iGEM 2025 — Team bnuzh-china

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{% block title %}Notebook{% endblock %}
{% block lead %}Documenting the iterative process of our project's development.{% endblock %}
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# Notebook

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Indigo
6,6'-Dibromoindigo
4,4'-Dinitroindigo

![](https://static.igem.wiki/teams/5858/images/template/template-sidenav-top.webp)

### [Blue-light Control](#indigo-s1)

* [July Week 1](#indigo-s1-w1)
* [July Week 2](#indigo-s1-w2)
* [July Week 3](#indigo-s1-w3)
* [July Week 4](#indigo-s1-w4)
* [July Week 5](#indigo-s1-w5)
* [August Week 6](#indigo-s1-w6)
* [August Week 7](#indigo-s1-w7)
* [August Week 8](#indigo-s1-w8)
* [August Week 9](#indigo-s1-w9)
* [September Week 10](#indigo-s1-w10)
* [September Week 11](#indigo-s1-w11)
* [September Week 12](#indigo-s1-w12)

### [Indigo Synthesis](#indigo-s2)

* [July Week 1](#indigo-s2-w1)
* [July Week 2](#indigo-s2-w2)
* [July Week 3](#indigo-s2-w3)
* [July Week 4](#indigo-s2-w4)
* [July Week 5](#indigo-s2-w5)
* [August Week 6](#indigo-s2-w6)
* [August Week 7](#indigo-s2-w7)
* [August Week 8](#indigo-s2-w8)
* [August Week 9](#indigo-s2-w9)
* [September Week 10](#indigo-s2-w10)
* [September Week 11](#indigo-s2-w11)
* [September Week 12](#indigo-s2-w12)

![](https://static.igem.wiki/teams/5858/images/template/template-sidenav-bottom.webp)

## Blue-light Control

### July Week 1 (7.1–7.6)

This week, we performed PCR amplification of the *PhlF* gene to obtain the target DNA fragment and learned relevant experimental techniques.

1. Using the commercially sourced [*YF1-fixJ-PhlF*]-pUC18 plasmid as a template, we amplified the *PhlF* gene via PCR. The *PhlF* gene was digested with restriction enzymes. The pCDFDuet-1 vector, which would be used in subsequent experiments, was transformed into *E. coli* DH5α for amplification and stored for future use. Additionally, we learned basic molecular biology techniques.

### July Week 2 (7.7–7.13)

This week, we re-amplified the *PhlF* gene via PCR and cloned it into the pCDFDuet-1 vector.

1. Using the [*YF1-fixJ-PhlF*]-pUC18 plasmid as a template, we performed PCR to amplify the *PhlF* gene, which was then digested with NdeⅠ and XhoⅠ restriction enzymes. Due to template degradation, no correct band was observed via agarose gel electrophoresis. Plasmid extraction via alkaline lysis was performed on *E. coli* strains containing pCDFDuet-1. The pCDFDuet-1 vector was also digested with NdeⅠ and XhoⅠ restriction enzymes.

### July Week 3 (7.14–7.20)

This week, we conducted the construction of the pCDFDuet-*PhlF* plasmid. The successful construction was verified by colony PCR and Sanger sequencing. The constructed plasmid was transformed into *E. coli* BL21(DE3) for expression. Positive strains identified by sequencing were preserved.

1. The pCDFDuet-1 vector and the PCR-amplified *PhlF* gene were digested with NdeⅠ and XhoⅠ restriction enzymes. T4 DNA ligase (from Vazyme) was used to ligate the digested *PhlF* fragment and the pCDFDuet-1 vector. The ligated pCDFDuet-*PhlF* plasmid was transformed into *E. coli* DH5α via heat shock transformation, followed by colony PCR. Plasmids extracted from colony PCR-positive strains were subjected to Sanger sequencing for verification. Glycerol stocks of the sequenced positive strains were prepared, and the extracted plasmids were transformed into *E. coli* BL21(DE3) for expression.

### July Week 4 (7.21–7.27)

This week, we performed expression validation of the PhlFgene. We learned basic operations for SDS-PAGE and Western Blot (WB) and prepared the corresponding reagents.

1. We prepared reagents required for SDS-PAGE and WB and standardized the formulations. *E. coli* strains harboring pCDFDuet*-PhlF* were induced with IPTG overnight. The cells were lysed, and both the pellet and supernatant were collected for SDS-PAGE and WB analysis.

### July Week 5 (7.28–8.3)

This week, we amplified the blue light-controlled *YF1* and *fixJ* genes and constructed the pCDFDuet-*YF1*-*fixj*-*PhlF* plasmid. Glycerol stocks of strains containing the pCDFDuet-1 plasmid were prepared.

1. Using primers *YF1*-*fixj*-F and *YF1*
-*fixj*-R, we amplified the *YF1-fixJ*
gene from the commercially obtained pUC18-*YF1*-*fixj*-*PhlF* plasmid via PCR. The *YF1-fixJ*
gene and the pCDFDuet-*PhlF*
vector were digested with BamHⅠ and HindⅢ restriction enzymes. T4 DNA ligase (from Vazyme) was used to ligate the digested *YF1-fixJ* gene and the pCDFDuet-*PhlF* vector. The ligated pCDFDuet-*YF1*-*fixj*-*PhlF*
plasmid was transformed into *E. coli* DH5α
via heat shock transformation, followed by colony PCR.
Plasmids extracted from colony PCR-positive strains were submitted for Sanger sequencing.
To ensure subsequent experiments,
glycerol stocks of strains containing the pCDFDuet-1 vector were prepared.

### August Week 6 (8.4–8.10)

This week, we began constructing the blue light-inducible validation plasmid, pRSFDuet-1-pPhlF-AcGFP.

1. Retrieved bacterial strain containing pET21a-GFPfrom the -80°C freezer, cultured it, and performed plasmid miniprep after the culture reached sufficient density.

Performed large-scale culture of the bacterial strain containing pRSFDuet-1, followed by plasmid miniprep to obtain and store pRSFDuet-1 for later use.

2. Amplified the GFPfragment via PCR using pET21a-GFPas the template.

3. Digested both the GFPfragment and the pRSFDuet-1 plasmid using the restriction enzymes HindIII and XhoI.

4. Ligated the digested pRSFDuet-1 vector and GFPfragment using T4 DNA Ligase (from Vazyme Biotech).

5. Transformed the ligated pRSFDuet-1-GFPplasmid into *E. coli* DH5α cells via heat shock transformation and screened transformants by colony PCR.

6. Amplified the pPhlF fragment via PCR using [*TnaA*-*MaFMO*]pUC57 as the template.

7. Digested both the pRSFDuet-1-GFPplasmid and the pPhlF fragment using the restriction enzymes HindIII and NcoI.

8. Ligated the digested pRSFDuet-1-GFPvector and pPhlF fragment using T4 DNA Ligase (from Vazyme Biotech).

9. Transformed the ligated pRSFDuet-1-pPhlF-GFPplasmid into *E. coli* DH5α cells via heat shock transformation and screened transformants by colony PCR.

10. Prepared a glycerol stock of the bacterial strain harboring pET28-GFP.

### August Week 7 (8.11–8.17)

This week, we continued the construction of the constitutively expressed blue light-responsive plasmid. Multiple attempts were made to ligate the *pPhlF* promoter with the pRSFDuet-1-AcGFP vector, but no positive results were obtained.

1. Using the empty pCDFDuet vector as a template,
we amplified the origin of replication and antibiotic resistance gene fragment (*ori-Sm*) via PCR.
Using pUC18-*YF1*-*fixj*-*PhlF* as a template,
we amplified the Pj23100-*YF1*-*fixj*-*PhlF*-B0015 fragment via PCR.
The *ori-Sm* and Pj23100-*YF1*-*fixj*-*PhlF*-B0015 fragments were digested with BamHⅠ and HindⅢ restriction enzymes.
T4 DNA ligase (from Vazyme) was used to ligate the digested *ori-Sm* and Pj23100-*YF1*-*fixj*-*PhlF*-B0015 fragments.
The ligated pCDFDuet-*YF1*-*fixj*-*PhlF* plasmid was transformed into *E. coli* DH5α and BL21(DE3) via heat shock transformation, followed by colony PCR. Plasmids extracted from colony PCR-positive strains were submitted for Sanger sequencing.
The results indicated incorrect ligation.
Using the pUC57-*MaFMO*-Ec*TnaA* plasmid as a template, we amplified the *pPhlF* promoter via PCR.
Subsequent digestion and ligation steps were performed,
but colony PCR showed no positive results.

### August Week 8 (8.18–8.24)

This week, we continued the construction of the constitutively expressed blue light-responsive plasmid.

1. The *ori-Sm* and Pj23100-*YF1*-*fixj*-*PhlF*-B0015 fragments were re-ligated and transformed. Plasmids were extracted and tested via PCR, which showed multiple non-specific bands.

### August Week 9 (8.25–8.31)

This week, we continued the construction of the constitutively expressed blue light-responsive plasmid.

1. The *ori-Sm* and Pj23100-*YF1*-*fixj*-*PhlF*-B0015 fragments were re-amplified via PCR, ligated, and transformed. Plasmids extracted from PCR-positive strains were submitted for Sanger sequencing.

### September Week 10 (9.1–9.7)

This week, we re-validated the protein expression of the pCDFDuet-*YF1*-*fixj*-*PhlF* plasmid.

1. The glycerol stock of the strain containing the pCDFDuet-*YF1*-*fixj*-*PhlF* inducible expression plasmid, stored at -80°C, was revived. The cells were lysed and centrifuged, and both the supernatant and pellet were analyzed via SDS-PAGE and WB. Incorrect band positions were observed. Further investigation revealed a reading frame shift issue in the plasmid construction.

### September Week 11 (9.8–9.14)

This week, we attempted to construct the constitutively expressed blue light-responsive plasmid using conventional restriction-ligation cloning.

1. The empty pCDFDuet-1 vector and the Pj23100-*YF1*-*fixj*-*PhlF*-B0015 fragment were digested with BamHⅠ and HindⅢ restriction enzymes. T4 DNA ligase (from Vazyme) was used to ligate the digested vector and the Pj23100-*YF1*-*fixj*-*PhlF*-B0015 fragment. The ligated pCDFDuet-*YF1*-*fixj*-*PhlF* plasmid was transformed into *E. coli* DH5α via heat shock transformation, followed by colony PCR. Plasmids extracted from colony PCR-positive strains were submitted for Sanger sequencing. The results indicated incorrect ligation.

### September Week 12 (9.15–9.21)

This week, we performed feasibility verification of the blue light-inducible plasmid.

The bacterial strain containing the correct plasmid was subjected to large-scale culture. A small portion of the bacterial culture was used to prepare a glycerol stock, and the remainder was used for plasmid extraction.

1. The blue light-inducible plasmid and the indigo synthesis plasmid were co-transformed into *E. coli* BL21(DE3) cells. The transformation mixture was spread on plates and cultured overnight.

2. Single colonies were selected for colony PCR. Clones yielding correct PCR results were expanded in culture. Indigo production was observed under blue light illumination. The results indicated that the experimental strain failed to produce indigo.

## Indigo Synthesis

### July Week 1 (7.1-7.6)

This week, we carried out the amplification and the purification of the related genes *EcTnaA* and *MaFMO* for indigo synthesis.

1. We prepared the LB medium, and cultivated *Escherichia coli* DH5α containing pUC57-*EcTnaA-MaFMO* vector (from *Tsingke*) and extracted the plasmids by alkaline lysis.

2. We successfully amplified and purified the *EcTnaA* gene, and stored it for further usage.

3. We attempted to amplify and purify the *MaFMO* gene but failed to obtain the target gene.

4. We transferred the plasmid pRSFDuet-1 separately into *E. coli* DH5α by heat shock transformation and extracted the plasmids by alkaline lysis.

### July Week 2 (7.7-7.13)

This week, we carried out the construction of the pRSFDuet-1-*EcTnaA* vector and the amplification and purification of *MaFMO*.

1. We re-conducted PCR using the optimized primers of the *MaFMO* gene, successfully amplified and purified the *MaFMO* gene.

2. We performed enzymatic cleavage of the *EcTnaA* gene and the pRSFDuet-1 plasmid using Not I restriction enzyme (from Vazyme) and Nco I restriction enzyme (from Vazyme).

3. We used T4 DNA ligase (from Vazyme) to ligate the enzymatically cleaved *EcTnaA* gene with the pRSFDuet-1 plasmid.

4. We transferred the pRSFDuet-1-*EcTnaA* vector into *E. coli* DH5α by heat shock transformation and conducted colony PCR and Sanger sequencing.

### July Week 3 (7.14-7.20)

This week, we carried out the preservation of some strains and the construction of the pRSFDuet-1-*EcTnaA-MaFMO* vector.

1. We have preserved the strains containing the following vectors: pUC57-*EcTnaA-MaFMO*, pRSFDuet-1,pRSFDuet-1-*EcTnaA*.

2. We extracted the pRSFDuet-1-*EcTnaA* plasmid by alkaline lysis, and performed enzymatic cleavage of the pRSFDuet-1-*EcTnaA* plasmid and the *MaFMO* gene using Xho I restriction enzyme (from Vazyme) and Bgl II restriction enzyme (from Vazyme).

3. We used T4 DNA ligase (from Vazyme)to ligate the enzyme-cleaved *MaFMO* gene with the pRSFDuet-1-*EcTnaA* plasmid.

4. We used the thermal recovery method to transfer the pRSFDuet-1-*EcTnaA*-*MaFMO* vector into *E.coli* DH5α and *Escherichia coli* BL21 (DE3), and conducted colony PCR and Sanger sequencing.

### July Week 4 (7.21-7.27)

This week, we carried out literature reading on indigo fermentation and made preparations for it.

1. We read the relevant literature on indigo fermentation and decided to produce indigo by the two-stage fermentation method, and wrote an experimental plan.

2. We prepared a glucose defined medium and stored it for further usage.

### July Week 5 (7.28-8.3)

This week, we tried to conduct two-stage fermentation of indigo, perform PCR with high-fidelity Taq enzyme to address the issue of gene mutations, and conduct SDS-PAGE and Western Blot to detect *EcTnaA* and *MaFMO* proteins.

1. We improved the preparation method of the glucose defined medium and sterilized tryptophan using a 0.22μm filter membrane.

