iGEM 2024 — Team dku

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# Lab Notebook

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## Our Journey

We have compiled three lab notebooks that document our experimental progress in detail. The general lab notebook provides an overview of our foundational work, including bacterial transformations, culture preparations, and plasmid extraction. The nisin group's lab notebook focuses on experiments involving *Lactococcus lactis*, such as preparing competent cells, transformations with the pMG36e plasmid, and subsequent protein analysis using Western blotting. Meanwhile, the H2O2 group's lab notebook records our efforts in testing hydrogen peroxide stress response in *Lactococcus lactis*, highlighting the growth conditions, electroporations, and H2O2 expression tests. Together, these notebooks capture our systematic approach and the progression of our wet lab experiments.

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Notebook

* [General Notebook](#section1)
* [Nisin Group](#section2)
* [H2O2 Group](#section3)

## General Notebook

## Nisin Group

## H2O2 Group

##

Our lab notebooks document all our experimental protocols and preliminary data, which evidences our contributions and could potentially provide insight for future research. Feel Free to download and use any of the protocols used.

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# Engineering

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## Innovate, Experiment, Repeat.

We tested promoters for AcGFP expression in *Lactococcus lactis*, identifying PnisA as the most effective through ELISA and Western blot. Using PPAP, we predicted promoter strength and validated it in vivo. Overexpressing nisRK enhanced PnisA activity, and CRISPR-Cas9 replaced weaker promoters to boost nisin production. Genes LJ\_0548 and LJ\_0549 overexpression in *L. johnsonii* increased hydrogen peroxide under anaerobic conditions, suggesting their role in oxidative metabolism. Protein docking provided insights into enzyme interactions.

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Engineering

* [**Nisin Solution**](#section1)
  + [Cycle 1](#section2)
  + [Cycle 2](#section3)
  + [Cycle 3](#section4)
  + [Cycle 4](#section5)
* [**H2O2 Solution**](#section6)
  + [Cycle 5](#section7)
  + [Cycle 6](#section8)
* [**References**](#section9)

## Nisin Solution

## Cycle 1: Promoter Screening and Testing

**• Description:**To quantitatively measure the strength of each candidate promoter, we did enzyme-linked immunosorbent assay (ELISA) and Western blot based on 6x His-tag and Flag-tag. Before that, 6xHis-and-Flag-tagged AcGFP reporter gene was inserted into plasmid vector and then electroporated these plasmids into *Lactoccoccus lactis* for expressing protein.

**• Design:**To consistently improve the expression level of nisin, we aim to verify strong Lactic Acid Bacteria promoters with experiment and choose the strongest one to express our target genes. *Lactococcus lactis* ATCC11454 strain was chosen as our model organism due to its capability to produce nisin. 14 strong promoters were selected from a wide range of literature concerning promoter strengths in *Lactococcus lactis*, and their strengths were evaluated with quantitative testing of a reporter AcGFP gene.

**• Build:**We inserted our candidate promoters and AcGFP reporter gene into the Lactic Acid Bacteria expression vector pMG36e backbone following Biobricks Assembly. 6x His-tag and Flag-tag were attached to the N- and C-terminals of AcGFP gene sequence respectively. The plasmids were then transformed into *Lactoccoccus lactis* with electroporation.

**• Test:**After electroporation, Erythromycin was applied to select successful transformants. Enzyme-linked immunosorbent assay (ELISA) was then performed on bacterial whole cell protein extract for quantitative testing of protein expression, where 6x His-tag and Flag-tag worked as reporter tags. Western Blot was also performed for auxiliary verification.

**• Learn:**From three biological replicates’ ELISA results, P3, P11, PTSIIC, PnisA, PnisR showed relatively stronger expression than the rest of promoters. From two rounds of Western Blot results, PTSIIC and PnisA showed consistent strong expression. Therefore, PnisA was selected as our target promoter.

## Cycle 2: Promoter Prediction

We developed a computational tool (Prokaryotic Motif-Based Promoter Strength Comparison Analysis Program, PPAP) to predict the strength of 13 bacterial promoters in *Lactococcus lactis* based on conserved motifs. Our goal was to identify key motifs, specifically the -10 (TATA box) and -35 (Sigma factor binding site) sequences, which play a crucial role in gene transcription. By analyzing GC content and motif placement, we created a custom scoring system to rank promoters based on predicted efficiency. This computational approach was intended to assist in selecting strong promoters for gene expression, minimizing the need for extensive lab work and experimental iterations.

