iGEM 2024 — Team jiashu-shanghai

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CONTENTS

[Daily Plan Summary Table (July 1, 2024 – August 20, 2024)](#a1)
[Experiment Plan Summary (July 1, 2024 – August 20, 2024)](#a2)

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Daily Plan Summary Table (July 1, 2024 – August 20, 2024)

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Date |

System |

Activities |

Results |

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July 1 |

System 1 |

Prepare plasmid with CrCPR, LjIFS, GmHID, perform codon optimization, and synthesize genes |

Plasmids ready for transformation into E. coli BL21 |

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July 2-4 |

System 1 |

Transform plasmid into E. coli BL21 and screen for positive clones on Amp LB plates |

Positive clones with plasmids obtained |

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July 5 |

System 1 |

sequencing to verify gene insertion |

Successful verification of gene insertion |

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July 6-8 |

System 1 |

Begin fermentation of BL21 strain in LB media |

Fermentation process established |

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July 9 |

System 1 |

Measure absorbance values of genistein standards to create the standard curve |

Standard curve established for calculating genistein concentration |

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July 10 |

System 1 |

Measure absorbance values from the production strain and calculate genistein concentrations |

Initial genistein production data obtained |

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July 11-12 |

System 1 |

Truncate the first 21 amino acids of LjIFS and compare genistein production before and after truncation |

Results show the impact of truncation on genistein production |

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July 13-14 |

System 1 |

Modify truncated LjIFS with N-terminal tags (17A, KKK, HHHH) and compare their effects on genistein yield |

Comparison of different tag effects on IFS activity |

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July 15 |

System 1 |

Test the addition of OmpAL to the N-terminus of CrCPR and measure its effect on genistein synthesis |

Impact of OmpAL on genistein production evaluated |

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July 16 |

System 2 |

Construct plasmids containing acsAB and acsCD, transform into E. coli BL21 and screen for positive clones |

BL21/AcsAB and BL21/AcsAB-AcsCD strains successfully constructed |

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July 17-18 |

System 2 |

Measure intracellular and extracellular cellulose production, compare the synthesis capabilities of both strains |

Analysis of cellulose production differences between the strains |

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July 19-21 |

System 2 |

Process the produced cellulose into facial masks, test water retention and tensile strength |

Data on water retention and tensile strength of cellulose masks collected |

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July 22-23 |

System 2 |

Analyze data from water retention and tensile strength tests |

Results show high water retention and strong tensile properties of cellulose masks |

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July 24 |

System 3 |

Construct blue light-inducible reporter system pSB-pDawn-mRFP, transform into E. coli DH5α |

Positive clones for reporter system obtained |

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July 25 |

System 3 |

Transform pSB-pDawn-mRFP，suicide system into E. coli BL21 |

BL21 system verification successful |

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July 26 |

System 3 |

Test the fluorescence expression of the blue light-inducible reporter strain |

Fluorescence data obtained to confirm blue light induction |

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July 27-29 |

System 3 |

Construct blue light-inducible suicide system pSB-pDawn-mazF, transform into E. coli DH5α and test |

DH5α system verification successful |

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July 30-31 |

System 3 |

Transform pSB-pDawn-mazF suicide system into E. coli BL21 and perform blue light exposure tests |

BL21 suicide system test results collected |

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August 1-2 |

System 1 |

Analyze the effects of LjIFS truncation, N-terminal modifications, and OmpAL addition on genistein production |

Optimized conditions for maximizing genistein yield determined |

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August 3-5 |

System 1 |

Summarize and organize experimental data on genistein production |

Final genistein production data compiled for report |

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August 6-7 |

System 2 |

Conduct final cellulose production experiments, collect and analyze cellulose yield data |

Final cellulose production data collected and evaluated |

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August 8-10 |

System 3 |

Summarize the results of blue light-induced suicide system tests and prepare visual data |

Report finalized with summarized results and graphical representations |

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August 11-12 |

All Systems |

Process data, create visual figures, and draft final report |

Complete data analysis and final report ready for submission |

Experiment Plan Summary (July 1, 2024 – August 20, 2024)

System 1: Construction and Optimization of Genistein-Producing Strain

Objective: Construct the genistein-producing strain BL21/IFS-CPR-HID by integrating plant-derived genes (CrCPR, LjIFS, GmHID) into E. coli, and enhance genistein production by truncating and modifying the N-terminal of LjIFS and CrCPR.

Procedure:

Construct a plasmid containing CrCPR, LjIFS, and GmHID, and transform it into E. coli BL21 to create the genistein-producing strain.

Use a genistein standard curve to measure absorbance values and calculate genistein concentrations from production strains.

To increase genistein yield, truncate the first 21 amino acids of LjIFS and compare the production before and after truncation.

Further modify the truncated LjIFS by adding N-terminal tags (17A, KKK, HHHH) and assess their impact on genistein yield while keeping CrCPR and GmHID constant.

Add the OmpAL tag to the N-terminus of CrCPR and evaluate whether it enhances genistein synthesis efficiency by improving CrCPR secretion.

Results: Investigate the impact of different N-terminal tags and truncation on genistein production, optimizing the expression and functionality of LjIFS and CrCPR.

**System 1:Laboratory notebook（Total 43 pages）**

System 2: Construction and Characterization of Cellulose-Producing Strain

Objective: Construct cellulose-producing strains BL21/AcsAB and BL21/AcsAB-AcsCD, and compare their cellulose synthesis capabilities.

Procedure:

Construct plasmids containing acsAB and acsCD cellulose synthase genes, and transform them into E. coli BL21 to form cellulose-producing strains.

Measure the cellulose production of the strains by comparing intracellular and extracellular cellulose yields.

Perform small-scale experiments to process the produced cellulose into facial masks and test their water retention and tensile strength.

Results: Compare the cellulose production between different strains, evaluate the performance of cellulose-based facial masks, and analyze intracellular and extracellular cellulose production.

**System 2：Laboratory notebook（Total 24 pages）**

System 3: Construction and Testing of Blue Light-Induced Systems

Objective: Construct and test blue light-inducible reporter and suicide systems.

Procedure:

Construct the blue light-inducible reporter strain pSB-pDawn-mRFP, first transform it into E. coli DH5α for verification, and then transform it into E. coli BL21 for final functional testing , and test its fluorescence expression to verify the effectiveness of blue light induction.