2. We transferred *E.coli* BL21(DE3) containing the pRSFDuet-1*-EcTnaA-MaFMO* vector into the glucose defined medium and carried out indigo fermentation using the two-stage fermentation method, but no indigo was produced.

3. We used high-fidelity 2x Phanta Max Master Mix enzyme (from Vazyme) to re-amplify and purify the *EcTnaA* gene and the *MaFMO* gene with the pUC57*-EcTnaA-MaFMO* vector as the template.

4. We performed enzymatic cleavage of the *EcTnaA* gene and the pRSFDuet-1 plasmid using Not I restriction enzyme (fromvazyme) and Nco I restriction enzyme (from vazyme).

5. We used T4 DNA ligase (from vazyme) to ligate the enzymally cleaved *EcTnaA* gene with the pRSFDuet-1 plasmid.

6. We conducted SDS-PAGE and Western Blot experiments on *E. coli* BL21(DE3) containing the pRSFDuet-1-*EcTnaA-MaFMO* vector. The results showed that the MaFMO protein was expressed and the molecular weight of the EcTnaA protein was abnormal.

### August Week 6 (8.4-8.10)

This week, we changed the temperature conditions for indigo fermentation and successfully produced indigo. We replaced the primary antibody used last time and conducted Western Blot and SDS-PAGE experiments again. We also attempted to use inverse PCR technology and homologous recombination technology to replace the first promoter of the vector pRSFDuet-1-*EcTnaA-MaFMO*.

1. We improved the temperature conditions for indigo production and successfully produced indigo. Subsequently, we prepared indigo samples and determined their concentrations.

2. We replaced the primary antibody of 6xHis-tag used in the former Western Blot experiment, re-conducted the SDS-PAGE experiment and the Western Blot experiment, and obtained the correct EcTnaA protein and MaFMO protein.

3. We linearized the vector pRSFDuet-1-*EcTnaA*-*MaFMO* using inverse PCR technology. Subsequently, we tried to ligate the promoter pPhlF amplified by PCR technology with the linearized vector using homologous recombination technology. Subsequent verification indicated that the ligation failed.

### August Week 7 (8.11-8.17)

This week We carried out the amplification,extraction of the relevant promoter-gene-terminator pPhlF-*EcTnaA*-*MaFMO*-L3S3P11 terminator for indigo synthesis and the vector pRSFDuet-1-pPhlF promoter-*EcTnaA*-*MaFMO*-L3S3P11 reminnator construction work and the indigo productiom.

1. We successfully amplified and purified the DNA sequence of pPhlF promoter*-EcTnaA-MaFMO-*L3S3P11 terminator.

2. We performed enzymatic cleavage of the pPhlF promoter-*EcTnaA-MaFMO*-L3S3P11 terminator sequence and the pRSFDuet-1 plasmid using Mlu I restriction enzyme (from Biolabs) and Bgl II restriction enzyme (from Vazyme).

3. We used T4 DNA ligase (from Vazyme) to ligate the enzyme-cleaved pPhlF promoter -*EcTnaA-MaFMO-*L3S3P11 terminator sequence with the pRSFDuet-1 plasmid.

4. We transferred the pRSFDuet-1-pPhlF promoter*-EcTnaA-MaFMO*-L3S3P11 terminator vector into *E.coli* DH5α using the thermal recovery method, and conducted colony PCR and sanger sequencing. The sanger sequencing indicated that the ligation was unsuccessful.

5. We re-carried out indigo fermentation, but it was not successful.

### August Week 8 (8.18-8.24)

This week, we repeated the indigo fermentation and the construction of the vector pRSFDuet-1-pPhlF promoter-*EcTnaA-MaFMO*-L3S3P11 terminator from last week, but still failed. We also tested the stability of indigo at different temperatures.

### August Week 9 (8.25-8.31)

This week, we repeated the indigo fermentation and the construction of the vector pRSFDuet-1-pPhlF promoter-*EcTnaA-MaFMO*-L3S3P11 terminator from last week, but still failed.

### September Week 10 (9.1-9.7)

This week, we replaced the restriction enzyme and re-constructed the vector pRSFDuet-1-pPhlF promoter-EcTnaA-MaFMO-L3S3P11 terminator.

1. We redesigned the PCR primers so that the pPhlF promoter-EcTnaA-MaFMO-L3S3P11 terminator sequence had two restriction restriction sites, NcoI and NotI, at both ends, and successfully amplified and purified this segment.

2. We used two restriction enzymes, Nco I and Not I (from Vazyme), to enzymatize this sequence with the pRSFDuet-1 plasmid.

3. We used T4 DNA ligase (from Vazyme) to ligate the pPhlF promoter -*EcTnaA-MaFMO*-L3S3P11 terminator gene after enzymatic cleavage with the pRSFDuet-1 plasmid.

4. We transferred the pRSFDuet-1-pPhlF promoter-*EcTnaA-MaFMO*-L3S3P11 terminator vector into *E.coli* DH5α using the thermal recovery method, and conducted colony PCR and sanger sequencing. The sanger sequencing indicated that the ligation was successful.

### September Week 11 (9.8-9.14)

1. We revived and induced the expression of the preserved indigo bacteria to produce indigo.

2. We used two restriction enzymes, Nco I and Not I (from Vazyme), to enzymatize this sequence with the pRSFDuet-1 plasmid.

3. We used T4 DNA ligase (from Vazyme) to ligate the pPhlF promoter -*EcTnaA-MaFMO*-L3S3P11 terminator gene after enzymatic cleavage with the pRSFDuet-1 plasmid.

4. We transferred the pRSFDuet-1-pPhlF promoter-*EcTnaA-MaFMO*-L3S3P11 terminator vector into *E.coli* DH5α using the thermal recovery method, and conducted colony PCR and sanger sequencing. The sanger sequencing indicated that the ligation was not successful.

### September Week 12 (9.15-9.21)

This week, we attempted to fit a standard curve of indigo concentration vs. absorbance, performed bacterial preservation, and conducted fermentation cultures.

1. We induced the expression of the original indigo-producing bacteria to produce and extract indigo, determined the indigo concentration, and prepared a series of standard indigo samples with gradient concentrations to fit the standard curve of indigo concentration vs. absorbance.

2. We preserved the *E. coli* DH5α T1 and 2-H-3 monoclonals that had successfully been sequenced and transformed with the pRSFDuet-1-pPhlF promoter-*EcTnaA-MaFMO*-L3S3P11 terminator plasmid.

3. We transformed the successfully constructed pRSFDuet-1-pPhlF promoter-*EcTnaA-MaFMO*-L3S3P11 terminator plasmid into *E. coli* BL21 (DE3) for fermentation culture. This confirmed the feasibility of the pPhlF promoter, with successful indigo production.

![](https://static.igem.wiki/teams/5858/images/template/template-sidenav-top.webp)

### [Red-light Control](#6brig-s1)

* [August Week 7](#6brig-s1-w1)
* [August Week 8](#6brig-s1-w2)
* [August Week 9](#6brig-s1-w3)
* [September Week 10](#6brig-s1-w4)
* [September Week 11](#6brig-s1-w5)
* [September Week 12](#6brig-s1-w6)
* [September Week 13](#6brig-s1-w7)
* [September Week 14](#6brig-s1-w8)

### [6,6'-Dibromoindigo Synthesis](#6brig-s2)

* [July Week 3](#6brig-s2-w1)
* [July Week 4](#6brig-s2-w2)
* [July Week 5](#6brig-s2-w3)
* [August Week 6](#6brig-s2-w4)
* [August Week 7](#6brig-s2-w5)
* [August Week 8](#6brig-s2-w6)
* [August Week 9](#6brig-s2-w7)
* [September Week 10](#6brig-s2-w8)
* [September Week 11](#6brig-s2-w9)
* [September Week 12](#6brig-s2-w10)
* [September Week 13](#6brig-s2-w11)
* [October Week 14](#6brig-s2-w12)

![](https://static.igem.wiki/teams/5858/images/template/template-sidenav-bottom.webp)

## Red-light Control

### August Week 7 (8.11-8.17)

This week, we conducted the expression verification experiment of pCDFDuet-1-*PadC4-BphO-YhjH-MrkH* vector, the construction work of pmrkA-*GFP* -pRSFDuet-1 vector.

1. We extracted the pCDFDuet-1-*PadC4-BphO-YhjH-MrkH* vector from puncture bacteria (from BGI) using the alkaline lysis method.

2. We used the thermal shock method to transfer the pCDFDuet-1-*PadC4-BphO-YhjH-MrkH* vector into *E.coli* BL21(DE3).

3. We conducted a Western Blot experiment to verify that the YhjH and MrkH proteins were correctly expressed, while the expression of PadC4 and BphO proteins was not detected.

4. We successfully amplified and purified the PmrkA promoter and RBS sequence. Enzymatic digestion of the PmrkA-RBS sequence and the pRSFDuet-1-*GFP* vector was performed using BamH I and Hind III restriction enzymes (from Vazyme).

5. We used T4 DNA ligase (from Vazyme) to ligate the PmrkA-RBS sequence and the pRSFDuet-1-*GFP* vector after enzymatic digestion, and conducted colony PCR and sanger sequencing.

### August Week 8 (8.18-8.24)

This week, we carried out the validation work of co-expression of two plasmids.

1. We used the thermal rest method to transfer the pRSFDuet-1-PmrkA-*GFP* vector into *E.coli* BL21(DE3) containing the pCDFDuet-1-*PadC4-BphO-YhjH-MrkH* vector and carried out fermentation under red light conditions. No GFP fluorescent protein was detected.

2. After verification, our pRSFDuet-1-PmrkA-*GFP* vector was not constructed successfully.

### August Week 9 (8.25-8.31)

This week, we repeated the construction of the vector pRSFDuet-1-PmrkA-*GFP* from last week, but still failed.

### September Week 10 (9.1-9.7)

This week, we repeated the expression verification experiment for the pCDFDuet-1-PadC4-BphO-YhjH-MrkH vector and also attempted to verify the expression of the pCDFDuet-1-PadC4-BphO vector.

1. We used the thermal recovery method to transfer the pCDFDuet-1-*PadC4*-*BphO-YhjH-MrkH* vector into *E.coli* BL21 (DE3).

2. We repeated the Western Blot experiment on E. coli BL21(DE3) harboring the pCDFDuet-1-PadC4-BphO-YhjH-MrkH vector and verified that the YhjH and MrkH proteins were correctly expressed, while the expression of PadC4 and BphO proteins remained undetectable.

3. We used the thermal recovery method to transfer the pCDFDuet-1-*PadC4*-*BphO* vector into *E.coli* BL21 (DE3).

4. We conducted a Western Blot experiment for the third time on E. coli BL21(DE3) harboring the pCDFDuet-1-PadC4-BphO vector, which showed that the PadC4 and BphO proteins continued to be undetectable.

### September Week 11 (9.8-9.14)

This week, we replaced the restriction enzyme and ligation sequences and re-constructed the vector pRSFDuet-1-RBS-GFP.

1. We redesigned the PCR primers to add an RBS sequence upstream of the GFP sequence, flanked by Salt and KpnI restriction sites, and successfully amplified and purified the resulting fragment.

2. We used two restriction enzymes, Salt and KpnI (from Biolabs), to enzymatize this sequence with the pRSFDuet-1 plasmid.

3. We used T4 DNA ligase (from Vazyme) to ligate the RBS-GFP gene after enzymatic cleavage with the pRSFDuet-1 plasmid.

4. We transferred the pRSFDuet-1-RBS-GFP into *E.coli* DH5α using the thermal recovery method, and conducted colony PCR and sanger sequencing. The sanger sequencing indicated that the ligation was unsuccessful.

### September Week 12 (9.15-9.21)

This week, we replaced the ligase for the construction of the vector pRSFDuet-1-RBS-GFP and performed the expression verification of the pCDFDuet-1-*PadC4*-*BphO-YhjH-MrkH* vector.

1. We reused the 5min Universal Ligation Mix (from Vazyme) to ligate the digested RBS-GFP gene and the pRSFDuet-1 plasmid.

2. We transferred the pRSFDuet-1-RBS-GFP into *E.coli* DH5α using the thermal recovery method, and conducted colony PCR and sanger sequencing. The sanger sequencing indicated that the ligation was successful.

3. We used the thermal recovery method to transfer the pCDFDuet-1-*PadC4*-*BphO* vector into *E.coli* BL21 (DE3).

4. We conducted a Western Blot experiment for the fourth time on *E. coli* BL21(DE3) harboring the pCDFDuet-1*-PadC4-BphO* vector, and verified that the BphO protein was correctly expressed, while the expression of PadC4 proteins remained undetectable ultimately.

### September Week 13 (9.22-9.28)

This week, we carried out the construction work of pRSFDuet-1-PmrkA-RBS-*AcGFP1* vector.

1. We designed PCR primers targeting the PmrkA promoter and successfully amplified and purified this DNA fragment.

2. We used two restriction enzymes, SalI and MluI (from Biolabs), to enzymatize this sequence with the pRSFDuet-1-RBS-*AcGFP1* plasmid.

3. We used the 5min Universal Ligation Mix (from Vazyme) to ligate the digested RBS-*AcGFP1* gene with the pRSFDuet-1 plasmid.

4. We transformed the pRSFDuet-1-PmrkA-RBS-*AcGFP1* vector into *E. coli* DH5α using the heat shock method, and performed colony PCR. The colony PCR results confirmed successful ligation. In parallel, we extracted the plasmid via alkaline lysis and performed restriction digestion with SalI and MluI enzymes, both of which confirmed successful ligation.

### September Week 14 (9.29-10.4)

This week, we performed the dual-plasmid transformation to validate the pRSFDuet-1-PmrkA-RBS-*AcGFP1* and pCDFDuet-1-*PadC4-BphO-YhjH-MrkH* constructs.

1. We co-transformed the pRSFDuet-1-PmrkA-RBS*-AcGFP1* and pCDFDuet-1-*PadC4-BphO-YhjH-MrkH* plasmids into *E. coli* BL21(DE3) using the heat shock method. The appearance of colonies on agar plates containing streptomycin and kanamycin confirmed successful transformation.

2. We picked single colonies, performed expansion culture, and verified the expression of AcGFP1 protein under red light induction.

