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Blue-light Control

Indigo Synthesis

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-EcTnaA 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.

Red-light Control

6,6'-Dibromoindigo Synthesis

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.

Green-light Control

4,4'-Dinitroindigo Synthesis

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.