After constructing the pSB-pDawn-mazF blue light-inducible suicide system, first transform it into E. coli DH5α for verification, and then transform it into E. coli BL21 for final functional testing under blue light exposure to monitor cell death.

Results: The blue light-inducible suicide system effectively induces cell death under blue light exposure in BL21, significantly reducing cell survival compared to control groups.

**System 3：Laboratory notebook（Total 34 pages）**

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CONTENTS

[Abstract](#a1)
[Cycle 1-1: Construction of Genistein-producing Strain](#a2)
[Cycle 1-2: Construction of Truncated LjIFS Genistein-producing Strain](#a3)
[Cycle 1-3: Construction of Different Tags on IFS Genistein-producing Strain](#a4)
[Cycle 1-4: Construction of OmpAL Tag on CPR Genistein-producing Strain](#a5)
[Cycle 2: Construction of Cellulose-Producing Strain](#a6)
[Cycle 3-1: Construction of Blue Light-Induced Reporter Strain](#a7)
[Cycle 3-2 : Construction of Blue light-induced suicide system Testing](#a8)
[Problem solving and optimisation](#a9)
[Applications and Impact](#a10)
[Future work](#a11)
[Reference：](#a12)

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Abstract

We successfully constructed engineered strains for genistein production, bacterial cellulose synthesis, and a blue light-induced suicide system. The genistein-producing strain, optimized through N-terminal truncation of LjIFS and adding OmpAL tag to CrCPR, achieved a yield of 176.97 ± 15.30 μM. The bacterial cellulose strain, expressing acsAB and acsCD, produced 177.70 ± 31.62 mg/L of extracellular cellulose, demonstrating excellent water retention and tensile strength in mask applications. Additionally, the blue light-induced suicide system, using the MazF gene, effectively triggered bacterial cell death under blue light. These results validate our systems’ functionality, with future improvements focused on optimizing efficiency and exploring broader applications.

![](https://static.igem.wiki/teams/5481/engineering/img1.png)

Cycle 1-1: Construction of Genistein-producing Strain

Design:

Genistein is a natural phytoestrogen. It not only regulates skin moisture balance but also participates in the synthesis of collagen and elastin, helping to delay skin aging in menopausal women. To achieve the biosynthesis of genistein, we selected three key enzymes: cytochrome P450 reductase (CrCPR) from Catharanthus roseus, 2-hydroxyisoflavone synthase (LjIFS) from Lotus japonicus, and 2-hydroxyisoflavanone dehydratase (GmHID) from Glycine max. These three enzymes are essential for the conversion of naringenin to genistein.

![](https://static.igem.wiki/teams/5481/engineering/img2.png)

Figure 1. Synthetic pathway of naringenin conversion to genistein

In the first engineering phase, our main goal is to extract and test genistein production from the engineered E. coli BL21 strain. To this end, we designed and constructed the plasmid pSB-LjIFS-CrCPR-GmHID, integrating the genes of these three key enzymes into the plasmid and transforming it into E. coli BL21 for expression. In the presence of the substrate naringenin, the engineered strain will convert naringenin into genistein. Through this design, we aim to achieve efficient biosynthesis of genistein, verify the functionality of the engineered strain, and lay the foundation for future optimization of production yields.

![](https://static.igem.wiki/teams/5481/engineering/img3.png)

Figure 2. plasmid map of pSB-LjIFS-CrCPR-GmHID

Build：

We optimized the codons of the three key enzyme genes (LjIFS, CrCPR, GmHID) and outsourced their synthesis to a biotech company. The plasmid vector pSB1A3 was selected, and a polycistronic sequence was constructed. A dual promoter system was introduced, with one promoter driving the expression of IFS and CPR, while the other promoter specifically drives the expression of HID. Finally, the constructed plasmid pSB-LjIFS-CrCPR-GmHID was successfully transformed into E. coli BL21.

![](https://static.igem.wiki/teams/5481/engineering/img4.png)

Figure 3. Gel electrophoresis of CrCPR, LjIFS, GmHID gene

The results of the agarose gel electrophoresis clearly show that we obtained the gene fragments of the expected size (CrCPR~2142 bp; LjIFS~1554 bp; GmHID 957 bp).

Test:

Inoculate the engineered bacteria at a 1:100 ratio into 5 mL of LB medium (supplemented with 50 µg/mL ampicillin) and incubate overnight at 37°C. Wash once with PBS. Transfer the culture into 50 mL of M9 medium (supplemented with 10 g/L glucose and 0.5 mM naringenin). Adjust the initial OD600 to 1 and incubate at 30°C for 24 hours.

After fermentation, collect 1 mL of the bacterial culture and add an equal volume of ethyl acetate. Vortex for 2 hours. Centrifuge at 15,000 rpm for 20 minutes, let it stand for 5 minutes, and collect the supernatant organic phase. Transfer 200 µL of the supernatant into an HPLC vial and evaporate it to dryness in a 40°C oven for 3-5 hours. Resuspend the residue in 200 µL of methanol. Measure the absorbance at 250 nm using a microplate reader.

![](https://static.igem.wiki/teams/5481/engineering/img5.png)

Figure 4. Genistein standaed curve

Figure 4 presents the standard curve for genistein, illustrating the linear relationship between absorbance and genistein concentration. The regression equation obtained is Y = 0.02631X + 0.3105 , with a correlation coefficient (R) of 0.9831, indicating a strong linear correlation. This standard curve can be used to accurately determine the concentration of genistein in unknown samples by measuring their absorbance.

![](https://static.igem.wiki/teams/5481/engineering/img6.png)

Figure 5. Genistein production in genistein-producing strain

Figure 5 shows the genistein concentrations in different bacterial strains. BL21 and BL21/pSB1A3 were used as blank and negative controls, respectively, while the experimental group consisted of the engineered genistein-producing strain BL21/LjIFS-CrCPR-GmHID. The results indicate that there is no significant difference in genistein concentration between the two control strains, BL21 and BL21/pSB1A3. However, the genistein concentration in the experimental group was significantly higher, reaching 1.97 ± 0.38 μM. This confirms that the engineered strain is capable of producing genistein. The detection of genistein in the control groups is likely due to experimental error or the absorbance of other compounds at 250 nm, which may have influenced the measurements.

