH-rrnB T1 terminator

A positive clone (lane 11) shows a band of the expected size (2494 bp), confirming the presence of the correct plasmid.

Protein Expression Verification

The successfully constructed plasmid, pRSFDuet-1-Fre-L3-SttH-rrnB T1 terminator was transformed into E. coliBL21(DE3) cells. The transformed cells were then induced with different concentrations of IPTG (0 mM, 0.3 mM, 0.5 mM, and 0.7 mM) and incubated at 16°C for 16 hours. Protein expression was subsequently analyzed by Western blot.

Figure41: Western blot Analysis of the Fre-L3-SttH Protein (6xHis-tag).

A band was detected at the expected molecular weight of approximately 85.3 kDa, confirming the expression of the target fusion protein.

Plasmids construction

We constructed a red light-responsive expression vector, pCDFDuet-1-PadC4-BphO-YhjH-MrkH，synthesised by Sangon Biotech. In this construct, the PadC4 and BphO genes are driven by the constitutive promoter pJ23106 and terminated by the rrnB T1 terminator, whereas the YhjHand MrkH genes are under the transcriptional control of the pJ23109 promoter and the same rrnB T1 terminator. This rationally designed plasmid serves as the core genetic circuit for sensing and responding to red light stimulation.

Figure42: Plasmid map of pCDFDuet-1-PadC4-BphO-YhjH-MrkH.

Figure43: Plasmid map of pRSFDuet-1-RBS-AcGFP1.

Figure44: Plasmid map of pRSFDuet-1-PmrkA-RBS-AcGFP1.

Moreover, we constructed two plasmids, pRSFDuet-1-RBS-AcGFP1 and pRSFDuet-1-PmrkA-RBS-AcGFP1, using the green fluorescent protein (AcGFP1) as a reporter to validate the activity of the PmrkA promoter.

Successful transformation verification

1.Plasmid pCDFDuet-1-PadC4-BphO-YhjH-MrkH

We transformed E. coli BL21(DE3) with the plasmid pCDFDuet-1-PadC4-BphO-YhjH-MrkH via heat shock. The appearance of distinct single colonies on streptomycin-containing agar plates confirmed successful transformation.

Figure45：Transformation of plasmid pCDFDuet-1-PadC4-BphO-YhjH-MrkH in E. coli BL21(DE3).

2.Plasmid pRSFDuet-1-RBS-AcGFP1

We obtained the RBS-AcGFP1 gene fragment from a cloning vector and selected SalI and KpnI as restriction sites. We successfully inserted RBS-AcGFP1 into the pRSFDuet-1 plasmid using restriction digestion and ligation. Experimental results confirmed the correct assembly of the pRSFDuet1-RBS-AcGFP1 construct.

Figure46: PCR amplification of the RBS-AcGFP1 gene fragment

The target band corresponds to the expected length of 720 bp.

Figure47: Colony PCR verification of pRSFDuet-1-AcGFP1 transformation in E. coli DH5α

All bands represent positive clones and align with the expected 720 bp fragment length.

3.Plasmid pRSFDuet-1-PmrkA-RBS-AcGFP1

We PCR-amplified the PmrkA gene fragment from a cloning vector. Using the pre-constructed pRSFDuet-1-RBS-AcGFP1 plasmid as the backbone, we selected MluI and SalI restriction sites to insert the PmrkA promoter. This strategy intentionally disrupted the original T7 promoter while simultaneously creating a system to validate PmrkA promoter activity. Experimental results confirmed the successful construction of the pRSFDuet-1-PmrkA-RBS-AcGFP1 plasmid.

Figure48: PCR amplification of the PmrkA gene fragment

The target band aligns with the expected length of 215 bp.

Figure49: Colony PCR verification of pRSFDuet1-PmrkA-RBS-AcGFP1 transformation in E. coli DH5α

All bands represent positive clones and correspond to the expected 215 bp fragment length.

We simultaneously transformed the red light-controlled plasmids pCDFDuet-1-PadC4-BphO-YhjH-MrkH and pRSFDuet-1-PmrkA-RBS-AcGFP1 into E. coliBL21(DE3) via heat shock method and plated them on agar medium containing streptomycin and kanamycin. Colony growth confirmed successful transformation. We then expanded single colonies and,assessed the intensity of green fluorescence to verify AcGFP1 expression under red light illumination.

However, no AcGFP1 expression was detected in two independent red light-induced fermentation experiments, suggesting a potential failure in the incorporation of the PmrkA promoter. But it performed normally in our subsequent red-light and 6,6'-dibromoindigo system, exhibiting PmrkA stable expression and biological function.

Figure 50. Transformation of successful dual-plasmid transformation

Protein expression verification

We transformed the plasmid into E. coliBL21(DE3) and selected single colonies on streptomycin-containing agar plates. We then expanded the cultures until the OD₆₀₀ reached 0.8-1 and performed Western blot analysis to validate heterologous protein expression. Our results confirmed successful expression of all three target genes (MrkH, YhjH, and BphO), demonstrating functional production of the encoded proteins. Although the PadC4 protein exhibited an aberrant band position, implying potential structural alterations, it demonstrated normal function in our subsequent red-light and 6,6'dibromoindigo system, with the red light system being stably expressed, confirming that its biological activity remained largely unaffected.