## 6,6'-Dibromoindigo Synthesis

### July Week 3 (7.14-7.20)

This week, we carried out the amplification and extraction of the fre-sttH gene (related to haloperoxidase synthesis), as well as the restriction enzyme digestion, ligation, and transformation of the fre-sttH gene with the pET-28a(+) plasmid. We also performed colony PCR and Sanger sequencing.

1. We prepared LB medium, conducted the expansion culture of the stab culture containing the pMV-fre-sttH vector, and extracted the plasmid using the alkaline lysis method.

2. We successfully amplified and extracted the fre-sttH gene.

3. We digested the fre-sttH gene and the pET-28a(+) plasmid with restriction enzymes Nco I (from Vazyme Biotech) and Hind III (from Vazyme Biotech).

4. We ligated the digested fre-sttH gene with the pET-28a(+) plasmid using T4 DNA Ligase (from Vazyme Biotech) and 5min Universal Ligation Mix (from Vazyme Biotech), respectively.

5. We transformed the pET-28a(+) plasmid into E. coli BL21 via the heat shock method, performed colony PCR using 2× Rapid Taq Master Mix, and conducted Sanger sequencing.

### July Week 4 (7.21-7.27)

The sequencing results from last week were not ideal. This week, we reconstructed the pET28a-fre-sttH vector, introduced the reconstructed plasmid into DH5α, and performed colony PCR and Sanger sequencing.

1. We digested the fre-sttH gene and the pET-28a(+) plasmid with restriction enzymes Nco I (from Vazyme Biotech) and Hind III (from Vazyme Biotech).

2. We ligated the digested fre-sttH gene with the pET-28a(+) plasmid using T4 DNA Ligase (from Vazyme Biotech).

3. We transformed the pET28a-fre-sttH vector into E. coli DH5α via the heat shock method. Meanwhile, we performed colony PCR using 2× Rapid Taq Master Mix and conducted Sanger sequencing.

4. We preserved the bacterial strain containing the pET28a-fre-sttH vector.

### July Week 5 (7.28-8.3)

This week, we re-amplified and extracted the fre-sttH gene (related to haloperoxidase synthesis), and carried out the restriction enzyme digestion, ligation, and transformation of the fre-sttH gene with the pET-28a(+) plasmid. We also performed colony PCR and Sanger sequencing.

### August Week 6 (8.4-8.10)

This week, we verified whether the fre-sttH gene was successfully ligated with the pET-28a(+) plasmid, re-conducted the ligation and transformation, performed IPTG induction, and carried out SDS-PAGE and Western Blot to detect protein expression.

1. We extracted the plasmid from the transformed DH5α for PCR verification. After gel electrophoresis, it was found that the brightness of the target band was very low, while the brightness of non-specific bands was very high.

2. We re-amplified the fre-sttH gene and completed the restriction enzyme digestion, ligation, and transformation of the fre-sttH gene with the pET-28a(+) plasmid.

3. We performed SDS-PAGE and Western Blot experiments on E. coli BL21 (DE3) that we believed contained the pET28a-fre-sttH vector, but the results showed that the bands were incorrect.

### August Week 7 (8.11-8.17)

This week, we found that all experiments from last week were contaminated. Therefore, we re-conducted the amplification and extraction of the fre-sttH gene, as well as the restriction enzyme digestion, ligation, and transformation of the fre-sttH gene with the pET-28a(+) plasmid. However, the final colony PCR results still showed ligation failure. Starting from this week, while constructing the inducible expression vector pET28a-fre-sttH, we also began constructing the red light-regulated expression vector pET28a-PmrkA-fre-sttH.

1. We performed PCR verification on all bacterial liquid samples used last week, and the verification showed that both the pMV plasmid and the pET-28a(+) plasmid contained the pRSFDuet-1-EcTnaA-MaFMO plasmid.

2. We re-amplified and extracted the fre-sttH gene (related to haloperoxidase synthesis), and carried out the restriction enzyme digestion, ligation, and transformation of the fre-sttH gene with the pET-28a(+) plasmid. In addition, we performed colony PCR verification on the DH5α into which the plasmid was transformed, and the results confirmed that the ligation between the plasmid and the gene failed.

3. We prepared LB medium, conducted the expansion culture of the stab culture containing the pMV-PmrkA-fre-sttH vector, and extracted the plasmid using the alkaline lysis method.

4. We successfully amplified and extracted the PmrkA-fre-sttH gene by PCR using the high-fidelity 2x Phanta Max Master Mix enzyme (from Vazyme Biotech).

5. We digested the PmrkA-fre-sttH gene and the pET-28a(+) plasmid with restriction enzymes Bgl II (from Vazyme Biotech) and Hind III (from Vazyme Biotech).

6. We ligated the digested PmrkA-fre-sttH gene with the pET-28a(+) plasmid using T4 DNA Ligase (from Vazyme Biotech).

7. We transformed the pET-28a(+) plasmid into E. coli DH5α via the heat shock method, performed colony PCR using 2× Rapid Taq Master Mix, and conducted Sanger sequencing. The sequencing results showed that the ligation was normal and no gene mutation occurred.

### August Week 8 (8.18-8.24)

This week, in response to the failed colony PCR results from last week, we re-extracted the plasmid for plasmid PCR verification, and adjusted the experimental design to address the issue of low concentration of digested products. Meanwhile, we co-transformed the pET28a-PmrkA-fre-sttH plasmid and the red light-regulated pCDFDuet-1-PadC4-BphO-YhjH-MrkH plasmid into BL21(DE3), and verified whether the genes on the two plasmids could be normally expressed simultaneously and whether the expression of the fre-sttH gene was regulated by red light through SDS-PAGE and Western Blot experiments.

1. We re-amplified and extracted the fre-sttH gene (related to haloperoxidase synthesis) using Taq enzyme and high-fidelity enzyme, and carried out the restriction enzyme digestion, ligation, and transformation of the fre-sttH gene with the pET-28a(+) plasmid. In addition, we performed colony PCR verification on the DH5α into which the plasmid was transformed, and the results confirmed that the ligation between the plasmid and the gene failed.

2. We re-digested the fre-sttH gene and the pET-28a(+) plasmid with Nco I and Hind III restriction enzymes, extended the gene digestion time to 1 hour and 40 minutes, and increased the gene dosage (more than 1500 ng). Furthermore, we re-ligated the plasmid and the gene at ratios of 1:3 and 1:5 using T4 DNA Ligase and 5min Universal Ligation Mix enzyme, respectively, and transformed the ligation products into DH5α.

3. We co-transformed the pET28a-PmrkA-fre-sttH plasmid and the red light-regulated pCDFDuet-1-PadC4-BphO-YhjH-MrkH plasmid into BL21(DE3). Through SDS-PAGE and Western Blot experiments, we found that the haloperoxidase gene fre-sttH, and the red light-controlled system genes PadC4, BphO, YhjH, and MrkH could all be normally expressed, and the expression of fre-sttH was regulated by red light.

### August Week 9 (8.25-8.31)

This week, we learned the design of annealing gradient PCR, identified the low specificity of the downstream primer for the target gene fre-sttH and the uneven distribution of the gene's GC content, and proposed a backup strategy of modifying the primer. Meanwhile, we preserved the E. coli BL21(DE3) strain into which the pET28a-PmrkA-fre-sttH plasmid and the red light-regulated pCDFDuet-1-PadC4-BphO-YhjH-MrkH plasmid were introduced.

### September Week 10 (9.1-9.7)

This week, experiments focused on the double enzyme digestion verification of the vector, sequencing analysis of the empty plasmid issue. Based on the results, we adjusted the PCR conditions (changing enzymes and primers) and ligation ratios, and conducted a new batch of ligation, transformation, and verification. Meanwhile, we prepared competent cells of E. coli BL21-ΔTnaA and co-transformed the haloperoxidase plasmid and the red light plasmid into E. coli BL21-ΔTnaA cells.

1. We extracted the plasmid ligated with 5min Universal Ligation Mix enzyme and performed double enzyme digestion verification with Nco I and Hind III. The experimental results showed no two target bands that met the expectations.

2. We extracted the plasmid ligated with T4 DNA Ligase and performed double enzyme digestion verification with Nco I and Hind III using an 8 μL system and a 3-hour reaction time; however, no two target bands that met the expectations were still observed.

3. We re-picked single colonies from the medium plate of E. coli DH5α into which the plasmid ligated with T4 DNA Ligase was introduced, and performed colony PCR using Taq enzyme, but no correct results were obtained.

4. Following the teacher's suggestion (to solve the problem of PCR band singularity), we switched to using high-fidelity 2x Phanta Max Master Mix enzyme + a new downstream primer for the fre-sttH gene for PCR.

5. We performed double enzyme digestion on the new gene and plasmid with Nco I and Hind III; ligated them at ratios of 1:3 and 1:5 using 5min Universal Ligation Mix and T4 DNA Ligase, respectively, and transformed the ligation products into DH5α.

6. We performed colony PCR on the strain into which the plasmid ligated with 5min Universal Ligation Mix was introduced. Since the positive control group in the results did not amplify a band consistent with the length of the target gene, we decided to use double enzyme digestion verification in subsequent experiments.

7. We extracted the plasmid ligated with 5min Universal Ligation Mix and performed double enzyme digestion verification with Nco I and Hind III; after replacing the downstream primer, the theoretical length of the recombinant plasmid was 7734 bp, but no target band was detected.

### September Week 11 (9.8-9.14)

This week, focusing on vector construction, we carried out verification after ligation of the plasmid and gene, plasmid extraction, and enzyme digestion. We analyzed the empty plasmid issue, adjusted the scheme (changing the vector, optimizing the recovery method), and promoted the ligation and transformation experiments of the new vector. Meanwhile, we began exploring bromide fermentation.

1. We extracted the plasmid from E. coli DH5α into which the plasmid ligated with T4 DNA Ligase (from last week) was introduced and performed PCR verification. However, the PCR results showed poor band singularity; we extracted the pRSFDuet plasmid in preparation for subsequent vector replacement and ligation.

2. We ligated the fre-sttH gene with the pET-28a(+) plasmid using T4 DNA Ligase and 5min Universal Ligation Mix enzyme, respectively, completed the transformation, extracted the plasmid for sequencing. However, the sequencing results showed that all the submitted plasmids were empty plasmids. It was speculated that after digestion of the original vector pET-28a, the distance between the recognition sites of Nco I and Hind III was only 123 bp, and the plasmid recovery adopted the liquid recovery method, which caused the cut small fragments to be more easily ligated than the 2488 bp target gene, resulting in most of the plasmids transferred into the competent cells being empty vectors.

3. We selected a new vector pRSFDuet-1, whose distance between the cleavage sites of Nco I and Hind III exceeds 1000 bp, and the integrity of double enzyme digestion can be accurately identified by electrophoresis.

4. We performed colony PCR on the strain after ligation and transformation, and conducted Sanger sequencing on the plasmid extracted from the strain.

5. We irradiated the bacterial suspension with red light to induce the expression of the *fre-sttH* gene, which produces haloperoxidase. Following the method described in the literature, we attempted halogenated fermentation.

### September Week 12 (9.15-9.21)

This week's work focused on analyzing sequencing results and addressing new technical challenges.Meanwhile, we explored the optimal duration for bromide fermentation.

1. Sequencing analysis of the positive clone plasmids from the previous week revealed a large fragment deletion in the target gene. This indicated that despite the initial positive colony PCR results, the obtained plasmids were not suitable for subsequent protein expression experiments.

2. It was hypothesized that unstable regions might exist within the target gene sequence, or that recombination and deletion occurred during strain subculture. To obtain the full-length gene, we decided to re-streak the original positive clone culture for single colonies. Individual monoclonal colonies were then picked for renewed colony PCR screening to identify clones containing the intact target gene.

3. Since the desired purple pigment was not obtained last week—instead, indigo was produced—we conducted a gradient experiment on the duration of bromide fermentation.

### September Week 13 (9.22--9.28)

This week was dedicated to confirming the correct clone and preparing the engineered strain for protein expression.We prepared a new buffer for halogenated fermentation. Meanwhile, we explored the optimal concentration of bromide ions in halogenated fermentation.

1. The single colonies picked after re-streaking were expanded in culture, and their plasmids were extracted for sequencing identification.

2. Sequence analysis confirmed that the plasmid from the initial positive clone (strain #11) contained the full-length *Fre-L3-SttH*-*rrnB T1 terminator* target gene sequence without mutations, successfully validating the construct.

3. The verified correct recombinant plasmid, pRSFDuet-1-*Fre-L3-SttH*-*rrnB T1 terminator*, was successfully transformed into the protein expression host, *E. coli* BL21(DE3), preparing it for subsequent induction experiments.

4. Using the new buffer, we performed fermentation with different concentrations of bromide ions and obtained varying results. We also observed that the haloperoxidase appears to be capable of chlorination in addition to bromination.

### October Week 14 (9.29--10.5)

The core task this week was to induce target protein expression in the expression host and perform verification.Meanwhile, we explored the optimal chloride ion concentration and fermentation time for chlorinated fermentation.

1. The engineered *E. coli* BL21(DE3) strain harboring the successfully constructed pRSFDuet-1-*Fre-L3-SttH-rrnB T1 terminator* plasmid was induced for 16 hours at 16°C using different concentrations (0 mM, 0.3 mM, 0.5 mM, 0.7 mM) of the inducer IPTG, aiming to promote soluble protein production.

2. After induction, bacterial samples were harvested, and protein expression was analyzed via Western Blot. The results clearly showed a specific band at the expected molecular weight of ~85.3 kDa, which corresponded perfectly with the theoretical size of the Fre-L3-SttH fusion protein. This successfully confirmed the expression of the target protein in the engineered strain.

3. We conducted fermentation with different concentrations of chloride ions and investigated the impact of fermentation time.

![](https://static.igem.wiki/teams/5858/images/template/template-sidenav-top.webp)

### [Green-light Control](#44d-s1)

* [August Week 8](#44d-s1-w1)
* [August Week 9](#44d-s1-w2)
* [September Week 10](#44d-s1-w3)
* [September Week 11](#44d-s1-w4)
* [September Week 12](#44d-s1-w5)
* [September Week 13](#44d-s1-w6)

### [4,4'-Dinitroindigo Synthesis](#44d-s2)

* [October Week 14](#44d-s2-w1)

![](https://static.igem.wiki/teams/5858/images/template/template-sidenav-bottom.webp)

## Green-light Control

### August Week 8 (8.18-8.24)

This week, we attempted to construct the pRSFDuet-PcpcG2-AcGFPplasmid.