**• Design:**We aimed to develop a computational tool to predict the strength of 13 bacterial promoters in *Lactococcus lactis* based on conserved motifs. Our goal was to identify key motifs, like the -10 and -35 sequences, which play a crucial role in gene transcription. By analyzing GC content and motif placement, we created a custom scoring system to rank promoters based on predicted efficiency. This computational approach was intended to assist in selecting strong promoters for gene expression, minimizing the need for extensive lab work and experimental iterations.

**• Build:**We implemented the analysis by running our script on 13 selected promoters. Each promoter’s GC content and the presence of conserved motifs were calculated, and these features were integrated into a scoring function. Promoters were then ranked by strength, with nisA\_op scoring the highest at 13.28. A promoter with a higher custom score should, theoretically, be more effective in initiating transcription and thus be a stronger promoter.

**• Test:**To validate our computational predictions, we plan to test these *Lactococcus lactis* promoters in vivo using BCA Assay and enzyme-linked immunosorbent assay (ELISA). Most of the promoter strength results were in line with our predictions with the absolute p-value lower than 0.1, despite two exceptions observed in nisF and P32 promoters (with p-values of 0.166256 and 0.10916 respectively), concretely validate the promoter prediction model.

**• Learn:**Through this project, we have learned that bioinformatics tools can significantly streamline the process of promoter selection. Our custom scoring system offers a computational approach to predict promoter strength based on conserved motifs and GC content. (Results for prediction: nisA\_op: 13.28 P48: 12.50 P11: 11.00 cc: 9.39 nisR: 8.96 nisR\_op: 8.94 P5: 8.77 P3: 8.73 nisA: 8.32 P8: 8.28 nisF: 6.36 PTCIIC-celA: 0.36 P32: 0.00)

## Cycle 3: NisRK Overexpression

Overexpression of nisRK with plasmid vectors can promote PnisA activity by utilizing the self-regulation of nisin in NICE system, thereby increasing nisin yield. This scheme not only can verify the feasibility of our genome editing scheme which aims to edit the nisR promoter, but also can be used as a simple and independent way to increase the expression of nisin.

**• Design：**The membrane-located histidine kinase NisK senses the inducing signal nisin and autophosphorylates, then transfers phosphorous group to intracellular response regulator protein NisR which activates nisA promoter to express the downstream gene.

**• Build:**We added Myc-tagged nisR CDS and HA-tagged nisK CDS into the pMG36e plasmid with PnisA-GFP, aiming to detect the effect of nisRK on nisA promoter activity through changes in GFP expression level, and verifying nisRK expression through detection of respective tags.

**• Test:**We aim to detect expression of both AcGFP reporter gene and nisK through Western blot, targeting Flag tag and HA tag, respectively. As a semi-quantitative method, Western Blot can verify the expression of nisRK regulation system while also providing some clues on the changes in nisA promoter activity in the presence of nisRK.

**• Learn:**The experimental results show that nisRK was successfully expressed. In addition, the increase in GFP production caused by overexpression of nisRK was detected, which can indicate possible changes in nisin production guided by the same promoter, verifying the feasibility to increase nisin production by increasing nisRK expression through genome editing. Also, this solution alone can also be used as a simple method to increase nisin yield.

## Cycle 4: Genome Editing

In Genomic editing engineering cycle, we discuss the design of approach to improve nisin production. In the design phase, original promoters are designed to be replaced with strong constitutive promoters, utilizing CRISPR-Cas9 for precise genome editing. The build phase focuses on constructing an appropriate plasmid (pMG-Cas) for transformation into *Lactococcus lactis*. Testing evaluates nisin yield via qPCR and protein assays, with GFP reporters to assess promoter activity.

**• Design:**Our goal is to enhance nisin production in *Lactococcus lactis* by replacing weak or inducible promoters in the nisin gene cluster with strong constitutive promoters. Specifically, PnisR, the promoter responsible for expression of the nisRK regulatory pathway, will be substituted with a stronger constitutive promoter to increase transcriptional activity and thus nisin yield. Homology arms for genome editing was obtained based on whole genome sequence of the *Lactococcus lactis* strain ATCC11454. The genome editing will be performed using a Red/ET assisted CRISPR-Cas9 system. The plasmid pMG-Cas, which incorporates pMG36e’s pWV01 ori and Ery resistance gene into the CRISPR/Cas9 plasmid pCas, was design be used as the CRISPR delivery vehicle, ensuring stability and compatibility with the host system.