Figure 51: Western blot analysis of MrkH gene expression.

The observed band aligns with the expected molecular weight of 28.7 kDa.

Figure 52: Western blot analysis of YhjH gene expression.

The observed band aligns with the expected molecular weight of 30.9 kDa.

Figure 53: Western blot analysis of BphO gene expression.

The observed band aligns with the expected molecular weight of 22.8kDa.

Figure 54: Western blot analysis of PadC4 gene expression.

The observed band presents a slight discrepancy compared to the expected molecular weight of 78.8 kDa.

System construction

We co-transformed the plasmids pCDFDuet-1-PadC4-BphO-YhjH-MrkH and pET-28a-PmrkA-Fre-L3-SttH into Escherichia coli BL21(DE3) strain, and screened using double-resistant agar plates containing streptomycin and kanamycin. Correctly transformed strains were obtained.

Protein expression verification

Results (Figure 55 and Figure 56) showed that clear target protein bands could be detected in the red light group, while no obvious bands were observed in the dark group. This indicates that the halogenase is only expressed under red light.

Figure 55: SDS-PAGE Electrophoresis result figure

Figure 56: Western blot result figure of the Fre-L3-SttHgene

Effect verification

1.Fermentation effect of 6BrIG

1.1Experimental design and objective

To optimize the synthesis efficiency of the purple pigment 6,6'-dibromoindigo (6BrIG), red light-regulated halogenase-engineered bacteria were used as the research object. Bromide ion concentration gradients (150 mM, 300 mM, 600 mM) and fermentation time gradients (24 h, 36 h, 48 h) were set up. The optimal fermentation conditions were screened by observing the color of the fermentation broth and the color of the pigment after DMSO dissolution. First, we determined the optimal halogenation fermentation concentration, and then explored the influence of bromide ion concentration.

1.2 Analysis of fermentation results

1.2.1 Effect of fermentation time on 6BrIG synthesis (fixed KBr concentration: 300 mM)

24 h group: The halogenated fermentation broth showed light red (Figure 57A), which was lighter than that of the 36 h group, indicating insufficient fermentation time and less accumulation of intermediate products. The pigment fermentation broth appeared blue (Figure 57B), which was determined to be due to incomplete bromination of tryptophan, leading to direct synthesis of indigo pigment.

36 h group: The halogenated fermentation broth showed the deepest light red (Figure 57C), and the pigment fermentation broth appeared intense purple (Figure 57D). The color reached its peak, making this the optimal fermentation time.

48 h group: The light red color of the halogenated fermentation broth faded slightly (Figure 57E), and the pigment fermentation broth showed pale purple (Figure 57F). It is speculated that prolonged fermentation leads to oxidative degradation of part of 6BrIG or a decrease in bacterial metabolic activity.

Figure 57: Results of 6BrIG under different fermentation times

1.2.2 Effect of KBr concentration on 6BrIG synthesis (fixed fermentation time: 36 h)

150 mM KBr group: The bacterial broth showed light pink (Figure 58A) with a pale color, indicating a small amount of 6BrIG intermediate products. After dissolving the precipitate of the pigment fermentation broth with DMSO, the solution appeared pale red (Figure 58B), with a low pigment concentration.

300 mM KBr group: The bacterial broth showed red (Figure 58C), indicating high halogenase activity and sufficient tryptophan bromination reaction. After dissolving the centrifuged precipitate of the fermentation broth with DMSO, the solution appeared intense purple (Figure 58D).

600 mM KBr group: The bacterial broth showed very light pink (Figure 58E). After dissolving the centrifuged precipitate of the fermentation broth with DMSO, the solution appeared pale red (Figure 58F). It is speculated that high-concentration Br⁻ inhibits the growth of E. coli, resulting in reduced pigment synthesis.

Figure 58: Results of 6BrIG under different KBr concentrations

1.2.3 Summary of optimal fermentation conditions for 6BrIG

The optimal condition was 300 mM KBr + 36 h fermentation. At this condition, the accumulation of 6BrIG intermediate products was the highest, and the final pigment concentration was the highest.

2.Fermentation effect of 6ClIG

2.1 Experimental design and objective

Based on the chlorination activity of halogenase discovered in the 6BrIG experiment, the halogen donor was replaced with NaCl. Chloride ion concentration gradients (150 mM, 300 mM, 600 mM) and fermentation time gradients (24 h, 36 h, 48 h) were set up. The synthesis effect of the pale red pigment 6,6'-dichloroindigo (6ClIG) was evaluated by observing the color of the fermentation broth and the color of the pigment after DMSO dissolution, so as to screen the optimal conditions. First, we determined the optimal halogenation fermentation concentration, and then explored the influence of chloride ion concentration.