Learn:

We successfully constructed an engineered strain capable of producing genistein; however, the concentration of genistein synthesized by the strain was lower than expected. We conducted further research and exploration on how to increase the yield of genistein.

Cycle 1-2: Construction of Truncated LjIFS Genistein-producing Strain

Design:

We discovered that the hydrophobic structure at the N-terminus of the IFS protein could cause protein aggregation, significantly reducing the yield of genistein. To mitigate the interactions between these hydrophobic structures, we decided to alter a segment of the DNA sequence. Our research identified that the first 21 amino acids at the N-terminus of LjIFS were the primary factors contributing to aggregation. To avoid affecting the expression of other genes while reducing this impact, we employed a substitution method, replacing these 21 amino acids with alanine. Alanine has minimal impact on protein structure and function, thus achieving our goal.

Build:

We optimized the codons of the three key enzyme genes (LjIFS, CrCPR, GmHID) and outsourced their synthesis to a biotech company. The N-terminal 21 amino acids of LjIFS were truncated and replaced with alanine, resulting in a truncated version of LjIFS(BBa\_K5481004). In this modified system, segments of LjIFS were replaced while retaining the GmHID and CrCPR genes to enhance genistein biosynthesis. The constructed plasmid pSB-LjtIFS-CrCPR-GmHID was successfully transformed into E. coli BL21.

.

![](https://static.igem.wiki/teams/5481/engineering/img7.png)

Figure 6 Gel electrophoresis of truncated LjIFS(LjtIFS)

Test:

After cultivating the engineered strain at 30°C for 24 hours, we measured the concentration of genistein by detecting the absorbance at 250 nm.The bacterial culture was collected and treated with ethyl acetate to dissolve the genistein. After centrifugation, the organic phase supernatant was collected, concentrated, and resuspended. The absorbance at 250 nm was then measured using a microplate reader. Finally, we converted the results into the two sets of data shown in the figure below using a standard curve.

![](https://static.igem.wiki/teams/5481/engineering/img8.png)

Figure 7. Genistein production in the truncated LjIFS strain

Experimental results showed that the genistein concentration of truncated LjIFS strain (LjtIFS) was significantly higher than the wild-type LjIFS strain, reaching 17.33 ± 2.37 μM. This indicates that truncating the N-terminal sequence of LjIFS and replacing it with alanine can significantly increase genistein production.

Learn:

By truncating the N-terminal sequence of LjIFS, we successfully resolved the issue of IFS enzyme activity and increased the production of genistein.This method demonstrates the potential of optimizing biosynthetic yields through fine adjustments in protein structure.

Cycle 1-3: Construction of Different Tags on IFS Genistein-producing Strain

Design:

The hydrophobic structure at the N-terminus of IFS may cause protein aggregation, which significantly affects IFS activity. To enhance the solubility and functionality of LjIFS, the N-terminus was modified by adding hydrophilic (KKK, HHHH) or hydrophobic tags.

Build:

We tested four variants of LjIFS: a truncated version without a tag, and three modified versions where the N-terminal 21 amino acids were replaced with hydrophilic tags—17A (BBa\_K5481006), KKK (BBa\_K5481007), and HHHH (BBa\_K5481008). These gene variants were synthesized and used to construct recombinant plasmids. The resulting plasmids were transformed into E. coli BL21, and positive clones were selected on LB (Luria Bertani) agar plates containing 50 μg/mL ampicillin (Amp). The selected clones were then verified through DNA sequencing to confirm the successful construction of the engineered strains.

![](https://static.igem.wiki/teams/5481/engineering/img9.png)

Figure 8. A-Gel electrophoresis of different tags on LjIFS. B-Gel electrophoresis-2 of different tags on LjIFS.

Test:

These engineered strains were cultured at 30°C for 24 hours, and the genistein concentration produced by each strain was measured.

![](https://static.igem.wiki/teams/5481/engineering/img10.png)

Figure 9. Genistein production in engineered strains with different LjIFS tags

The results demonstrate varying effects on genistein production among the different LjIFS variants. The truncated LjIFS with the 17A tag showed a decrease in genistein concentration, producing 11.35 ± 1.28 μM of genistein, compared to the truncated LjIFS(LjtIFS) baseline of 17.44 ± 5.62 μM. In contrast, the variants with KKK and HHHH tags exhibited significant increases in genistein production. Notably, the LjIFS strain with the KKK tag produced the highest concentration, reaching 130.66 ± 14.68 μM, representing a substantial improvement.These findings suggest that adding hydrophilic tags such as KKK and HHHH to the N-terminus of LjIFS enhances its solubility and activity. Among these, the KKK tag proved to be the most effective.

Learn:

Based on these results, we selected the KKK-tagged LjIFS for further optimization and genistein production studies.By modifying the structure of LjIFS to improve its solubility and protein functionality, we successfully increased the concentration of genistein produced. A comparison of LjIFS with different tags revealed varying effects on protein modification. Based on these findings, we aim to apply a similar approach to CPR to enhance its stability.

Cycle 1-4: Construction of OmpAL Tag on CPR Genistein-producing Strain

Design:

Initially, we designed a project to use CrCPR in E. coli to convert naringenin into genistein. However, we discovered that this gene is difficult to stabilize and maintain activity in E. coli, potentially due to its hydrophobic N-terminal tail, which functions as a membrane anchor in plant cells. In E. coli, the absence of an endoplasmic reticulum may reduce protein solubility and activity. The specific method we decided to use is to employ OmpAL (bacterial outer membrane protein A) as the signal peptide to enhance the activity and stability of CrCPR, so that CrCPR can play its role more completely.

Build:

In this modified system, CrCPR were ligated to OmpAL tag while retaining the GmHID and LjIFS genes to enhance genistein biosynthesis. Finally, the constructed plasmid was successfully transformed into E. coli BL21.

![](https://static.igem.wiki/teams/5481/engineering/img11.png)

Figure 10. Gel electrophoresis of OmpAL-CPR

Test:

Using the same method, we tested two versions of CPR—one with the OmpAL sequence and one without—under identical cultivation conditions. The final results demonstrated that the CPR with the OmpAL sequence significantly increased genistein production, reaching 176.97 ± 15.30 μM, compared to the lower yield from the CPR without the OmpAL sequence (Figure 11).