**• Build:**The plasmid pMG-Cas was be constructed by replacing the pUC ori and Kan resistance gene from pCas with the pWV01 ori and Ery resistance gene from pMG36e. This allows the plasmid to function effectively in *Lactococcus lactis*. The homology arms flanked the strong constitutive promoter, and a Cas9-sgRNA system was designed to target PnisR for substitution. Following plasmid assembly, *Lactococcus lactis* cells would be transformed with the pMG-Cas plasmid. The cells would be cultured at 30°C to maintain plasmid stability. PCR amplification can then confirm successful integration of the promoter at the desired locus, followed by sequencing to ensure the accuracy of the modification.

**• Test:**Whether the knock-in was successful will be determined by PCR after the transformed plasmid is lost. Nisin production levels was designed to be measured post-transformation to assess the effectiveness of the promoter substitution. A secondary experiment would utilize GFP as a reporter to monitor the activity of the new constitutive promoter in real-time, providing an indirect measure of its strength compared to the original inducible promoter. This would help identify any discrepancies in promoter activity or expression levels.

**• Learn:**Data from PCR and nisin production assays would be analyzed to determine whether the new promoter was successfully knocked in and whether it significantly enhances expression. If successful, the promoter substitution can be considered validated. Possible bottlenecks still need to be identified such as low CRISPR efficiency or plasmid instability, which influence the selection of promoter or the construction of plasmids.

## H2O2 Solution

One team is working on enhancing the hydrogen peroxide production of *L. johnsonii* to inhibit *S. aureus*, which is linked to toxic shock syndrome (TSS). We aim to engineer *L. johnsonii* by overexpressing two genes, LJ\_0548 and LJ\_0549, which encode subunits of FMN reductase (FRedA/B). These genes were found to significantly impact H2O2 production, with overexpression increasing it. The research was done through the following DBTL (Design, Build, Test, Learn) Cycles. Using the pMG36E plasmid, we cloned the genes under the control of a strong P32 promoter and transformed the plasmid into *L. johnsonii* (ATCC 33200). To test expression, we tagged LJ\_0548 with Flag and LJ\_0549 with 6xHis Tag. The engineered strain could potentially reduce TSS risk when introduced into tampon products.

## Cycle 5: H2O2 Enhancement

**• Design:**pMG36E is a lactic acid bacteria expression vector that contains the P32 promoter, an erythromycin resistance gene and a multiple cloning site. (van de Guchte, Van der Vossen, Kok and Venema) We designed our plasmid based on this backbone.

The plasmid is designed to overexpress a protein dimer FMN reductase, which consists of FRedA and FRedB, thus increasing the production of hydrogen peroxide of *L. johnsonii.*

**• Build:**We introduced genes encoding the proteins FredA and FredB into the backbone pMG36E. Both coding sequence were optimized using Genscript’s codon optimization service Gensmart. To detect facilitate testing detect the expression of these two genes, we attached Flag tag and 6xHis tag respectively.

![](https://static.igem.wiki/teams/5076/des4.png)

**Fig. 1 pMG36E with gene LJ\_0548 and LJ\_0549.**

**• Test:**

![](https://static.igem.wiki/teams/5076/figure-for-engineering-cyc-g2.jpg)

**Fig.  2 H2O2 expression test and protein expression level test of *L. johnsonii*.**

a) Description of the four different groups about their strain chosen and culturing environment. pMG36E stands for the strain with the plasmid backbone and pMG36E\_LJ0548-0549 stands for the overexpression strain.

b) H2O2 production measured using phenol red assay for four groups.

c) Western Blot result for Flag Tag (up) and 6xHis tag (down). For each group, 2 replicates were done.

d) Result of ELISA test for Flag tag which is attached to the gene LJ\_0548 after culturing for 16h. Group B2 was ignored because of too low protein concentration.