2.2 Analysis of fermentation results

2.2.1 Effect of fermentation time on 6ClIG synthesis (fixed NaCl concentration: 150 mM)

24 h group: The bacterial broth showed pale milky white (Figure 59A); the pigment fermentation broth appeared blue (Figure 59B). It is speculated that insufficient fermentation time led to incomplete chlorination of part of tryptophan, resulting in the synthesis of a small amount of indigo pigment.

36 h group: The bacterial broth showed pale red (Figure 59C); the pigment fermentation broth appeared reddish brown (Figure 59D) with a high pigment concentration.

48 h group: The bacterial broth showed light gray (Figure 59E); the pigment fermentation broth appeared dark blue (Figure 59F). It is speculated that prolonged fermentation leads to oxidative degradation of part of 6ClIG or a decrease in bacterial metabolic activity.

Figure59: Results of 6ClIG under different fermentation times

2.2.2 Effect of NaCl concentration on 6ClIG synthesis (fixed fermentation time: 36 h)

150 mM NaCl group: The bacterial broth showed light brown (Figure 60A) with a small amount of flaky crystals. After dissolving the centrifuged precipitate with DMSO, the solution appeared pink (Figure 60B) with a high pigment concentration.

300 mM NaCl group: The bacterial broth showed pale gray (Figure 60C); after DMSO dissolution, the solution appeared extremely pale red (Figure 60D). It is speculated that excessively high NaCl concentration slightly inhibits the chlorination activity of halogenase.

600 mM NaCl group: The bacterial broth showed pale milky white (Figure 60E); after DMSO dissolution, the solution appeared colorless (Figure 60F), which confirms that high-concentration NaCl completely inhibits 6ClIG synthesis.

Figure 60: Results of 6ClIG under different NaCl concentrations

2.2.3 Summary of optimal fermentation conditions for 6ClIG

The optimal condition was 150 mM NaCl + 36 h fermentation. At this condition, 6ClIG had the highest synthesis efficiency, and the final pigment showed intense color and good stability.

Plasmid construction

We constructed a plasmid, pRSFDuet-1-txtE-txtD-Fdr-Fdx, to achieve direct regiospecific 4-nitration of L-tryptophan, thereby obtaining L-4-nitro-trp as the substrate for the production of 4,4'-dinitroindigo.

Figure61：Plasmid map of pRSFDuet-1-txtE-txtD-Fdr-Fdx

Protein expression verification

We co-transformed the green light-regulated plasmid pCDFDuet-1-ho1-pcyA-mini-CcaS-CcaR and the target plasmid pRSFDuet-1-txtE-txtD-Fdr-Fdx into E. coli BL21(DE3). Positive clones were selected using double-antibiotic plates containing streptomycin and kanamycin.

Western blot analysis of the experimental groups—green light-exposed versus dark-maintained negative controls—revealed successful expression of FdR and Fdx proteins. In contrast, TxtE and TxtD expression was undetectable. Notably, significant leaky expression was observed, likely due to suboptimal light blocking.

Figure62：Western blot result of Fdx protein

The red box highlights the target protein region，corresponded to the expected size 12.6KDa

Figure63：Western blot result of FdR protein

The red box highlights the target protein region，corresponded to the expected size 46.9KDa

Figure64：Western blot result of TxtD protein

Figure65：Western blot result of TxtE protein

Plasmid construction

Plasmid pCDFDuet-1-ho1-pcyA-mini-CcaS-CcaR (Photosensitive Plasmid)

This plasmid constitutes the core photosensitive module of the green light system, containing four key functional genes.

ho1 (heme oxygenase) and pcyA (ferredoxin oxidoreductase): These two genes are driven by a constitutive promoter to catalyze the conversion of endogenous heme in E. coli to phycocyanobilin (PCB) — the essential photosensitive chromophore for the mini-CcaS protein.

mini-CcaS: Binds to PCB to form an active photosensitive complex; under 520 nm green light, it undergoes a conformational change and gains kinase activity.

CcaR (response regulator): Phosphorylated by activated mini-CcaS (forming CcaR-P), which then specifically binds to the PcpcG2-172 promoter to activate downstream gene expression. In the dark, mini-CcaS maintains low kinase activity, keeping CcaR unphosphorylated and PcpcG2-172 in a repressed state.

Figure66:Genetic Pathway image of the Green Light System

Figure 67: Plasmid map of pCDFDuet-1-ho1-pcyA-miniCcaS-CcaR

Protein expression verification

The photosensitive plasmid pCDFDuet-1-ho1-pcyA-mini-CcaS-CcaR was transformed into E. coli BL21(DE3) for protein expression verification. Western Blot analysis was performed using specific antibodies against HO1, PcyA, CcaR, and mini-CcaS.

HO1: A clear band was detected at the expected molecular weight (~29.0 kDa).

Figure 68: Western Blot analysis of HO1 protein

Figure 69: Western Blot analysis of PcyA protein

Figure 70: Western Blot analysis of CcaR protein

Figure 71: Western Blot analysis of mini-CcaS protein