![](https://static.igem.wiki/teams/5481/engineering/img12.png)

Figure 11. Genistein production in CrCPR strains with and without OmpAL Tag

Learn:

By introducing the OmpAL sequence , the efficiency of CPR in converting naringenin in E. coli has significantly improved. The increased output was in line with expectations, so we started building a second system

Cycle 2: Construction of Cellulose-Producing Strain

Design:

Bacterial cellulose has excellent water retention capacity, biocompatibility, and biodegradability, making it an ideal primary ingredient for facial masks. Therefore, we designed a second system specifically for the production of bacterial cellulose. acsAB and acsCD are key genes involved in bacterial cellulose synthesis. acsAB encodes the core protein of the cellulose synthase complex, responsible for polymerizing glucose molecules into cellulose. acsC forms pores to secrete cellulose, while acsD controls the crystallization of cellulose into nanofibers. By constructing a cellulose-producing strain containing acsAB and acsCD, we will test its cellulose yield. At the same time, we will use the cellulose produced to create facial masks and evaluate their water content and tensile strength.

![](https://static.igem.wiki/teams/5481/engineering/img13.png)

Figure 12. The plasmid map of pSB-acsAB-acsCD

Build：

We selected the synthetic cellulose synthase-encoding genes acsAB and acsCD and added them to the bacterium, and codon optimization was performed for E. coli. Multiple cis-trans sequences were constructed using pSB1A3 as the vector and J23100-B0034 as the promoter-RBS sequence. Transform the recombinant plasmid into E. coli BL21.

![](https://static.igem.wiki/teams/5481/engineering/img14.png)

Figure 13. Gel electrophoresis of acsAB and acsCD

Test:

To assess cellulose synthesis, the engineered BL21 strain was inoculated into 100 mL of fresh LB medium and cultured at 30°C and 180 rpm for 48 hours. The 100 mL culture was collected and centrifuged at 8,000 rpm for 5 minutes. The supernatant (20 mL) was collected as the extracellular sample. The bacterial pellet was resuspended in an equal volume of pre-chilled PBS (pH 7.4), and then sonicated on ice to lyse the bacteria. Next, the lysate was centrifuged at 15,000 × g for 20 minutes to remove cell membranes and debris, and the supernatant was collected as the intracellular sample.

Both the 20 mL intracellular and extracellular samples were mixed with 20 mL of 4M NaOH and incubated at 80°C for 2 hours to dissolve non-cellulose components and precipitate the cellulose. After incubation, the samples were centrifuged at 15,000 × g at 4°C for 30 minutes to collect the cellulose pellet. The pellet was washed repeatedly with distilled water until the pH reached 7.0. The washed cellulose was dried at 60°C to a constant weight, and the dry weight was measured to determine the cellulose content.

![](https://static.igem.wiki/teams/5481/engineering/img15.png)

Figure 14. Cellulose production. A Extracellular sample. B Intracellular sample

The results demonstrated a clear difference in cellulose production between the strains. The strain expressing only acsAB produced 9.99 ± 3.25 mg/L of extracellular cellulose, while the strain BL21/acsAB-acsCD significantly increased extracellular production to 177.70 ± 31.62 mg/L. This confirms the critical role of acsCD in exporting cellulose out of the cell. In terms of intracellular cellulose, the strain expressing acsAB alone accumulated a higher amount (524.61 ± 96.39 mg/L), whereas BL21/acsAB-acsCD reduced intracellular cellulose to 208.97 ± 40.40 mg/L, further supporting the role of acsCD in facilitating cellulose export.

To test the moisture content of our final mask product and compare it with common masks on the market:First, the mask was folded and soaked in serum for 0.5 hours to allow it to fully absorb the serum. Then, it was weighed using a balance and the weight was recorded. After drying the mask until no surface moisture remained, a second weight was measured. The difference between the two readings was used to determine the mask’s moisture content. This method was repeated to measure the moisture content of conventional market masks. According to the results, the maximum water content of our generated masks is much higher than that of the common masks in the market, which is 5 times of their moisture content. This means that our masks do not need to add the water retention agents that are added to regular masks to achieve better water retention than they do.

![](https://static.igem.wiki/teams/5481/engineering/img16.png)

Figure 15. Our product-bacterial cellulose mask VS commercial mask

After successfully producing the bacterial cellulose material required for our project, we conducted a tensile strength test to evaluate the mechanical properties of the bacterial cellulose-based mask. The testing method involved suspending a foam box filled with water, weighing approximately 2 kg, using multiple layers of tape wrapped around the box and then hanging the tape from the bacterial cellulose sheet. One end of the bacterial cellulose sheet was tied and secured to a wooden stick to hold the weight. A single sheet of bacterial cellulose, stretched to approximately the thickness of a 1-yuan coin (about 2 mm), was able to support the weight of the foam box filled with water for an extended period of time. This indicates that bacterial cellulose exhibits excellent tensile strength, making it a durable material suitable for mask production.

![](https://static.igem.wiki/teams/5481/engineering/img17.png)

Figure 16. Tensile strength test of bacterial cellulose mask

Learn:

We successfully constructed a bacterial cellulose-producing strain. The acsAB gene effectively produces cellulose intracellularly, while the acsCD gene enables the secretion of cellulose to the extracellular space. The final cellulose yield met our expectations. In addtion,these findings confirm that the bacterial cellulose mask not only provides effective hydration but also possesses strong mechanical properties, further validating its potential as a robust and practical product in the skincare industry. Considering safety concerns, we need to design a suicide system next.

Cycle 3-1: Construction of Blue Light-Induced Reporter Strain

Design:

To test whether the established pDawn promoter and Pr promoter can activate the blue light-inducible system properly under blue light induction. To achieve this goal, we constructed a test system that places the red fluorescent protein (mRFP) gene downstream of the pDawn promoter with the aim of verifying the functionality of the blue light-inducible system.

![](https://static.igem.wiki/teams/5481/engineering/img18.png)

Figure 17. plasmid map of pSB-pDawn-mRFP

Build:

To ensure the success of our genetic construct, we selected the pSB1A3 plasmid as the vector for cloning the pDawn promoter and the mRFP gene between the XbaI and SpeI restriction sites. The pDawn promoter is light-sensitive and can be activated under blue light to drive the expression of the mRFP gene. After successfully constructing the recombinant plasmid pDawn-mRFP, we transformed it into E. coli DH5α.