1. Using the pRSFDuet-txtE-txtDplasmid as a template, we obtained the promoter PcpcG2via PCR.

2. The PcpcG2promoter fragment and the pRSFDuet-1 vector were digested using BamHI and HindIII restriction enzymes.

3. The digested promoter fragment and vector were ligated using T4 DNA ligase (from Vazyme Biotech).

4. The ligated pRSFDuet-PcpcG2-GFPplasmid was transformed into E. coliDH5α cells via heat shock transformation. Colony PCR was performed, but no positive results were obtained.

5. Selected clones were sent to a biotechnology company for second-generation sequencing analysis. The sequencing results indicated no insertion of the PcpcG2promoter fragment or the GFPgene within the pRSFDuet vector.

### August Week 9 (8.25-8.31)

This week, we continued attempts to construct the pRSFDuet-PcpcG2-AcGFPplasmid and began preparations for verifying the optogenetic proteins.

1. Changed the restriction sites on the PCR primers. Used the new primers to PCR-amplify the GFPgene fragment and the PcpcG2promoter fragment.

2. The GFPfragment and the pRSFDuet-1 vector were digested using BglII and XhoI restriction enzymes. The digested GFPfragment and vector were ligated using T4 DNA ligase. The pRSFDuet-GFPplasmid was transformed into E. coliDH5α via heat shock transformation. Colony PCR was performed, and a positive clone (Strain 1) was selected for culture expansion.

3. Performed PCR to amplify the genes for the four green light optogenetic proteins – ho1, pcyA, mini-CcaSand CcaR– respectively. The obtained gene fragments were prepared for subsequent single-protein verification.

### September Week 10 (9.1-9.7)

This week, we continued the construction of the pRSFDuet-PcpcG2-AcGFPplasmid, began verifying the expression of the optogenetic genes, and initiated stepwise validation.

1. The PcpcG2promoter fragment and the pRSFDuet-GFPvector were digested using NcoI and BglII restriction enzymes. The digested promoter fragment and pRSFDuet-GFPwere ligated using T4 DNA ligase. The pRSFDuet-PcpcG2-GFPplasmid was transformed into E. coliDH5α via heat shock transformation. Colony PCR was performed, and a positive clone was selected. The plasmid was extracted and sent for second-generation sequencing.

2. The green light optogenetic plasmid was transformed into E. coliBL21, and a Western Blot experiment was performed. The results showed that ho1and CcaRwere expressed normally, while verification for pcyAand mini-CcaSfailed.

3. The ho1-pcyAand mini-CcaSgene fragments and the pRSFDuet-1 vector were digested using NdeI and XhoI restriction enzymes. The digested fragments and vector were ligated using T4 DNA ligase. The two recombinant plasmids were transformed into E. coliDH5α via heat shock transformation. Colony PCR was performed, but no successfully transformed colonies were obtained.

### September Week 11 (9.8-9.14)

This week, we completed the construction of the pRSFDuet-PcpcG2-AcGFPplasmid and continued the stepwise verification of the ho1-pcyAand mini-CcaSoptogenetic genes.

1. Re-performed plasmid miniprep from the strain containing the pRSFDuet-PcpcG2-GFPplasmid and verified it by plasmid PCR. The plasmid showing the correct band was sent for sequencing, and a successfully sequenced fragment was finally obtained.

2. Prepared glycerol stocks of the strain containing the successfully sequenced plasmid.

3. The ho1-pcyAand mini-CcaSgene fragments and the pRSFDuet-1 vector were digested using NdeI and XhoI restriction enzymes. The digested fragments and vector were ligated using T4 DNA ligase. The two recombinant plasmids were transformed into E. coliDH5α via heat shock transformation. Colony PCR was performed, but no successfully transformed colonies were obtained.

4. Performed another Western Blot experiment using E. coliDH5α containing the green light optogenetic plasmid. The results indicated that verification for mini-CcaSfailed.

### September Week 12 (9.15-9.21)

This week, we tested the feasibility of the green light optogenetic system.

1. The plasmid was extracted from E. coliDH5α containing the green light optogenetic plasmid.

2. The pRSF-PcpcG2-GFPplasmid and the green light optogenetic plasmid were transformed into BL21, plated, and incubated overnight at a constant temperature.

3. Single colonies were picked for colony PCR. The colony PCR results were incorrect. The bacteria were irradiated with green light overnight but failed to express GFP.

### September Week 13 (9.22-9.28)

This week, we tested the feasibility of the green light optogenetic system again.

1. Another sample of lyophilized plasmid containing the green light optogenetic genes was processed and transformed into E. coliDH5α, plated, and incubated overnight.

2. Single colonies were picked and cultured for expansion, followed by plasmid extraction.

3. The pRSF-PcpcG2-GFPplasmid and the green light optogenetic plasmid were transformed into BL21, plated, and incubated overnight at a constant temperature.

4. Single colonies were picked for colony PCR. Strains with correct colony PCR results were cultured for expansion. After overnight irradiation with green light, GFP expression was not successfully induced.

## 4,4'-Dinitroindigo Synthesis

### October Week 14 (9.29-10.5)

This week, we expanded the stab culture of *E. coli* DH5α containing the pCDFDuet-1-*txtE-txtD-Fdr-Fdx* plasmid and performed co-transformation with both the pRSFDuet-1-*ho1-pcyA-mini-CcaS-CcaR* and pCDFDuet-1-*txtE-txtD-Fdr-Fdx* plasmids. Western blot was conducted to validate the expression of proteins associated with the synthesis of the green pigment 4,4'-dinitroindigo.

1. We picked colonies from a commercial stab culture of *E. coli* DH5α harboring the pCDFDuet-1-*txtE-txtD-Fdr-Fdx* plasmid and performed culture expansion for subsequent plasmid extraction.

2. The pCDFDuet-1*-txtE-txtD-Fdr-Fdx* plasmid was extracted using the alkaline lysis method, and both this plasmid and the pRSFDuet-1-*ho1-pcyA-mini-CcaS-CcaR* plasmid were co-transformed into *E. coli* BL21(DE3) competent cells.

3. Single colonies were selected from solid medium and cultured overnight, followed by inoculation into 50 mL conical flasks. When the OD600 reached 0.8–1.0, one flask was incubated under 520nm green light at 16 °C for 16 hours, while the other was kept in the dark under the same temperature and duration.

4. Western blot was performed to assess the expression of TxtE, TxtD, FdR, and Fdx proteins.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5858/images/template/banner-flower.svg)

# Engineering

![](https://static.igem.wiki/teams/5858/images/template/banner-flower.svg)

Indigo
6,6'-Dibromoindigo
4,4'-Dinitroindigo

![](https://static.igem.wiki/teams/5858/images/template/template-sidenav-top.webp)

![Engineering Cycle Base 2](https://static.igem.wiki/teams/5858/images/engineer/cycle-bg-2.webp)
![design highlight](https://static.igem.wiki/teams/5858/images/engineer/design-final-2.webp)
![build highlight](https://static.igem.wiki/teams/5858/images/engineer/build-final-2.webp)
![test highlight](https://static.igem.wiki/teams/5858/images/engineer/test-final-2.webp)
![learn highlight](https://static.igem.wiki/teams/5858/images/engineer/learn-final-2.webp)

Design

Build

Test

Learn

click
the
cycle!

### [Indigo synthesis](#sub1-1)

* [IPTG-induced expression for indigo production](#sub1-1-1)
  + [Cycle1—IPTG-induced expression](#sub1-1-1-1)
  + [Cycle1.2 Induction conditions modification](#sub1-1-1-2)
* [Promoter modification](#sub1-1-2)
  + [Cycle 2.1 Via Homologous Recombination](#sub1-1-2-1)
  + [Cycle 2.2 Via Restriction Enzyme Digestion and Ligation](#sub1-1-2-2)

### [Blue light-controlled system](#sub1-2)

* [Cycle 1 IPTG-induced expression](#sub1-2-1)
* [Cycle 2 Promoter modification](#sub1-2-2)

### [Dual-plasmid co-expression](#sub1-3)

* [Blue-light control the synthesis of indigo](#sub1-3-1)

### [References](#sub1-4)

![](https://static.igem.wiki/teams/5858/images/template/template-sidenav-bottom.webp)

## Indigo synthesis

### IPTG-induced expression for indigo production

#### Cycle1——IPTG-induced expression for indigo production

##### Design

To biosynthesize indigo, we selected gene *EcTnaA* and gene *MaFMO* based on literature. Although EcTnaA (tryptophanase) is native to *Escherichia coli*, its low endogenous expression required exogenous introduction. EcTnaA cleaves L-tryptophan to indole, which is then oxidized by MaFMO to 3-hydroxyindole. Spontaneous condensation of 3-hydroxyindole forms indigo with water release.[[1]](#ref1-1)

We cloned these genes into the pRSFDuet-1 vector existing in the lab under a T7 promoter for IPTG-induced expression, testing whether this dual-enzyme system could produce indigo in *E.coli.*

##### Build

All cloning procedures were performed in SnapGene. The two genes were assembled into the plasmid backbone by restriction–ligation. *EcTnaA* was inserted with NcoI and NotI sites, whereas *MaFMO* was inserted with BglII and XhoI sites. After sequential digestion and ligation, two genes were seamlessly inserted into the vector.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/img1-png.avif)

Figure 1: History record of pRSFDuet-1-EcTnaA-MaFMO plasmid

##### Test

The assembled plasmid was transformed into *Escherichia coli* BL21(DE3). Cells were first cultured in glucose-containing fermentation medium and induced with 0, 0.2, 0.4, or 0.6 mM IPTG. After 24 h and 48 h, concentrated glucose and tryptophan solutions were fed to the cultures.[[2]](#ref1-2)Both induction and fermentation were carried out at 30 °C. No visible color change was observed at 48 h (Figure 2).

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image002.avif)

Figure 2: The state of the bacterial liquid after 48 hours

Protein expression was subsequently assessed by SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) (Figure 3) and Western blot. In the Figures, 0, 0.2, 0.4, and 0.6 denote IPTG induction concentrations of 0 mM, 0.2 mM, 0.4 mM, and 0.6 mM, respectively.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image003.avif)

Figure 3: SDS-PAGE electrophoresis result image

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image004.avif)

Figure 4: Western blot result image of protein EcTnaA

Western blot showed that *EcTnaA* was not expressed as expected; the observed band was at the wrong molecular weight.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image005.avif)

Figure 5: Western blot result image of the protein MaFMO

Western blot showed that *MaFMO* was expressed as expected; the observed band was at the right molecular weight（56.0KDa）.

##### Learn

After consulting relevant materials, we learned that indole is toxic to cells, and the function of the EcTnaA protein is to convert L-tryptophan into indole. If the induced expression temperature is too high, it will cause the EcTnaA protein to fold incorrectly and form inclusion bodies. Our experiment adopted an induction condition of 30°C. Therefore, we believe that the excessively high induction temperature led to the incorrect expression of EcTnaA protein, thus failing to produce our target product, indigo.

In addition, we have also learned that the glucose in the glucose fixation medium used for bacterial culture will undergo the Maillard reaction with tryptophan at high temperatures, thus being unable to be correctly utilized by bacteria. Moreover, the structure of tryptophan will also change at high temperatures.

#### Cycle1.2 Induction conditions modification

##### Redesign

We adjusted the temperature and time for induction and enzyme activity, and used LB medium to cultivate the bacteria. We added 0.2mM IPTG and 0.4mM IPTG to induce expression, and re-conducted Western Blot experiments to verify the expression of the protein EcTnaA. After confirming the correct expression of the protein, we added glucose concentrate and tryptophan concentrate to the culture medium and re-fermented and cultured to produce indigo.

##### Rebuild

We added 0.2mM and 0.4mM concentrations of IPTG to the culture medium respectively to induce expression, and incubated at 16°C and 190rpm for 16 hours. Then, glucose concentrate and tryptophan concentrate are added, and the bacteria are cultured through two-stage fermentation.

##### Retest

After induction at 16°C, blue bubbles were clearly visible in the culture medium (Figure 6).

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image006.avif)

Figure 6: 0.2 mM IPTG (left), 0.4 mM IPTG (right), 16 °C induction, 16 h

We conducted a Western Blot experiment, and the results indicated that the EcTnaA protein was correctly folded and expressed (Figure 7).

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image007.avif)

Figure 7: Western blot results of EcTnaA protein

After adding glucose and tryptophan, the blue color significantly darkened after 24 hours of culture at 30°C and 200rpm. Moreover, blue particles can be observed in the culture medium (Figure 8).

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image008.avif)

Figure 8: 0.2 mM IPTG (left), 0.4 mM IPTG (right), 30 °C fermentation, 24 h

After adding glucose and tryptophan and continuing to culture for 24 hours, it can be seen that the number of blue particles increases and the color of the culture medium darkens (Figure 9).

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image009.avif)

Figure 9: 0.2 mM IPTG (left), 0.4 mM IPTG (right), 30 °C fermentation, 48 h

##### Relearn

From failure to success in indigo fermentation, we accumulated the experience of failure, adjusted the induction temperature and fermentation conditions, and achieved engineering success. During the experiment, we noticed that tryptophan and glucose would react at high temperatures, and the structure of tryptophan would denature at high temperatures. Therefore, we did not use glucose medium but adopted the method of preparing glucose concentrate and tryptophan concentrate to provide the raw materials for fermentation.

At the same time, we flexibly adjusted the induction temperature and fermentation temperature of IPTG, hoping to first allow*E.coli* to express the corresponding enzyme at 16°C and achieve the best activity of the enzyme at 30°C, so as to better ferment indigo.

Furthermore, by setting different IPTG induction concentrations, we found that the protein expression was the best and the indigo yield was the highest under the induction of 0.2mM concentration IPTG.

### Promoter modification

#### Cycle 2.1 Via Homologous Recombination

##### Design

Since our ultimate goal is to produce indigo under blue light irradiation, we need to modify the promoter of the previously constructed pRSFDuet-1-*EcTnaA-MaFMO* plasmid, as the target genes in this plasmid are driven by the T7 promoter and T7 terminator. We decided to use homologous recombination and inverse PCR methods to replace the T7 promoter and T7 terminator with the pPhlF promoter and L3S3P11 terminator.