Four groups with different strains and done under different conditions were examined for H2O2 production and gene expression level. (Fig. 2a) The overexpression of the two genes which encode the FMN protein dimer did not show significant difference in aerobic groups, but showed a notable increases in H2O2 production in anaerobic groups after growth of 12 hours. (Fig. 2b)

For the Western Blot result of gene LJ\_0548, no significant differences were observed for anaerobic groups and the result was not trustworthy due to too much unspecific binding of proteins whose length did not march our target (46.29kD). (Fig. 2c) Under aerobic conditions, the expression levels of the protein FRedB encoded by gene LJ\_0549 were moderately increased. Although this result means gene LJ\_0549 in the plasmid successfully expressed, it is not significantly elevated.

To validate protein expression, ELISA was carried out and the outcome revealed a significant downregulation of protein levels in the insertion groups under both conditions, aligning with the WB observations for the 6xHis tag. (Fig. 2d)

**• Learn:**Our data suggest that anaerobic conditions favor higher H2O2 production, and the overexpression of LJ\_0548 and LJ\_0549 amplifies this effect. Aerobic conditions, on the other hand, limit H2O2 production, regardless of gene overexpression. The insertion of the two genes leads to a notable increase in H2O2 production in anaerobic environment after certain period of growth time.

Upon analyzing the relationship between H2O2 production and protein expression, we observed a negative correlation. While H2O2 levels increased following gene insertion, the corresponding protein levels for LJ\_0548 and LJ\_0549 decreased. We hypothesize that this reduction in protein levels may be due to the consumption or degradation of these enzymes after H2O2 production. Additionally, this may represent a regulatory mechanism by *L. johnsonii* to prevent excessive H2O2 accumulation, protecting the bacteria and its environment from oxidative damage.

## Cycle 6: Protein Docking Analysis

**• Design:**FMN (flavin mononucleotide) reductase is a protein dimer which is made up of the 2 proteins we inserted (nfr1/2, or FRedA/B). Because the function of the two protein is still not fully understood and research showed that the loss of the two proteins would disable the bacteria’s ability to produce hydrogen peroxide, we use protein function prediction tool and protein docking online services to predict the function of this protein dimer. We hoped that the result could give us some insight on the mechanism of hydrogen peroxide production in *Lactobacillus johnsonii.*

**• Test:**First, we uploaded our 2 protein sequences (nfr1/2) to STRING, a database of protein-protein interactions.

In our current organism *L. johnsonii*, 9 predicted functional partners of nfr1 and nfr2 were chosen for analysis (Fig. 3).

![](https://static.igem.wiki/teams/5076/screenshot-2024-10-01-at-6-36-29-am.png)

**Fig. 3 Nine predicted functional partners for our reductase which were found by STRING. AOG26301.1 is nfr1.**

ClusPro, a web-based tool was used for protein docking for conducting our analysis of the 9 predicted functional partners. The 9 partners which were found by STRING were performed protein docking with the FMN reductase.

For the FMN complex and Oxidoreductase 1 of *L. johnsonii* (BBP16\_00775), the protein docking result shows that the surface representations of them fit together well, indicating good structural complementarity. (Fig. 4) The highlighted residues (red and yellow) indicate key interaction sites and interacting surfaces, contributing to the stability and specificity of the interaction.

![](https://static.igem.wiki/teams/5076/screenshot-2024-10-01-at-6-41-04-am.png)

**Fig. 4 The docking result between the FMN complex (in cyan) and the Oxidoreductase 1
(in magenta).**

Also, ClusPro clusters the docking results to identify similar binding modes and provides scores for these clusters. (Table. 1) Among all the partners we found in Fig. 3, this cell surface protein shows the lowest energy. This suggests that the docking model is the most stable among the 9 partners and biologically relevant.

| Cluster | Members | Representative | Weighted Score |
| --- | --- | --- | --- |
| 0 | 98 | Center | -771.3 |
|  |  | Lowest Energy | -820.3 |

**Table. 1 The scores provided by ClusPro for the protein docking result for FMN complex and Oxidoreductase 1.**

**• Learn:**The docking result between FMN complex and the Oxidoreductase 1 of *L. johnsonii* (BBP16\_00775) shows significant surface contact. FMN may play a role in catalyzing the transfer from reductants to oxidant and work with oxidoreductase inside *L. johnsonii* to deal with the problem of hydrogen peroxide accumulation. The structural complementarity observed suggests that the docking model is plausible and may provide hint for the analysis of metabolism chain.