Test:

The transformed blue light-inducible reporter strain was inoculated at a 1:100 ratio into 5 mL of LB medium containing 50 μg/mL ampicillin. The test tubes were wrapped in aluminum foil to prevent light exposure, and the culture was incubated overnight in a shaker at 180 rpm and 37°C. The next day, 1 mL of the culture was collected and centrifuged at 10,000 rpm for 1 minute to pellet the cells, followed by one wash with PBS (pH 7.4). The cells were then transferred into 50 mL of fresh M9 medium (supplemented with 10 g/L glucose and ampicillin). A 5 mL aliquot of the culture was placed into 33 mm petri dishes, with the experimental group exposed to blue light in a light box, while the control group was shielded from light with aluminum foil. The cultures were incubated statically at 30°C for 12 hours. After incubation, 200 μL of the culture was sampled, and fluorescence (excitation wavelength 584 nm, emission wavelength 607 nm) along with OD600 values were measured using a microplate reader. The normalized fluorescence ratio (Fluorescence/OD600) was then calculated.

![](https://static.igem.wiki/teams/5481/engineering/img19.png)

Figure 18. The standardized fluorescence ratio of blue light-induced reporter strain

The results showed minimal fluorescence in both the BL21/pSB1A3 and BL21/pDawn strains, indicating low background signal. However, the BL21/pDawn-mRFP strain displayed a significant increase in fluorescence under blue light, with an average standardized fluorescence ratio of 702.03 ± 140.88, confirming successful activation of the pDawn promoter and strong mRFP expression. These findings demonstrate that the blue light induction system functions effectively.

Learn:

During the experiment, we successfully constructed and validated the pDawn-mRFP system. The results showed that the blue light receptor system could activate the expression of the mRFP gene under appropriate conditions, proving the functionality of the blue light-induced system. Through this experiment, we verified the effectiveness of the blue light-induced system and provided a solid foundation for the subsequent development of the blue light-induced suicide system. In subsequent experiments, we replaced the red fluorescent protein mRFP with MazF, the main body of the suicide system, for further validation and application development.In the future, we plan to optimise the blue light induced suicide system and further test its stability and effectiveness under different conditions. Through further research, we hope to develop an efficient and reliable blue light-induced suicide system that will provide new solutions for biosafety and control applications.

Cycle 3-2 : Construction of Blue light-induced suicide system Testing

Design:

the mRNA synthesis disrupting enzyme mazF gene was replaced with the red fluorescent protein mRFP and inserted into the blue light sensing promoter pDawn. The effect of bacterial suicide was detected by testing the OD600 value of bacteria.

![](https://static.igem.wiki/teams/5481/engineering/img20.png)

Figure 19. Plasmid map of pSB-pDawn-mazF

Build:

The BL21/pDawn-MazF and BL21/pDawn-mRFP strains were constructed by transforming pDawn-mazF and pDawn-mRFP vectors into BL21 strains, respectively, by molecular cloning. The transformed strains were screened and validated to ensure successful transformation.

![](https://static.igem.wiki/teams/5481/engineering/img21.png)

Figure 20. Gel electrophoresis of mazF

Test:

100 μL of the overnight culture was inoculated into 5 ml of LB medium containing ampicillin (50 μg/ml) and incubated for 2 hours at 37°C and 220 rpm with oscillation until the OD600 value reached 0.4 .The culture solution was poured into 33 mm Petri dishes and the dishes were irradiated with 488 nm blue light at 30°C. A 200 μL sample was taken at 2 hours, 4 hours, 6 hours and 12 hours to determine the OD600 value.

![](https://static.igem.wiki/teams/5481/engineering/img22.png)

Figure 21 Effect of blue light-induced mazF system on bacterial growth

The results demonstrate distinct differences in the growth of the strains: BL21/pSB1A3 and BL21/pDawn-mRFP strains exhibited normal growth throughout the 12-hour period. At 12 hours, their OD600 values reached approximately 1.5, indicating healthy bacterial growth. In contrast, the BL21/pDawn-MazF strain showed a significant reduction in OD600 over time. At 2 hours, there was a slight decrease in growth compared to the control strains. By 6 hours, the OD600 values had dropped considerably, and at 12 hours, the OD600 was near zero, indicating almost complete bacterial death. The results demonstrated that the blue light-induced system connected to mazF can be induced by blue light, leading to bacterial cell death.

Learn：

In the future, we will improve our light-induced suicide system in the following aspects：

Problem solving and optimisation

Optimisation of blue light irradiation time and intensity: Determine the optimal irradiation time and intensity to ensure maximum expression of suicide genes and efficient killing of bacteria.

Stability of gene components: adjust the expression vector to improve the stability and expression efficiency of the gene in bacteria.

Cell protection mechanism: introduce protective genes to prevent non-target bacteria from being affected by blue light irradiation.

Applications and Impact

Medical field: for precise control of drug-resistant bacterial infections and reducing the use of antibiotics.

Agricultural field: used to control crop diseases and reduce the use of chemical pesticides.

Environmental protection: used to deal with harmful bacterial pollution in the environment and safeguard ecological safety.

Future work

Integration of multiple control elements: Combine other light-sensitive elements to develop a suicide system with multiple light controls to improve the flexibility and application range of the system.

Large-scale application testing: Conduct large-scale testing in real environments to evaluate the stability and practicality of the system.

Commercialisation and promotion: Collaborate with relevant companies to promote the commercialisation of blue light-induced suicide systems, providing a new approach to solving the problem of drug-resistant bacteria.

Reference：

[1]Liu X, Li L, Zhao G R. Systems metabolic engineering of Escherichia coli coculture for de novo production of genistein[J]. ACS Synthetic Biology, 2022, 11(5): 1746-1757.

[2]Scott E E, Spatzenegger M, Halpert J R. A truncation of 2B subfamily cytochromes P450 yields increased expression levels, increased solubility, and decreased aggregation while retaining function[J]. Archives of Biochemistry and Biophysics, 2001, 395(1): 57-68.

[3]Hwang Y, Noh M H, Jung G Y. Recent advancements in flavonoid production through engineering microbial systems[J]. Biotechnology and Bioprocess Engineering, 2024: 1-14.

[4]Quattrone A, Czajka A, Sibilla S. Thermosensitive hydrogel mask significantly improves skin moisture and skin tone; bilateral clinical trial[J]. Cosmetics, 2017, 4(2): 17.