##### Build

We designed inverse PCR primers to linearize the plasmid(Figure 10), aiming to remove the first T7 promoter before the MCS1 region. Meanwhile, we used designed homologous recombination primers to amplify the pPhlF promoter from the cloning vector pUC57-*MaFMO-EcTnaA* by PCR.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image010.avif)

Figure 10: Results of plasmid inverse PCR. Lanes 1, 2, 4, and 5 show bands consistent with the expected size of 5113bp.

We used Vazyme's ClonExpress MultiS One Step Cloning Kit (C113-01) to perform homologous recombination ligation between the promoter fragment and the linearized plasmid fragment. To enhance experimental rigor, we set up a blank control group where reaction products without the homologous recombinase Exnase MultiS were transformed into *E.coli* DH5α for culture.

##### Test

Our culture results did not match expectations, as the number of colonies on the negative control plate was not significantly fewer than that on the experimental group plate(Figure 11). We therefore adopted colony PCR to further verify whether the recombinant vector was successfully introduced into *E.coli* DH5α.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image011.avif)
![](https://static.igem.wiki/teams/5858/images/engineeringfig/image012.avif)

Figure 11: Growth of bacteria on plates from homologous recombination experiment. Left(a): Experimental group; Right(b): Negative control group.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image013.avif)

Figure 12: Results of colony PCR

The colony PCR results showed that our homologous recombination reaction failed(Figure 12), and we were unable to successfully ligate the promoter with the linearized plasmid.

##### Learn

We began to review the experiment and finally speculated that primer dimerization was severe. Additionally, with the promoter sequence being only 51bp, the amplified fragment was similar in length to the primer dimer fragments, making it difficult to determine whether the promoter was successfully amplified. Therefore, we used Oligo software for theoretical verification(Figure 13) and designed experiments for practical validation(Figure 14). We performed PCR with only forward and reverse primers without adding the plasmid template to observe if a sequence around 100bp would appear.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image014.avif)

Figure 13: Oligo analysis showing severe self-dimerization of the reverse primer

![](https://static.igem.wiki/teams/5858/images/engineeringfig/img15-png.avif)

Figure 14: Results of experimental verification. Template√ indicates the addition of plasmid template; Template× indicates no plasmid template added.

Based on the above verification, our method of modifying the promoter via homologous recombination failed because the promoter sequence was too short, and our primer design range was also limited. Although this experiment failed, it ruled out a less feasible method for our subsequent systematic promoter modification.

#### Cycle 2.2 Via Restriction Enzyme Digestion and Ligation

##### Redesign

After the failure of the homologous recombination method, we consulted our experimental instructor and decided to amplify the entire target gene fragment pPhlF-*MaFMO-EcTnaA*-L3S3P11 terminator, then insert it into the vector pRSFDuet-1 using restriction enzyme digestion and ligation.

##### Rebuild

We operated in SnapGene, using the restriction enzymes NcoI and NotI to insert the target gene into the vector.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image016.avif)

Figure 15: History record of constructing the pRSFDuet-1-MaFMO-EcTnaA-L3S3P11 vector on SnapGene

##### Retest

We introduced the constructed expression vector into *E.coli* DH5α. Colonies showing positive results in colony PCR were subjected to Sanger sequencing. Sequence alignment confirmed that our target gene was successfully ligated into the vector without mutations.

When our promoter pPhlF is bound to the PhlF protein, it will be unable to drive the expression of the target gene. However, when the blue light light-controlled plasmid is not introduced and the PhlF protein cannot be expressed in the bacteria, the promoter pPhlF will not be inhibited and will normally drive the expression of downstream genes. So we introduced the constructed recombinant plasmid into *E.coli* BL21 (DE3) for fermentation. After 24 hours of culture, we clearly saw indigo pigment particles produced in the culture medium (Figure 16).

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image017.avif)
![](https://static.igem.wiki/teams/5858/images/engineeringfig/img18-png.avif)

Figure 16: Results of plasmid introduction into *E.coli* BL21 (DE3) for 24h after promoter modification (a, b)

##### Relearn

This experimental cycle taught us that when the promoter sequence is too short, making PCR amplification difficult, and when sequences such as the T7 promoter are difficult to remove from the vector, we can consider amplifying the entire segment containing the promoter and using restriction enzyme digestion and ligation to connect it to the vector. Moreover, it is not necessary to completely remove the T7 promoter, Lac operator, and Lac sequences.

## Blue light-controlled system

#### Cycle 1 IPTG-induced expression for blue light-controlled system

##### Design

We constructed a pathway capable of sensing blue light stimulation, utilizing the endogenous flavin mononucleotide (FMN) in *E.coli* as the photosensitive chromophore. In the absence of light, the FixJ protein remains phosphorylated and activates the expression of the repressor protein PhlF, thereby inhibiting indigo pigment synthesis. When exposed to 470 nm blue light, the fusion protein YF1 loses its phosphorylation activity. FixJ-P is then naturally degraded, turning off PhlF expression. By linking the target gene downstream of the pPhlF promoter, an inverter was constructed to achieve gene expression upon blue light induction.[[3]](#ref1-3)

##### Build

*YF1*, *fixJ*, and *PhlF* were cloned into the IPTG-inducible vector pCDFDuet-1 via restriction-ligation cloning. Ribosome binding sites (RBSs) were incorporated between the genes to ensure proper translation initiation.

##### Test

After induction with IPTG at 16°C overnight, the cells were ultrasonically lysed. The supernatant and pellet fractions were separately collected and analyzed by SDS-PAGE to detect the presence of the target proteins (Figure 17).

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image019.avif)

Figure 17: SDS-PAGE result of protein PhlF

##### Learn

Induction experiments confirmed that the blue light-controlled PhlF protein could be correctly expressed in *E.coli*, providing a foundation for subsequent integrated expression. However, due to modifications in the empty vector (insertion of an additional sumo-tag), the reading frame was shifted, resulting in a premature stop codon within the original gene. This prevented normal expression and validation of YF1 and FixJ proteins. Given time constraints, we proceeded directly to constructing a constitutively expressed plasmid.

#### Cycle 2 Promoter modification

##### Design

To enable continuous expression of the blue light control system in *E.coli* for on-demand blue light induction under various conditions, the inducible promoter on the original plasmid needed to be replaced with a constitutive promoter. Considering that conventional restriction-ligation cloning would result in a large plasmid with non-essential segments such as lacI and T7 promoters—further increasing the metabolic burden on the host—we adopted a two-fragment assembly strategy to reconstruct an expression plasmid.

##### Build

Using primers ori-Sm-F and ori-Sm-R with added BamHⅠ and HindⅢ restriction sites, the SmR resistance gene and CloDF13 ori replicon from the original pCDFDuet-1 empty vector were amplified via PCR to obtain the basic plasmid backbone. Primers Y-f-P-F and Y-f-P-R were used to amplify the *YF1*, *fixJ*, and *PhlF* fragments along with the constitutive promoter from the pUC18-*YF1-fixJ-PhlF* plasmid (commercially sourced) in a single Phanta High-Fidelity PCR. The resulting PCR fragments were digested with BamHⅠ and HindⅢ and subsequently ligated(Figure 18,Figure 19).

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image020.avif)

Figure 18: Construction of pCDFDuet-1-PJ23100-YF1-fixJ-PhlF-B0015

![](https://static.igem.wiki/teams/5858/images/engineeringfig/img21-png.avif)

Figure 19: Map of pCDFDuet-1-PJ23100-YF1-fixJ-PhlF-B0015

##### Test

Colony PCR was performed for validation using primer pairs PhlF-F/ori-Sm-R and YF1-fixJ-F/YF1-fixJ-R to verify gene insertion and correct sequence order. Agarose gel electrophoresis confirmed bands of expected sizes (Figure 20). Further sequencing demonstrated successful assembly of the spliced plasmid. This plasmid will be co-transformed with the indigo synthase plasmid into the same bacterial strain for dual-plasmid expression.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image022.avif)

Figure 20: Colony PCR shows that proper gene had been insert in ON1-2/ON1-3/ON1-5

##### Learn

We employed a novel plasmid construction method to reduce non-target fragments in the constitutive expression plasmid, thereby lowering the metabolic burden on the host and facilitating normal expression of the target genes. The assembled plasmid meets our expression requirements, with the target genes correctly linked to the designated constitutive promoter.

## Dual-plasmid co-expression

#### Blue-light control the synthesis of indigo

##### Design

Since our ultimate goal is to achieve blue-light-controlled indigo synthesis, we designed a dual-plasmid expression system. After modifying the promoters on both the blue-light-inducible plasmid and the indigo synthesis plasmid, we decided to proceed with this step.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image023.avif)

Figure 21: Plasmid map of pCDFDuet-1-pJ23100-YF1-fixJ-PhlF-B0015

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image024.avif)

Figure 22: Plasmid map of pRSFDuet-1-pPhlF-MaFMO-EcTnaA-L3S3P11

##### Build

We employed a standard heat shock method to co-transform the two plasmids at a 1:1 molar ratio into *E.coli* BL21(DE3) cells. Positive clones were selected on solid LB medium containing both Kanamycin (Kan) and Streptomycin (Sm).

##### Test

Colonies grew on the double-antibiotic plates. To further confirm successful co-transformation, colony PCR was performed. The results showed the expected target bands: a 2954 bp band for the blue-light-inducible gene and a 3038 bp band for the indigo synthesis gene, indicating the dual-plasmid system was successfully constructed（Figure 23）.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image025.avif)

Figure 23: Colony PCR Analysis of the Two-Plasmid System

The experimental group was irradiated with 470 nm blue light, while the negative control was kept in darkness. After 16 hours of blue-light induction, glucose and tryptophan were added for fermentation. However, no indigo production was observed after 24 hours.

However, through our modeling work, we have theoretically demonstrated the efficacy of the blue light-controlled system(Figure 24 and Figure 25).

Figure 24 showed that PhlF protein is produced in the dark but is effectively suppressed upon light illumination.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/img26-png.avif)

Figure 24: Results for PhlF under Continuous Conditions

Figure 25 indicated that PhlF protein levels increased rapidly during the dark phase and decreased following light exposure.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/img27-png.avif)

Figure 25: Time-course analysis of PhlF protein levels under varying light conditions

##### Learn

Our results indicate that the two-plasmid system was successfully assembled, yet no indigo production was observed. We hypothesize that this may be attributed to either insufficient endogenous flavin mononucleotide (FMN) levels in *E.coli*, resulting in inadequate light sensing, or an incomplete conformational change in YF1. The latter could allow FixJ to remain phosphorylated, leading to continued PhlF expression and its subsequent binding to the pPhlF promoter, thereby suppressing the expression of the indigo-synthesizing enzymes MaFMO and EcTnaA.

[[1]AMERIA S P L, JUNG H S, KIM H S, et.al. Characterization of a flavin-containing monooxygenase from Corynebacterium glutamicum and its application to production of indigo and indirubin[J/OL]. Biotechnology Letters, 2015, 37(8): 1637-1644. DOI:10.1007/s10529-015-1824-2.](https://www.doi.org/10.1007/s10529-015-1824-2)

[[2]LU R, CHEN R, LI Z, et.al. Metabolic engineering of Escherichia coli BL21(DE3) for efficient production of indigo using Methylophaga aminisulfidivorans flavin-containing monooxygenase[J/OL]. Dyes and pigments, 2025, 236. DOI:10.1016/j.dyepig.2025.112682.](https://www.doi.org/10.1016/j.dyepig.2025.112682)

[[3]MÖGLICH A, AYERS R A, MOFFAT K. Design and Signaling Mechanism of Light-Regulated Histidine Kinases[J/OL]. Journal of molecular biology, 2009, 385(5): 1433-1444. DOI:10.1016/j.jmb.2008.12.017.](https://www.doi.org/10.1016/j.jmb.2008.12.017)

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![Engineering Cycle Base 1](https://static.igem.wiki/teams/5858/images/engineer/cycle-bg-1.webp)
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Design

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### [6,6'-Dibromoindigo synthesis](#sub2-1)

* [Cycle1: IPTG-induced expression](#sub2-1-1)

### [Red light-controlled system](#sub2-2)

* [Cycle 1: Verifying protein expression](#sub2-2-1)
* [Cycle 2: AcGFP expression vector](#sub2-2-2)

### [Dual-plasmid co-expression](#sub2-3)

* [Cycle 1: Halogenase-Red Light System (6BrI G)](#sub2-3-1)
* [Cycle 2: Halogenase-Red Light System (6Cl I G)](#sub2-3-2)

### [References](#sub2-4)

![](https://static.igem.wiki/teams/5858/images/template/template-sidenav-bottom.webp)

## 6,6'-Dibromoindigo synthesis

#### Cycle1: IPTG-induced expression for 6,6'-Dibromoindigo production

##### Design

6,6'-Dibromoindigo, which exhibits a purple color, was selected as the target product for Pathway 2. Tryptophan 6-halogenase (SttH) catalyzes the addition of halogen atoms to the C6 position of tryptophan. *Escherichia coli* flavin reductase (Fre) regenerates FADH₂ to facilitate the halogenase reaction. [1]We constructed a fusion protein, Fre-L3-SttH, comprising tryptophan 6-halogenase and flavin reductase, to achieve tryptophan halogenation via a multienzyme cascade. The conventional pRSFDuet-1 plasmid was chosen for inducible expression to assess construct feasibility.

##### Build

The synthetic *Fre-L3-SttH*-rrnB T1 terminator gene was cloned into the pRSFDuet-1 vector via restriction-ligation for IPTG-inducible expression(Figure 26). To address primer specificity, we optimized the PCR protocol by systematically increasing the annealing temperature. This enhancement promoted stricter primer binding and effectively prevented off-target amplification. For the ligation step, the plasmid vector pRSFDuet-1 (a kanamycin-resistant plasmid previously engineered by Professor Cao in our laboratory) was digested with restriction enzymes. The resulting linearized backbone was then isolated and purified using agarose gel electrophoresis. This critical purification step ensured the removal of any undesired small DNA fragments prior to ligation, thereby increasing the likelihood of successful and correct recombinant plasmid assembly.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image028.avif)

Figure 26: Plasmid construction of pRSFDuet-1-Fre-L3-SttH-rrnB T1 terminator.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image029.avif)

Figure 27: Plasmid map of pRSFDuet-1-Fre-L3-SttH-rnnB T1 terminator.

##### Test

Following transformation, positive clones were selected on Kanamycin plates and screened by colony PCR. Analysis of the PCR products via agarose gel electrophoresis identified colonies exhibiting strong and correct-sized amplification bands(Figure 28). The successfully constructed plasmid, pRSFDuet-1-*Fre-L3-SttH*-rrnB T1 terminator, was transformed into *E.coli* BL21(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(Figure 29).

![](https://static.igem.wiki/teams/5858/images/engineeringfig/img30-png.avif)

Figure 28: 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.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image031.avif)

Figure 29: 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.

##### Learn

Through the optimization of key technical parameters in the plasmid assembly process, we achieved efficient and precise ligation of the target gene into the vector. The constructed recombinant plasmid fully met our design requirements for protein expression. Following the transformation of the plasmid into *E.coli*, IPTG induction successfully triggered the expression of the target protein in the host cells.

## Red light-controlled system

#### Cycle 1: Verifying protein expression

##### Design

We identified a recent research paper on optogenetic systems.[[2]](#ref2-2) After reviewing relevant literature and considering the feasibility of our experiment, we designed our red light optogenetic system by modifying the red light system described in this paper.

##### Build

We selected the pCDFDuet-1 plasmid containing a SUMO sequence (previously modified by our laboratory leader) as our vector. Since we needed to express four genes, requiring four tags to detect the target proteins, we selected four commonly used tag proteins. We chose the pJ23106 and pJ23119 promoters from the pJ23 series, used in the RGB system, to drive the expression of two genes each respectively. The recombinant plasmid was designed using SnapGene software, and the map was sent to Sangon Biotech for synthesis. We received the synthesized plasmid approximately one month later.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image032.avif)

Figure 30: Plasmid map of pCDFDuet-1-PadC4-BphO-YhjH-MrkH

##### Test

We transformed the plasmid received from the company into E.coli BL21(DE3). Single colonies were picked and inoculated into 5 mL test tubes containing liquid LB medium and grown overnight. The cultures were then transferred to conical flasks containing 20 mL of liquid LB medium and grown until OD600 reached 0.8-1.0, after which samples were prepared for Western Blot (WB) analysis.

The WB results indicated that MrkH, YhjH, and BphO proteins were expressed correctly, producing visible bands upon reaction with their primary antibodies.

The band position for the PadC4 protein showed some deviation from the expected position of 78.9 kDa. However, subsequent validation using a two-plasmid system for red light-controlled synthesis of 6,6'-Dibromoindigo pigment confirmed that the red light system functioned normally.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image033.avif)

Figure 31: Western blot result image of MrkH protein

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image034.avif)

Figure 32: Western blot result image of YhjH protein

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image035.avif)

Figure 33: Western blot result image of BphO protein

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image036.avif)

Figure 34: Western blot result image of PadC4 protein

##### Learn

Based on subsequent experimental results, we believe that the four proteins of our red light-controlled system can be correctly expressed.

#### Cycle2: AcGFP expression vector

##### Design

We constructed the plasmid pRSFDuet1-PmrkA-RBS*-AcGFP1*, placing the AcGFP green fluorescent protein gene under the control of the PmrkA promoter. This design allows us to validate whether the PmrkA promoter can be regulated by the red light-sensitive plasmid system, thereby assessing the effectiveness and precision of the red light control mechanism.

##### Build

Using SnapGene software, we first selected SalI and KpnI as restriction sites for inserting the RBS-*AcGFP1* gene fragment, followed by the use of MluI and SalI sites for subsequent incorporation of the PmrkA promoter. The first construct, pRSFDuet1-RBS-*AcGFP1,* was sequence-verified by a commercial service to confirm proper gene insertion. For the second plasmid, pRSFDuet1-PmrkA-RBS-*AcGFP1*, although not subjected to commercial sequencing due to time constraints, successful assembly was preliminarily confirmed by colony PCR.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image037.avif)

Figure 35: Plasmid map of pRSFDuet1-PmrkA-RBS-AcGFP1 showing the key regulatory elements.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image038.avif)

Figure 36: Construction process of the pRSFDuet1-PmrkA-RBS-AcGFP1 plasmid.

##### Test

The constructed plasmid pRSFDuet1-PmrkA-RBS-*AcGFP1* was co-transformed with the red-light expression vector pCDFDuet-1-*PadC4-BphO-YhjH-MrkH* into *E.coli* BL21(DE3) and plated on agar medium containing both streptomycin and kanamycin. Visible colony formation confirmed successful transformation.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image039.avif)

Figure 37: 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.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image040.avif)

Figure 38: Colony PCR analysis of the pRSFDuet1-PmrkA-RBS-AcGFP1 plasmid transformed into ***E.coli*** DH5α. All observed bands represent positive clones and correspond to the expected fragment length of 215 bp.

Single colonies were selected and subjected to the following culture protocol: primary culture in 5 mL LB liquid medium with dual antibiotics overnight, followed by secondary culture in 25 mL LB medium. When the OD₆₀₀ reached 0.8-1, cultures were exposed to red light illumination for 12 h at 30°C with 200 rpm shaking.

After induction, cultures were further incubated for 4 h under dark conditions at 200 rpm to monitor *AcGFP* expression. Control groups with and without red-light exposure were established and cultured under identical conditions. Comparative analysis of *AcGFP* expression levels between groups could demonstrate the efficacy and precision of the red-light control system. Restriction analysis and colony PCR from two independent experiments confirmed the successful construction of the plasmids and the efficiency of the dual-plasmid transformation system.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image041.avif)

Figure 39: Transformation of successful dual-plasmid transformation.

However, in two separate rounds of red light-induced fermentation, no green fluorescent protein expression was detected. This suggests that the PmrkA promoter may not have been successfully incorporated into the plasmid, thus failing to initiate transcription of the downstream gene.

##### Learn

In this cycle, the plasmid pRSFDuet1-RBS-*AcGFP1* was successfully constructed. However, no green fluorescent protein expression was detected under red light-induced fermentation conditions. The lack of AcGFP1 expression indicated a construction failure in the pRSFDuet1-PmrkA-RBS-*AcGFP1* plasmid, likely due to unsuccessful incorporation of the PmrkA promoter that prevented functional validation. However, the subsequent dual-plasmid system containing both the red light-sensing and 6,6'dibromoindigo components demonstrated clear light-dependent production of 6,6'dibromoindigo pigment. These results collectively confirm that the PmrkA promoter retains full biological activity and exhibits strict light-responsive regulation.

## Dual-plasmid co-expression

#### Cycle 1: Halogenase-Red Light System for Chlorination: 6BrI G

##### Design

To achieve light-regulated synthesis of the purple pigment (6,6'-dibromoindigo), we upgraded the regulation mode of halogenase from traditional T7 induction to precision regulation by red light (710nm), thereby driving halogenase expression. Continuing with the previously validated and effective Fre-L3-SttH-6xHis fusion protein, we placed it downstream of the PmrkA promoter to achieve red light-dependent halogenase expression. Ultimately, the purple pigment is produced through the pathway "tryptophan → 6-bromotryptophan → 6-bromoindole → 6,6'-dibromoindigo". We tested KBr concentrations (150 mM, 300 mM, 600 mM) and fermentation times (24 h, 36 h, 48 h) to optimize yield, confirming 300 mM KBr + 36 h as optimal.

##### Build

Through restriction enzyme digestion and ligation, the PmrkA promoter and *Fre-L3-SttH* were ligated into the pET-28a vector. An RBS sequence was inserted between the genes to ensure correct gene expression.

##### Test

**1. Halogenase Expression Verification**

The red light-controlled plasmid and the halogenase plasmid were co-transformed into *Escherichia coli* BL21 (DE3). After 12 hours of red light irradiation, the bacterial cells were ultrasonically disrupted. Verification was performed by SDS-PAGE (Figure 40, to detect the ~90 kDa Fre-L3-SttH fusion protein) and Western Blot (Figure 41, with anti-6xHis tag antibody).

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image042.avif)

Figure 40: SDS-PAGE electrophoresis result image

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image043.avif)

Figure 41: Western Blot result image of gene Fre-L3-SttH

Results showed that clear target protein bands could be detected in the red light group, while no obvious bands were observed in the dark group, indicating that halogenase is only expressed under red light.

**2. 6Br I G Optimal Yield Confirmation**

300 mM KBr group's fermentation liquid (Figure 42) showed the the deepest light red (intermediate color during 6Br I G synthesis), indicating active pigment production.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image044.avif)

Figure 42: 6Br I G fermentation liquid (300 mM KBr, 36 h)

We used DMSO to dissolve the bacterial pellet after co-culturing with indigo bacteria (Figure 43), which shows intense purple, confirming successful conversion to the final pigment.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image045.avif)

Figure 43: DMSO-dissolved centrifuged precipitate

##### Learn

**Key Success Points:**

The red light system enables "on-off" expression of halogenase with high specificity. It requires no chemical inducers, thus reducing the metabolic burden on bacterial cells. The dual-plasmid system has good compatibility; the photosensitive module and halogenase expression module work synergistically, stably driving the synthesis of 6-bromotryptophan.

**Areas for Optimization:**

There is leaky expression in the dark group, which is speculated to be related to the expression level of YhjH. In the future, we can enhance YhjH activity by screening for stronger RBS sequences to further reduce c-di-GMP levels in the dark. There is still room for improvement in the yield of 6-bromotryptophan. We can attempt to optimize the strength of the PmrkA promoter or the linker sequence of *Fre-L3-SttH* (e.g., replacing it with a shorter GGS linker) to improve the catalytic efficiency of the fusion protein.

**Discoveries:**

300 mM KBr + 36 h is optimal; 600 mM KBr inhibits growth.

SttH can catalyze chlorination for Cycle 3.

#### Cycle 2: Halogenase-Red Light System for Chlorination: 6Cl I G

##### Design

Based on SttH's chlorination activity (discovered in Cycle 1), we synthesized pale red 6,6'-dichloroindigo (6Cl I G) using NaCl as the donor. After testing concentrations (150 mM, 300 mM, 600 mM) and times, 150 mM NaCl + 36 h was confirmed as optimal.

##### Build

Reused the dual-plasmid system from Cycle 1 (no modification); only replaced KBr with NaCl.

##### Test

150 mM NaCl group (Figure 44) showed the most stable pale red, indicating highest 6ClG yield.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image046.avif)

Figure 44: 6ClIG fermentation liquid (150 mM NaCl, 36 h)

We used DMSO to dissolve the bacterial pellet after co-culturing with indigo bacteria (Figure 45).

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image047.avif)

Figure 45: DMSO-dissolved bacterial pellet centrifuged precipitate

##### Learn

SttH catalyzes both bromination and chlorination, expanding the "microbial paintbrush" color palette.

150 mM NaCl + 36 h is optimal for 6ClIG.

[[1]LEE J, KIM J, SONG J E, et.al. Production of Tyrian purple indigoid dye from tryptophan in Escherichia coli[J/OL]. Nature chemical biology, 2021, 17(1): 104-112. DOI:10.1038/s41589-020-00684-4.](https://www.doi.org/10.1038/s41589-020-00684-4)

[[2]RYU M H, GOMELSKY M. Near-infrared Light Responsive Synthetic c-di-GMP Module for Optogenetic Applications[J/OL]. ACS synthetic biology, 2014, 3(11): 802-810. DOI:10.1021/sb400182x.](https://www.doi.org/10.1021/sb400182x)

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![Engineering Cycle Base 3](https://static.igem.wiki/teams/5858/images/engineer/cycle-bg-3.webp)
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Design

Build

Test

Learn

click
the
cycle!

### [4,4'-Dinitroindigo synthesis](#sub3-1)

* [Cycle1: Verifying protein expression](#sub3-1-1)

### [Green light-controlled system](#sub3-2)

* [Cycle 1: Verifying protein expression](#sub3-2-1)

### [References](#sub3-4)

![](https://static.igem.wiki/teams/5858/images/template/template-sidenav-bottom.webp)

## 4,4'-Dinitroindigo synthesis

#### Cycle1: Verifying protein expression

##### Design

Based on our review of the relevant literature, we selected TxtE[[1]](#ref3-1), a novel cytochrome P450 enzyme, to achieve direct regiospecific 4-nitration of L-tryptophan, thereby generating the substrate required for 4,4'-dinitroindigo production. Since the nitration activity of TxtE depends on NO, O₂, and electrons[[2]](#ref3-2), we also incorporated nitric oxide synthase TxtD[[3]](#ref3-3), ferredoxin reductase FdR, and the small iron-sulfur protein ferredoxin Fdx to supply NO and electrons.

##### Build

We selected the pRSFDuet-1 plasmid as our expression vector. Following the design principle of the RGB system, we incorporated the DT25 terminator. To facilitate subsequent verification of protein expression, we appended common epitope tags (S-tag, 6×His tag, HA-tag, and FLAG-tag) after each gene. The constructed plasmid map was sent to a commercial supplier for synthesis, and the synthesized plasmid was received near the end of this year's competition season.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image048.avif)

Figure 46: Plasmid map of pRSFDuet-1-txtE-txtD-Fdr-Fdx

##### Test

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. After obtaining positive monoclonal colonies, we established experimental and negative control groups: the experimental group was exposed to green light, while the negative control group was kept in the dark.

Western blot analysis showed that FdR(Figure 48) and Fdx(Figure 47) proteins were correctly expressed, whereas TxtD(Figure 49) and TxtE(Figure 50) proteins failed to express properly. In addition, relatively severe leaky expression was observed, which may be attributed to incomplete light exclusion.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image049.avif)

Figure 47: Western blot result of Fdx protein. The red box highlights the target protein region（12.6KDa）

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image050.avif)

Figure 48: Western blot result of FdR protein. The red box highlights the target protein region（46.9KDa）

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image051.avif)

Figure 49: Western blot result of TxtD protein

![](https://static.igem.wiki/teams/5858/images/engineeringfig/img52-png.avif)

Figure 50: Western blot result of TxtE protein

##### Learn

Upon further literature review, we identified a 2022 study reporting that heterologous expression of TxtD in bacterial hosts such as *E.coli* remained unsuccessful. The authors proposed that overexpression of nitric oxide synthase in vivo may elevate intracellular NO levels to cytotoxic concentrations, which likely explains the observed poor expression or insoluble aggregation of TxtD protein. Through this additional investigation, we have clarified the probable cause of the failed protein expression.