[5]Marbun N, Fhitriana S, Leny L, et al. Preparation and evaluation of moisturizing sheet mask of Lady Finger (Abelmoschus esculentus (L.) Moench) extract[J]. Journal of Drug Delivery and Therapeutics, 2022, 12(3): 14-19.

[6]Wang H, Zhou B. Development and performance study of a natural silk fiber facial mask paper[J]. Journal of Engineered Fibers and Fabrics, 2020, 15: 1558925020975756.

[7]Fan X, Bai X, Tan L, et al. A Transparent Additive‐Free Fibrous Facial Mask for Skin Moisturizing[J]. Macromolecular Rapid Communications, 2023, 44(17): 2300180

[8]Mazraeh D, Di Ventura B. Synthetic microbiology applications powered by light[J]. Current Opinion in Microbiology, 2022, 68: 102158.

[9]Chattopadhyay G, Bhasin M, Ahmed S, et al. Functional and biochemical characterization of the MazEF6 toxin-antitoxin system of Mycobacterium tuberculosis[J]. Journal of Bacteriology, 2022, 204(4): e00058-22.

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CONTENTS

[Construction of Genistein-Producing Strains](#a1)
[Analysis of Genistein Content in Engineered Strains Using Microplate Reader](#a2)
[Construction of Cellulose-Producing Strains](#a3)
[Testing the Cellulose Synthesis Ability of the Strains](#a4)
[Moisture Content Test:](#a5)
[Tensile Strength Test of Bacterial Cellulose Mask:](#a6)
[Construction of blue light-inducible reporter strain](#a7)
[Testing the blue light-inducible reporter strain](#a8)
[Construction of blue light-inducible suicide system](#a9)
[Testing the blue light-inducible suicide system](#a10)
[Statistical analysis](#a11)
[Related kit instructions：R21648 Glucose Test Kit (GOD-POD Microplate method)](#a12)

---

Construction of Genistein-Producing Strains

Cytochrome P450 enzyme reductase (CrCPR) from Catharanthus roseus (Madagascar periwinkle), 2-hydroxyisoflavone synthase (2-HIS), also known as isoflavone synthase (LjIFS) from Lotus japonicus (bird's-foot trefoil), and 2-hydroxyisoflavanone dehydratase (GmHID) from  Glycine max (soybean) were synthesized. Codon optimization for Escherichia coli  was performed, and EcoRI, XbaI, SpeI, and PstI restriction sites were eliminated to comply with RFC#10 standards (Genewiz, USA). Using pSB1A3 as the vector and J23100-B0034 as the promoter-RBS sequence, a polycistronic sequence was constructed. A dual-promoter design was employed to enhance mRNA expression efficiency. The recombinant plasmid was transformed into E. coli  BL21, and positive clones were selected on LB (Luria Bertani) plates containing 50 μg/mL ampicillin (Amp). Sequencing verification was performed (Qingke, Beijing, China) to obtain the engineered strains. The engineered strains were cultured at 37 °C, inoculated and expanded in LB liquid medium. Bacterial growth was monitored by measuring optical density (OD) at 600 nm using a spectrophotometer (GenStar, China).

![](https://static.igem.wiki/teams/5481/experiments/img1.png)

Figure 1. Gene circuit for synthesizing genistein from naringenin

To enhance the solubility and function of LjIFS, the N-terminus of LjIFS was modified, including truncation (removal of the first 21 amino acids) and addition of hydrophilic (KKK, HHHH) or hydrophobic tags. For CrCPR, OmpAL (localization sequence of the OmpA membrane protein) was added at the N-terminus. OmpAL acts as a signal peptide, guiding the fused exogenous protein to be transported to the cell membrane or periplasmic space via the Sec pathway.

Table 1. N-terminal sequence modifications. Underlines indicate truncated N-termini, replaced with alanine, 17A, KKK, and HHHH.

![](https://static.igem.wiki/teams/5481/experiments/img2.png)

Analysis of Genistein Content in Engineered Strains Using Microplate Reader

10 mM naringenin was purchased from Macklin (DMSO; N796909). The engineered bacteria were inoculated into 5 mL of LB medium (containing 50 μg/mL ampicillin) at a 1:100 ratio and cultured overnight at 37 °C. On the second day, 1 mL of the culture was transferred into 100 mL of fresh LB medium containing ampicillin. After culturing at 220 rpm and 37 °C for 12 hours, the cells were collected by centrifugation at 10,000 rpm for 1 minute. The bacterial pellet was washed once with PBS (pH 7.4). Then, the cells were transferred into 50 mL of fresh M9 medium containing 50 μg/mL ampicillin (Na₂HPO₄ 3.0 g/L, KH₂PO₄ 0.5 g/L, NaCl 1.0 g/L, NH₄Cl 1.0 g/L, MgSO₄ 5.0 mM, and CaCl₂ 0.1 mM; supplemented with 10 g/L glucose and 0.5 mM naringenin). The initial OD₆₀₀ was adjusted to 1, and fermentation was carried out at 220 rpm and 30 °C for 24 hours to produce genistein. After fermentation, 1 mL of the bacterial culture was collected and mixed with an equal volume of ethyl acetate, then vortexed for 2 hours. Centrifugation was performed at 15,000 rpm for 20 minutes, and after standing for 5 minutes, the organic phase supernatant was collected. A 200 μL aliquot of the organic supernatant was transferred into HPLC vials and evaporated to dryness in a 40 °C oven for 3–5 hours. The residue was then resuspended in 200 μL of methanol. The absorbance at 250 nm was measured using a microplate reader (Multiskan GO, Thermo Fisher Scientific).

Construction of Cellulose-Producing Strains

The cellulose synthase coding genes acsAB and acsCD were synthesized. Codon optimization for E. coli  was performed, eliminating EcoRI, XbaI, SpeI, and PstI restriction sites to comply with RFC#10 standards (Genewiz, USA). Using pSB1A3 as the vector and J23100-B0034 as the promoter-RBS sequence, a polycistronic sequence was constructed. The recombinant plasmid was transformed into E. coli  BL21, and positive clones were selected on LB (Luria Bertani) plates containing 50 μg/mL ampicillin (Amp) and verified by sequencing (Qingke, Beijing, China) to obtain engineered strains. The engineered strains were cultured at 37 °C, inoculated and expanded in LB liquid medium. Bacterial growth was monitored by measuring optical density (OD) at 600 nm using a microplate reader (GenStar, China).