## Green light-controlled system

#### Cycle 1: Verifying protein expression

##### Design

To achieve light-controlled synthesis of the green pigment (4,4'-dinitroindigo) in the "microbial paintbrush", we designed a light-controlled pathway centered on the CcaS/R green light sensing system[[4]](#ref3-4), with the core logic of "green light activation - no light deactivation". This pathway is coupled with a nitrating enzyme module to ensure precise regulation of green pigment synthesis.[[5]](#ref3-5)

The *ho1* and *pcyA* genes (encoding heme oxygenase and phycocyanobilin/ferredoxin oxidoreductase, respectively) were introduced and their expression was driven by a constitutive promoter. These genes catalyze the conversion of heme in Escherichia coli to the photosensitive chromophore phycocyanobilin (PCB), providing the essential chromogenic basis for mini-CcaS.[[6]](#ref3-6)

Upon irradiation with 520nm green light, PCB-bound mini-CcaS undergoes a conformational change, phosphorylating CcaR (forming CcaR-P). CcaR-P specifically binds to the optimized output promoter PcpcG2-172. In the absence of light, mini-CcaS maintains low kinase activity, CcaR remains unphosphorylated, the PcpcG2-172 promoter stays in a closed state, and downstream genes are not expressed, achieving the regulatory effect of "no light, no expression".

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image053.avif)

Figure 51: Plasmid map of pCDFDuet-1-ho1-pcyA-mini-CcaS-CcaR

##### Build

Using pCDFDuet-1 as the vector, component assembly was completed via restriction enzyme digestion and ligation.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image054.avif)

Figure 52: Plasmid construction history of pCDFDuet-1-ho1-pcyA-mini-CcaS-CcaR

##### Test

Engineered bacteria were subjected to green light/no light treatments, followed by detection via Western Blot.

The results showed that, except for HO1 (Figure 53), PcyA (Figure 54), CcaR (Figure 55) and mini-CcaS proteins (Figure 56) were all normally expressed.

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image055.avif)

Figure 53: Western Blot result image of protein HO1

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image056.avif)

Figure 54: Western Blot result image of protein PcyA

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image057.avif)

Figure 55: Western Blot result image of protein CcaR

![](https://static.igem.wiki/teams/5858/images/engineeringfig/image058.avif)

Figure 56: Western Blot result image of protein mini-CcaS

##### Learn

Although the dual-plasmid system is stable, future work could explore the single-plasmid integration strategy reported in literature (assembling photosensitive components and nitrating enzyme components into the same vector) to further simplify experimental operations.

[[1]LI S, DU L, BERNHARDT R. Redox Partners: Function Modulators of Bacterial P450 Enzymes[J/OL]. Trends in microbiology (Regular ed.), 2020, 28(6): 445-454. DOI:10.1016/j.tim.2020.02.012.](https://www.doi.org/10.1016/j.tim.2020.02.012)

[[2]BARRY S M, KERS J A, JOHNSON E G, et.al. Cytochrome P450–catalyzed L-tryptophan nitration in thaxtomin phytotoxin biosynthesis[J/OL]. Nature chemical biology, 2012, 8(10): 814-816. DOI:10.1038/nchembio.1048.](https://www.doi.org/10.1038/nchembio.1048)

[[3]TIAN X, LIU W Q, XU H, et.al. Cell-free expression of NO synthase and P450 enzyme for the biosynthesis of an unnatural amino acid L-4-nitrotryptophan[J/OL]. Synthetic and systems biotechnology, 2022, 7(2): 775-783. DOI:10.1016/j.synbio.2022.03.006](https://www.doi.org/10.1016/j.synbio.2022.03.006)

[[4]ONG N T, TABOR J J. A Miniaturized Escherichia coli Green Light Sensor with High Dynamic Range[J/OL]. Chembiochem : a European journal of chemical biology, 2018, 19(12): 1255-1258. DOI:10.1002/cbic.201800007](https://www.doi.org/10.1002/cbic.201800007)

[[5]NAMGUNG S, PARK H A, KIM J, et.al. Ecofriendly one-pot biosynthesis of indigo derivative dyes using CYP102G4 and PrnA halogenase[J/OL]. Dyes and pigments, 2019, 162: 80-88. DOI:10.1016/j.dyepig.2018.10.009.](https://www.doi.org/10.1016/j.dyepig.2018.10.009)

[[6]NAKAJIMA M, FERRI S, RÖGNER M, et.al. Construction of a Miniaturized Chromatic Acclimation Sensor from Cyanobacteria with Reversed Response to a Light Signal[J/OL]. Scientific reports, 2016, 6(1). DOI:10.1038/srep37595.](https://www.doi.org/10.1038/srep37595)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
{% block page\_content %}

## What Should this Page Contain?

---

Provide comprehensive, step-by-step protocols for all experiments conducted. Include a list of materials, reagents, and equipment used. Specify concentrations, volumes, incubation times, temperatures, and other critical parameters. Document any modifications or optimizations made to standard protocols.

Explain the purpose of each experiment and its relevance to your project goals. Describe the experimental design, including controls and replicates. Provide a clear rationale for the chosen methods and approaches.

Document any troubleshooting steps taken and optimizations made during the experimental process. Share any lessons learned that could benefit future teams.

## Inspirations

---

* [2024 Heidelberg](https://2024.igem.wiki/heidelberg/experiments)
* [2024 Marburg](https://2024.igem.wiki/marburg/experiments)
* [2024 EPFL](https://2024.igem.wiki/epfl/experiments)
* [2023 Estonia-TUIT](https://2023.igem.wiki/estonia-tuit/experiments)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5858/images/template/banner-flower.svg)

# Results

![](https://static.igem.wiki/teams/5858/images/template/banner-flower.svg)

Indigo
6,6'-Dibromoindigo
4,4'-Dinitroindigo

![](https://static.igem.wiki/teams/5858/images/template/template-sidenav-top.webp)

### [Indigo Synthesis](#sub1-1-1)

* [Preface](#sub1-1-1)
* [Plasmids construction](#sub1-1-2)
* [Successful transformation verification](#sub1-1-3)
* [Protein expression verification](#sub1-1-4)
* [Effect verification](#sub1-1-5)

### [Blue light-controlled System](#sub1-2-1)

* [Plasmids construction](#sub1-2-1)
* [Successful transformation verification](#sub1-2-2)
* [Protein expression verification](#sub1-2-3)

### [Dual-plasmid Co-expression](#sub1-3-1)

* [System construction](#sub1-3-1)
* [Effect verification](#sub1-3-2)

![](https://static.igem.wiki/teams/5858/images/template/template-sidenav-bottom.webp)

## Indigo synthesis

### Preface

We weighed 0.1g of the standard indigo sample powder, added 1mL of water, ethanol, isopropanol, DMSO, PBS and glycerol to each, dissolved them, and centrifuged at 12,000 rpm for 2 minutes. We took 500ul of the supernatant and then conducted high-temperature stability tests and indigo stability tests at room temperature.

We covered the samples with tin foil to control the variables and prevent light from affecting the indigo samples. We heated them at 40℃, 60℃, 80℃ and 100℃ for 30 minutes each. The results showed that the color of the samples did not change, but the volume of the samples decreased. It indicates that indigo does not decompose at high temperatures and has good stability. The order of solvents added to the samples in Figures 1 to 5 is water, ethanol, isopropanol, DMSO, PBS and glycerol.

![](https://static.igem.wiki/teams/5858/images/results/indigo-figure1.webp)

Figure 1:the indigo samples before heating

![](https://static.igem.wiki/teams/5858/images/results/indigo-figure2.webp)

Figure 2: the indigo samples 40℃ heated for 30 minutes

![](https://static.igem.wiki/teams/5858/images/results/indigo-figure3.webp)

Figure 3: the indigo samples 60℃ heated for 30 minutes

![](https://static.igem.wiki/teams/5858/images/results/indigo-figure4.webp)

Figure 4: the indigo samples 80℃ heated for 30 minutes

![](https://static.igem.wiki/teams/5858/images/results/indigo-figure5.webp)

Figure 5: the indigo samples 100℃ heated for 30 minutes

On August 19th, we wrapped another batch of indigo samples in tin foil and placed them at room temperature. We took them out for observation on October 4th and found no obvious fading. This indicates that when placed at room temperature for 46 days, indigo does not undergo obvious degradation or color change. The order of solvents added to the samples in Figure 6 is DMSO, glycerol, water, PBS, ethanol and isopropanol.

![](https://static.igem.wiki/teams/5858/images/results/indigo-figure6.webp)

Figure 6: Results of the observation taken on October 4

Because of the good stability of indigo, our team chose indigo as the main pigment for our experiment.

### Plasmids construction

Weconstructed two plasmids. The gene expression on the first plasmid pRSFDuet-1-**EcTnaA-MaFMO** are regulated by the T7 promoter and T7 terminator. The gene expression on the second plasmid pRSFDuet-1-pPhlF-*MaFMO-EcTnaA*-L3S3P11 are regulated by the promoter pPhlF and the terminator L3S3P11.

![](https://static.igem.wiki/teams/5858/images/results/indigo-figure7.webp)

Figure 7: The plasmid map of pRSFDuet-1-*EcTnaA-MaFMO*

![](https://static.igem.wiki/teams/5858/images/results/indigo-figure8.webp)

Figure 8: The plasmid map of pRSFDuet-1-pPhlF-*MaFMO-EcTnaA*-L3S3P11

The sequencing results from the company show that our target gene has indeed been successfully connected and no mutations have occurred.

### Successful transformation verification

#### 1.pRSFDuet-1*-EcTnaA-MaFMO*

We conducted the PCR amplified and purified*EcTnaA* and *MaFMO* genes from the cloning vector and ligated them to the expression vector pRSFDuet-1 respectively by enzymatic digestion.

![](https://static.igem.wiki/teams/5858/images/results/indigo-figure9.webp)

Figure 9:The PCR results of *EcTnaA* gene

The target band is in the red frame , which corresponds to the expected gene length of 1416bp.

![](https://static.igem.wiki/teams/5858/images/results/indigo-figure10.webp)

Figure 10: The colony PCR results of plasmid pRSFDuet-1-EcTnaA introduced into E.coli DH5α

There are positive clones 3, 4, 5 and 6, and the bands match the expected length of 1416bp.

![](https://static.igem.wiki/teams/5858/images/results/indigo-figure11.webp)

Figure 11:The PCR results of *MaFMO* gene

They correspond to the expected gene length of 1371bp.

![](https://static.igem.wiki/teams/5858/images/results/indigo-figure12.webp)

The colony PCR results of plasmid pRSFDuet-1-*EcTnaA-MaFMO* introduced into E.coli DH5α

There are positive clones 1 and 2, and the bands match the expected length of 1371bp.

The sequencing results from the company show that our target gene has indeed been successfully connected and no mutations have occurred.

#### 2.pRSFDuet-1-pPhlF-*MaFMO*-*EcTnaA*-L3S3P11

We cloned the entire target gene expression cassette pPhlF-*MaFMO-EcTnaA*-L3S3P11 from the cloning vector and inserted it into the pRSFDuet-1 vector using NcoI and NotI restriction enzymes.

![](https://static.igem.wiki/teams/5858/images/results/indigo-figure13.webp)

Figure 13:The PCR results of pPhlF-*MaFMO-EcTnaA*-L3S3P11 terminator

The PEMT in the figure refer to pPhlF-*MaFMO-EcTnaA*-L3S3P11 terminator.They correspond to the expected length of 3040bp.

![](https://static.igem.wiki/teams/5858/images/results/indigo-figure14.webp)

Figure 14: The colony PCR results of plasmid

pRSFDuet-1-pPhlF-*MaFMO-EcTnaA*-L3S3P11 introduced into E.coli DH5α There are positive clones T1 and the bands match the expected length of 3040bp.

#### Protein expression verification

We introduced the successfully constructed pRSFDuet-1-*EcTnaA-MaFMO* vector into *E.coli* BL21 (DE3) and induced it at 16 ℃ for 16 hours at IPTG concentrations of 0mM, 0.2mM, 0.4mM and 0.6mM, respectively. After induction,we conducted a Western Blot experiment to verify protein expression.

![](https://static.igem.wiki/teams/5858/images/results/indigo-figure15.webp)

Figure 15: Western blot results of *EcTnaA* protein (6xHis-tag)

The bands are consistent with the expected strip length of 53.8kDa.

![](https://static.igem.wiki/teams/5858/images/results/indigo-figure16.webp)

Figure 16:Western blot results of MaFMO protein (S-tag)

The bands are consistent with the expected strip length of 56.0kDa.

### Effect verification

After the low-temperature induction was completed, a distinct blue color was produced in the 0.2mM induced bacterial liquid, while the blue color was not obvious in the 0.4mM one.

After consulting the references, we added the corresponding amounts of L-trp and glucose to the bacterial liquid cultivated in a 50mL conical flask. The two-stage fermentation method produced a distinct blue color.

![](https://static.igem.wiki/teams/5858/images/results/indigo-figure17.webp)

Figure 17:Results of small conical bottle induction termination

(From left to right are 0mM,0.2mM and 0.4mM)

![](https://static.igem.wiki/teams/5858/images/results/indigo-figure18.webp)

Figure 18:Results of big conical bottle induction termination

(Left:0.2mM,right:0.4mM)

![](https://static.igem.wiki/teams/5858/images/results/indigo-figure19.webp)

Figure 19:the results of the first stage fermentation in a large conical flask for 24h

(Left:0.2mM,right:0.4mM)

![](https://static.igem.wiki/teams/5858/images/results/indigo-figure20.webp)

Figure 20:the results of the second stage fermentation in a large conical flask for 24h

(Left:0.2mM,right:0.4mM)

We took 1mL of the cultured bacterial liquid, washed the bacterial sediment with 1mL of anhydrous methanol, and then added 2 ml of DMSO to extract the indigo pigment.