Testing the Cellulose Synthesis Ability of the Strains

To assay cellulose synthesis, the BL21 engineered strain was inoculated into 100 mL of fresh LB medium containing 50 μg/mL Amp. The culture was incubated at 30 °C and 180 rpm for 48 hours. After incubation, 100 mL of the bacterial culture was collected and centrifuged at 8000 rpm for 5 minutes. A 20 mL aliquot of the culture supernatant was collected as the extracellular sample. The bacterial pellet was resuspended in an equal volume of pre-cooled PBS (pH 7.4). Under ice bath conditions, the cells were disrupted using an ultrasonic homogenizer (Biosafer1000, Saifei) with settings of 150 W power, 1 s ultrasonication, 3 s intervals, for a total of 20 minutes. Next, centrifugation was performed at 15,000 × g for 20 minutes to remove cell membranes and impurities, and the supernatant was collected as the intracellular sample. To each 20 mL of intracellular and extracellular samples, 20 mL of 4 M NaOH was added and incubated at 80 °C for 2 hours to further dissolve non-cellulose components and precipitate cellulose. Then, centrifugation was performed at 15,000 × g for 30 minutes at 4 °C to collect the cellulose precipitate. The precipitated cellulose was washed repeatedly with distilled water until the pH reached 7.0 to remove residual NaOH. The washed cellulose was dried at 60 °C to constant weight. The dried cellulose was weighed to determine the cellulose content.

Moisture Content Test:

To assess the moisture content of the bacterial cellulose mask, the mask was folded within its packaging to ensure full absorption of the serum and allowed to soak for 30 minutes. After saturation, the wet mask was weighed to obtain the initial weight. The mask was then air-dried until no surface moisture remained and weighed again to determine the dry weight. The moisture content was calculated by subtracting the dry weight from the initial wet weight. This procedure was also performed with a commercially available mask for comparison.

Tensile Strength Test of Bacterial Cellulose Mask:

To evaluate the mechanical properties of the bacterial cellulose mask, a tensile strength test was conducted. A bacterial cellulose sheet approximately 2 mm thick was prepared. A foam box filled with approximately 2 kg of water was suspended using tape attached to the bacterial cellulose sheet, which was secured at one end to a wooden stick. The setup was used to assess the mask’s ability to support weight under tension.

Construction of blue light-inducible reporter strain

The blue light-inducible promoter pDawn and the red fluorescent protein mRFP were synthesized. mRFP was placed downstream of pDawn. The mRFP sequence was codon-optimized for E. coli , eliminating EcoRI, XbaI, SpeI, and PstI restriction sites to comply with RFC#10 standards (Genewiz, USA). Using pSB1A3 as the vector, pDawn-mRFP was cloned into the XbaI and SpeI restriction sites. The recombinant plasmid was transformed into E. coli  DH5α, and positive clones were selected on LB (Luria Bertani) plates containing 50 μg/mL ampicillin (Amp) and verified by sequencing (Qingke, Beijing, China) to obtain the engineered strain. The engineered strain was cultured at 37 °C, inoculated and expanded in LB liquid medium. Bacterial growth was monitored by measuring optical density (OD) at 600 nm using a spectrophotometer (GenStar, China).

![](https://static.igem.wiki/teams/5481/experiments/img3.png)

Figure 2. Composition of the pDawn promoter. In the absence of blue light, the histidine kinase YF1 phosphorylates FixJ; the activated FixJ binds to Pfixk2 and initiates CI expression. CI represses the Pr promoter, preventing downstream gene expression. In the presence of blue light, the repression is lifted, and downstream genes are expressed.

Testing the blue light-inducible reporter strain

The blue light-inducible reporter strain was inoculated into 5 mL of LB medium containing 50 μg/mL Amp at a ratio of 1:100. Test tubes were wrapped with aluminum foil to prevent light exposure. Cultures were grown overnight in a shaking incubator at 180 rpm and 37 °C. The next day, 1 mL of bacterial culture was collected and centrifuged at 10,000 rpm for 1 minute to collect the bacterial pellet. The pellet was washed once with PBS (pH = 7.4). The cells were then transferred into 50 mL of fresh M9 medium containing 50 μg/mL ampicillin (Na2HPO4 3.0 g/L, KH2PO4 0.5 g/L, NaCl 0.5 g/L, NH4Cl 1.0 g/L, MgSO4 5.0 mM, and CaCl2 0.1 mM; supplemented with 10 g/L glucose). Five milliliters of the bacterial suspension were added to a 33 mm petri dish. The experimental group was exposed to blue light using a blue light box, while the petri dish in the control group was covered with aluminum foil. The cultures were incubated statically at 30 °C for 12 hours. Then, 200 μL of bacterial culture was taken, and fluorescence values (excitation wavelength 584 nm, emission wavelength 607 nm) and OD600 were measured using a microplate reader (Multiskan GO). The normalized fluorescence ratio (Fluorescence/OD600) was calculated.

Construction of blue light-inducible suicide system

The coding gene of the mRNA interferase mazF was inserted downstream of the pDawn promoter. The mazF sequence was codon-optimized for E. coli , eliminating EcoRI, XbaI, SpeI, and PstI restriction sites to comply with RFC#10 standards (Genewiz, USA). pDawn-mazF was cloned into the pSB1A3 plasmid (XbaI and SpeI restriction sites) and transformed into E. coli  DH5α.

Testing the blue light-inducible suicide system

A 100μL of overnight culture were inoculated into 5 mL of LB medium containing ampicillin (50 μg/mL) (protected from light using aluminum foil) and cultured at 37 °C with shaking at 220 rpm for 2 hours until OD600 reached 0.4. The culture was then poured into a 33 mm petri dish and exposed to blue light (488 nm) at 30 °C. Samples of 200 μL were taken at 2 h, 4 h, 6 h, and 12 h to measure the bacterial OD600.

Statistical analysis

Data were analyzed and plotted using GraphPad Prism software. Results are presented as mean ± standard deviation (SD). For comparisons among multiple groups, one-way analysis of variance (ANOVA) and Tukey's post hoc test were used for difference analysis. For comparisons between two groups, Student's t-test was used. A p-value less than 0.05 was considered statistically significant.

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CONTENTS

[Summary](#a1)
[Genistein system](#a2)
[Bacterial Cellulose System](#a3)
[Suicide System](#a4)

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Summary

Our project was dedicated to the application of genistein flavonoids, for which we designed several experiments and tests.We first successfully constructed an engineered strain to convert naringenin into genistein flavonoids, and experimentally verified the yield of genistein flavonoids.We modified the key enzymes, optimized the expression of the LjIFS gene, and added the OmpAL tag to CrCPR to improve its activity and significantly enhance enzyme efficiency.In addition, we engineered E. coli, which does not naturally produce bacterial cellulose, to both enhance its cellulose production and improve its secretion capacity. Experimental results showed that these strains were able to produce cellulose stably.Finally, we developed a blue light-induced suicide system and verified the suicide mechanism of bacteria under blue light irradiation.

The whole process demonstrated the feasibility, rationality of our project, and the overall result could not be achieved without the results and efforts of each experiment, and each part ensured the process.

Genistein system

The genistein production system successfully converted naringenin into genistein using Escherichia coliengineered with optimized LjIFS, CrCPR, and GmHID genes. The system produced significantly higher genistein than control strains, further enhanced by truncating LjIFS and adding KKK and HHHH tags, with the KKK-tagged variant showing the best results. The addition of the OmpAL tag improved enzyme efficiency, boosting genistein yield.

Test of Genistein-producing Strain

In order to verify whether the engineered bacteria we designed can produce genistein. We synthesized three key enzyme genes, cytochrome P450 reductase (CrCPR) from Catharanthus roseus , Isoflavone synthase (LjIFS) from Lotus japonicus , 2-hydroxyisoflavone dehydratase (GmHID) from Glycine max  .

![](https://static.igem.wiki/teams/5481/results/new-img1.png)

Figure 1. Gel electrophoresis of CrCPR, LjIFS, GmHID gene

We performed codon optimization for E. coli and chose to use pSB1A3 as a vector and constructed a multiple cis-trans sequence. A dual promoter system was added, one promoter for LjIFS(BBa\_K5481001) and CrCPR(BBa\_K5481000), and the other promoter for GmHID(BBa\_K5481002), which was used to improve the efficiency of mRNA expression. We chose BL21 as the host strain.

![](https://static.igem.wiki/teams/5481/results/img2.png)

Figure 2. plasmid map of pSB-LjIFS-CrCPR-GmHID

![](https://static.igem.wiki/teams/5481/results/img3.png)

Figure 3. E. coli  (produce genistein flavonoids) gene circuit

We incubated the engineered bacteria overnight at 37℃ in LB medium containing ampicillin antibiotics. It was washed using PBS. Subsequently transferred to 50 mL M9 medium. The OD600 value was adjusted to 1. The culture was incubated for 24 h at 30 ℃.

Since genistein has an absorption peak at 250 nm, we planned to detect the content of genistein by measuring the absorbance at 250 nm of the sample. Prior to this, we purchased a genistein standard and configured different concentrations of 0 μM, 50 μM, 100 μM, 150 μM and 200 μM. The absorbance values at 250 nm were detected for each concentration and the standard curve of genistein was plotted.

![](https://static.igem.wiki/teams/5481/results/img4.png)

Figure 4. Genistein standaed curve

Figure 4 presents the standard curve for genistein, illustrating the linear relationship between absorbance and genistein concentration. The regression equation obtained is Y = 0.02631X + 0.3105 , with a correlation coefficient (R) of 0.9831, indicating a strong linear correlation. This standard curve can be used to accurately determine the concentration of genistein in unknown samples by measuring their absorbance.

At the end of the fermentation of the engineered bacteria culture, we conducted an initial experiment and found that its concentration was too low for a successful detection. We planned to concentrate it 5-fold as follows: we collected 1 mL of the bacterial culture and added an equal volume of ethyl acetate and performed vortex shaking for 2 h to rupture all the engineered bacteria and to solubilize the genistein flavonoids. This was followed by centrifugation at 15,000 rpm for 20 min, at the end of which the organic layer supernatant was collected. The supernatant of the organic layer was poured into HPLC tubules and dried by evaporation in an oven at 40℃ for 3-5 h. The supernatant of the organic layer was then dried by evaporation. After that, it was resuspended with 200 μL of formaldehyde to concentrate it 5 times. And the absorbance at 250 nm was detected using an enzyme meter. Three sets of parallel experiments were performed to minimize the error.

![](https://static.igem.wiki/teams/5481/results/img5.png)

Figure 5. Genistein production in genistein-producing strain

Figure 5 shows the genistein concentrations in different bacterial strains. BL21 and BL21/pSB1A3 were used as blank and negative controls, respectively, while the experimental group consisted of the engineered genistein-producing strain BL21/LjIFS-CrCPR-GmHID. The results indicate that there is no significant difference in genistein concentration between the two control strains, BL21 and BL21/pSB1A3. However, the genistein concentration in the experimental group was significantly higher, reaching 1.97 ± 0.38 μM. This confirms that the engineered strain is capable of producing genistein. The detection of genistein in the control groups is likely due to experimental error or the absorbance of other compounds at 250 nm, which may have influenced the measurements.

Truncated LjIFS Enhances Genistein Production

Previous studies have shown that the N-terminal hydrophobic structure of the LjIFS protein may cause protein aggregation, significantly affecting its enzymatic activity. To reduce protein aggregation while maintaining sufficient genistein production, the N-terminal 21 amino acids of LjIFS were truncated and replaced with alanine, resulting in a truncated version of LjIFS(BBa\_K5481004). In this modified system, segments of LjIFS were replaced while retaining the GmHID and CrCPR genes to enhance genistein biosynthesis.

![](https://static.igem.wiki/teams/5481/results/imgx-1.png)

Figure 6 Gel electrophoresis of truncated LjIFS(LjtIFS).

The experimental procedure involved culturing the engineered strains at 30℃ for 24 hours. Following incubation, genistein concentration was determined by measuring absorbance at 250 nm. The bacterial culture was first collected and treated with ethyl acetate to extract genistein. After centrifugation, the organic phase supernatant was collected, concentrated, and re-suspended. Absorbance at 250 nm was measured using a microplate reader. The genistein concentration was then calculated using the standard curve derived from the regression equation.

![](https://static.igem.wiki/teams/5481/results/img6.png)

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