![](https://static.igem.wiki/teams/5858/images/results/indigo-figure21.webp)

Figure 21: Results of the dissolution of indigo in DMSO

(Left:0.2mM,right:0.4mM)

Using DSMO as the solvent, we prepared a series of indigo standard samples with concentration gradients. The absorbance of the samples at 610nm was tested using an enzyme-linked immunosorbent assay (ELISA) detector. Linear regression analysis was conducted on the absorbance and indigo concentration data, and the relationship curve between the two was fitted (Figure 16). The R2 of the linear regression analysis was close to 1, indicating that the image fitting was relatively good. We conducted an absorbance test on the indigo pigment we extracted ourselves, which was 0.635. According to the fitted formula, the concentration of the indigo pigment we extracted ourselves was approximately 0.03570mg/mL.

![](https://static.igem.wiki/teams/5858/images/results/indigo-figure22.webp)

Figure 22: The relationship between absorbance and indigo concentration

We used the fermentation liquid after our fermentation to dye the silk fabric. The dyeing effect was quite good. After dyeing, the fabric was left for a week under normal conditions, and the color slightly faded.

![](https://static.igem.wiki/teams/5858/images/results/indigo-figure23-a.webp)

Figure23-a

![](https://static.igem.wiki/teams/5858/images/results/indigo-figure23-b.webp)

Figure23-b

(The Figure23-b is 1 week after dyeing.)

When the vector of the modified promoter is not introduced with the blue light photocontrolled plasmid, the PhlF protein will not be produced in the bacteria, the promoter pPhlF will not be inhibited, and the expression of downstream genes can be correctly initiated. So we introduced the successfully constructed pRSFDuet-1-pPhlF-*MaFMO-EcTnaA*-L3S3P11 plasmid into *E.coli* BL21(DE3) for effect verification. After 24 hours of fermentation, obvious blue particles were produced in the culture medium.

![](https://static.igem.wiki/teams/5858/images/results/indigo-figure24-a.webp)

Figure 24-a

![](https://static.igem.wiki/teams/5858/images/results/indigo-figure24-b.webp)

Figure 24-b

Figure 24: Results of plasmid introduction into E.coli BL21 (DE3) for 24h after promoter modification

## Blue light-controlled system

### Plasmids construction

We constructed two plasmids. In the first plasmid, pCDFDuet-1-*YF1-fixJ-PhlF*, gene expression is regulated by the T7 promoter and T7 terminator and this plasmid is used to verify whether the optogenetic proteins can be expressed normally in *E.coli*. In the second plasmid, pCDFDuet-1-pJ23100*-YF1-fixJ-PhlF-*B0015, gene expression and termination are controlled by the constitutive promoter pJ23100 and the terminator B0015.​​

![](https://static.igem.wiki/teams/5858/images/results/blue-light-figure25.webp)

Figure 25:​​ Plasmid map of pCDFDuet-1-*YF1-fixJ-PhlF*.

![](https://static.igem.wiki/teams/5858/images/results/blue-light-figure26.webp)

Figure 2​​6: Plasmid map of pCDFDuet-1-PJ23100-*YF1-fixJ-PhlF*-B0015.

### Successful transformation verification

#### 1.pCDFDuet-1*-YF1-fixJ-PhlF*

The *YF1-fixJ* and *PhlF* fragments were obtained via PCR amplification from the cloning vectors and subsequently ligated into the MCS1 and MCS2 sites, respectively, of the pCDFDuet-1 vector via restriction enzyme digestion and ligation.​​

![](https://static.igem.wiki/teams/5858/images/results/blue-light-figure27.webp)

Figure 27:​​ PCR results for the *PhlF* gene fragment.The target band matches the expected size of 619 bp.

![](https://static.igem.wiki/teams/5858/images/results/blue-light-figure28.webp)

Figure 28:​​ Colony PCR results for pCDFDuet-1-PhlF transformed into *E. coli* DH5α.All bands are from positive clones and match the expected size of 619 bp.

![](https://static.igem.wiki/teams/5858/images/results/blue-light-figure29.webp)

Figure 29:​​ PCR results for the *YF1-fixJ* gene fragment.The target band matches the expected size of 1771 bp.

![](https://static.igem.wiki/teams/5858/images/results/blue-light-figure30.webp)

Figure 30:​​ Colony PCR results for pCDFDuet-1-*YF1-fixJ-PhlF* transformed into *E. coli* DH5α.Lanes 1, 2, 4, 6, and 7 show positive clones, with bands matching the expected size of 1771 bp.

#### pCDFDuet​​-1-pJ23100-*YF1-fixJ-PhlF-*B0015

The long fragment pJ23100-*YF1-fixJ-PhlF*-B0015, containing the constitutive promoter and terminator, was amplified via PCR using a high-fidelity DNA polymerase from the cloning vector. The vector replicon and streptomycin resistance gene (abbreviated as ori-Sm) were amplified via PCR from the empty pCDFDuet-1 vector using a high-fidelity DNA polymerase. The two fragments were assembled by restriction enzyme digestion and ligation to form the new plasmid.

![](https://static.igem.wiki/teams/5858/images/results/blue-light-figure31.webp)

Figure 31:​​ PCR results for the ori-Sm DNA fragment.The target band matches the expected size of 1995 bp.​​

![](https://static.igem.wiki/teams/5858/images/results/blue-light-figure32.webp)

Figure 32:​​ PCR results for the pJ23100-*YF1-fixJ-PhlF*-B0015 gene fragment.The target band matches the expected size of 3096 bp.

![](https://static.igem.wiki/teams/5858/images/results/blue-light-figure33.webp)

Figure 33: Colony PCR results for pCDFDuet-1-pJ23100-*YF1-fixJ-PhlF*-B0015 transformed into *E. coli* DH5α.Lanes 2, 3, and 5 show positive clones, with bands matching the expected sizes of 3096 bp and 1771 bp.

### Protein expression verification

We introduced the successfully constructed pCDFDuet-*YF1-fixJ-PhlF* vector into *E.coli* BL21 (DE3) and induced it at 16 ℃ for 16 hours at IPTG concentrations of 1mM, 0.8mM, 0.5mM, respectively. After induction,we conducted a SDS-PAGE experiment to verify protein expression.

![](https://static.igem.wiki/teams/5858/images/results/blue-light-figure34.webp)

Figure34: SDS-PAGE result for protein PhlF expression

## Dual-plasmid co-expression system

### System construction

The plasmids pCDFDuet-1-pJ23100-*YF1-fixJ-PhlF*-B0015 and pRSFDuet-1-pPhlF-*MaFMO-EcTnaA*-L3S3P11 were co-transformed into *E. coli* BL21(DE3) cells. Positive transformants were selected using double-antibiotic agar plates containing streptomycin and kanamycin.

Colony PCR confirmed the presence of both plasmids within the selected clones.

![](https://static.igem.wiki/teams/5858/images/results/blue-light-figure35.webp)

Figure35: Colony PCR analysis of *E. coli*BL21(DE3) after dual-plasmid transformation.

The band in lane YfP-1 corresponds to the expected size (2,964 bp) of the target gene from the blue light-controlled system, while the band in lane Ma-Ec-1 matches the predicted length (3,038 bp) of the target gene for indigo biosynthesis.

### Effect verification

Following induction with 470 nm blue light for 16 hours, the culture medium was supplemented with glucose and tryptophan for fermentation. After 24 hours of subsequent cultivation, no indigo production was observed.

Although time constraints prevented further investigation into the inability of the blue light-controlled system to facilitate indigo production, our computational modeling results indicate that the system is theoretically feasible and compatible with the pigment synthesis pathway.

![](https://static.igem.wiki/teams/5858/images/results/dual-plasmid1-figure36.avif)

Figure36: Results for PhlF under Continuous Conditions

These results demonstrate that PhlF is expressed in the dark, while its expression is suppressed under light illumination. Furthermore, the regulatory effect of the blue light-controlled system exhibits a time-dependent enhancement, demonstrating distinct and highly efficient "switch-like" characteristics.

![](https://static.igem.wiki/teams/5858/images/results/dual-plasmid1-figure37.avif)

Figure37:Time-course analysis of PhlF protein levels under varying light conditions

The results above demonstrate that regardless of the duration of dark pretreatment, PhlF protein levels increased rapidly during the dark phase and decreased following light exposure.

![](https://static.igem.wiki/teams/5858/images/template/template-sidenav-top.webp)

### [6,6'-Dibromoindigo Synthesis](#sub2-1-1)

* [Plasmid construction](#sub2-1-1)
* [Successful transformation verification](#sub2-1-2)
* [Protein expression verification](#sub2-1-3)

### [Red light-controlled system](#sub2-2-1)

* [Plasmids construction](#sub2-2-1)
* [Successful transformation verification](#sub2-2-2)
* [Protein expression verification](#sub2-2-3)

### [Dual-plasmid Co-expression](#sub2-3-1)

* [System construction](#sub2-3-1)
* [Protein expression verification](#sub2-3-2)
* [Effect verification](#sub2-3-3)

![](https://static.igem.wiki/teams/5858/images/template/template-sidenav-bottom.webp)

## 6,6'-Dibromoindigo synthesis

### 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.

![](https://static.igem.wiki/teams/5858/images/results/6-6-dibromoindigo-synthesis-figure38.webp)

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).

![](https://static.igem.wiki/teams/5858/images/results/6-6-dibromoindigo-synthesis-figure39.webp)

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.

![](https://static.igem.wiki/teams/5858/images/results/6-6-dibromoindigo-synthesis-figure40.webp)

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. coli*BL21(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.

![](https://static.igem.wiki/teams/5858/images/results/6-6-dibromoindigo-synthesis-figure41.webp)

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.

## Red light-controlled system

### 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 *YhjH*and *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.

![](https://static.igem.wiki/teams/5858/images/results/red-light-figure42.avif)

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

![](https://static.igem.wiki/teams/5858/images/results/red-light-figure43.avif)

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

![](https://static.igem.wiki/teams/5858/images/results/red-light-figure44.avif)

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.

![](https://static.igem.wiki/teams/5858/images/results/red-light-figure45.webp)

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.

![](https://static.igem.wiki/teams/5858/images/results/red-light-figure46.webp)

Figure46: PCR amplification of the RBS-*AcGFP1* gene fragment

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

![](https://static.igem.wiki/teams/5858/images/results/red-light-figure47.webp)

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.

![](https://static.igem.wiki/teams/5858/images/results/red-light-figure48.avif)

Figure48: PCR amplification of the PmrkA gene fragment

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

![](https://static.igem.wiki/teams/5858/images/results/red-light-figure49.webp)

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. coli*BL21(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.

![](https://static.igem.wiki/teams/5858/images/results/red-light-figure50.avif)

Figure 50. Transformation of successful dual-plasmid transformation

### Protein expression verification

We transformed the plasmid into *E. coli*BL21(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.

![](https://static.igem.wiki/teams/5858/images/results/red-light-figure51-jpeg.avif)

Figure 51: Western blot analysis of  *MrkH* gene expression.

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

![](https://static.igem.wiki/teams/5858/images/results/red-light-figure52.avif)

Figure 52: Western blot analysis of *YhjH* gene expression.

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

![](https://static.igem.wiki/teams/5858/images/results/red-light-figure53.avif)

Figure 53: Western blot analysis of *BphO*  gene expression.

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

![](https://static.igem.wiki/teams/5858/images/results/red-light-figure54.avif)

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.

## Dual-plasmid co-expression

### 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.

![](https://static.igem.wiki/teams/5858/images/results/dual-plasmid2-figure55.avif)

Figure 55: SDS-PAGE Electrophoresis result figure

![](https://static.igem.wiki/teams/5858/images/results/dual-plasmid2-figure56.avif)

Figure 56: Western blot result figure of the *Fre-L3-SttH*gene

### 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.

![](https://static.igem.wiki/teams/5858/images/results/dual-plasmid2-figure57.avif)

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.

![](https://static.igem.wiki/teams/5858/images/results/dual-plasmid2-figure58.avif)

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.

![](https://static.igem.wiki/teams/5858/images/results/dual-plasmid2-figure59.avif)

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.

![](https://static.igem.wiki/teams/5858/images/results/dual-plasmid2-figure60.avif)

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.

![](https://static.igem.wiki/teams/5858/images/template/template-sidenav-top.webp)

### [4,4'-Dinitroindigo Synthesis](#sub3-1-1)

* [Plasmid construction](#sub3-1-1)
* [Protein expression verification](#sub3-1-2)

### [Green light-controlled system](#sub3-2-1)

* [Plasmid construction](#sub3-2-1)
* [Protein expression verification](#sub3-2-3)

![](https://static.igem.wiki/teams/5858/images/template/template-sidenav-bottom.webp)

## 4,4'-Dinitroindigo synthesis

### 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.

![](https://static.igem.wiki/teams/5858/images/results/4-4-dinitroindigo-figure61.avif)

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.

![](https://static.igem.wiki/teams/5858/images/results/4-4-dinitroindigo-figure62.avif)

Figure62：Western blot result of Fdx protein

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

![](https://static.igem.wiki/teams/5858/images/results/4-4-dinitroindigo-figure63.avif)

Figure63：Western blot result of FdR protein

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

![](https://static.igem.wiki/teams/5858/images/results/4-4-dinitroindigo-figure64.avif)

Figure64：Western blot result of TxtD protein

![](https://static.igem.wiki/teams/5858/images/results/4-4-dinitroindigo-figure65.avif)

Figure65：Western blot result of TxtE protein

## Green light-controlled system

### 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.

![](https://static.igem.wiki/teams/5858/images/results/green-light-figure66.avif)

Figure66:Genetic Pathway image of the Green Light System

![](https://static.igem.wiki/teams/5858/images/results/green-light-figure67.avif)

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).

![](https://static.igem.wiki/teams/5858/images/results/green-light-figure68.avif)

Figure 68: Western Blot analysis of HO1 protein

![](https://static.igem.wiki/teams/5858/images/results/green-light-figure69.avif)

Figure 69: Western Blot analysis of PcyA protein

![](https://static.igem.wiki/teams/5858/images/results/green-light-figure70.avif)

Figure 70: Western Blot analysis of CcaR protein

![](https://static.igem.wiki/teams/5858/images/results/green-light-figure71.avif)

Figure 71: Western Blot analysis of mini-CcaS protein

{% endblock %}