## References

1. van de Guchte, M., Van der Vossen, J. M., Kok, J., & Venema, G. (1989). Construction of a lactococcal expression vector: Expression of hen egg white lysozyme in \**Lactococcus lactis*\* subsp. \*lactis\*. \*Applied and Environmental Microbiology, 55\*(1), 224-228.

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# Results

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## Our Findings

In our experiment, we transformed *Lactococcus lactis* with various plasmids, including pMG36e, using electroporation and chemical transformation. Competent cells were prepared, DNA uptake was facilitated, and transformants were cultivated on selective media. Verification was done via restriction enzyme digestion and gel electrophoresis. ELISA and Western blot analyses identified PnisA as the most effective promoter for AcGFP protein expression. Overexpressing nisRK with PnisA-guided genes enhanced GFP expression, confirming successful promoter amplification. Additionally, experiments on *L. johnsonii* showed increased H₂O₂ production under anaerobic conditions, with reduced protein expression, likely as a regulatory mechanism to prevent oxidative damage.

![Image placeholder](https://static.igem.wiki/teams/5076/resu1.png)

Results

* [**Nisin Solution**](#section1)
  + [Plasmid Transformation](#section2)
  + [Optimal Promoter Selection](#section3)
  + [NisRK Amplifies PnisA](#section4)
  + [Lactococcus CRISPR Build](#section5)
* [**H2O2 Solution**](#section6)
  + [Phenol Red Standard Curve](#section7)
  + [Bacterial Growth](#section8)
  + [H2O2 Production by Strain](#section9)
  + [Transformed H2O2 Output](#section10)
  + [Inserted Gene Expression](#section11)
  + [Protein Quantification by ELISA](#section12)
  + [H2O2-Protein Correlation](#section13)

## Nisin Solution

## Transformation of Test Plasmids into *Lactococcus lactis*

In this experiment, we successfully achieved the transformation of the target gene to *Lactococcus lactis* using various plasmids, including pMG36e. The process of transformation involves several steps, including competent cell preparation, electroporation, plasmid extraction, and verification through gel electrophoresis.

The experiment was started with the preparation of competent cells. The bacterial strain was cultivated until it reached the logarithmic growth phase, typically incubating at 37°C for 3-4 hours, until the OD600 reached 0.4-0.6. The cells were harvested by centrifugation at 5000 rpm and 4°C for 10 minutes. The cells were washed twice with pre-cooled 0.1 M CaCl2 solution to remove any residual medium. After washing, the cells were resuspended in 0.1 M CaCl2 solution and allowed to rest on ice for 30 minutes to ensure the formation of competent cells.

After the competent cells were prepared, two transformation processes were used in this experiment: electroporation and chemical transformation. In chemical transformation, plasmid DNA was added to competent cells, the mixture was incubated on ice for DNA uptake, the cells were heat-shocked, the cell membranes were stabilized, and incubation was performed for the expression of antibiotic-resistance genes. However, due to reasons of efficiency, electroporation was chosen for most genes.

For the process of electroporation, we transformed 9 plasmids, including CT(pMG36e), P3, P5, P8, P11, P32, P48, PTSIIC-celA, nisA, nisA-op, nisR, nisR-op, nisF, nisF-op. During the process, the frozen competent cells were thawed on ice, and 40 µL of cells were mixed with 1–2 µL of DNA in a polypropylene microcentrifuge tube. This mixture was carefully transferred to an ice-cooled electroporation cuvette, ensuring it was evenly distributed on the bottom to avoid air bubbles and minimize the risk of arcing during the pulse. The cells were electroporated with settings of 2.0 kV voltage, 25 µF capacitance, and 200 Ω resistance, aiming for a time constant of 4–5 ms. Immediately after pulsing, 0.96 mL of ice-cold SGM17 medium was added to the cuvette, and the mixture was transferred to a microcentrifuge tube and incubated at 30°C for 2 hours. The cell suspensions were spread onto SR or BSR plates containing the appropriate selective agent, such as erythromycin at 1 µg/mL or chloramphenicol at 5 µg/mL, to select transformants. The plates were incubated at 30°C for up to 3 days, with transformant colonies typically becoming visible after 1 day.

After cultivation on selective media, the transformants successfully grew on plates containing resistance markers, indicating that the transformation process was successful, and the target gene was successfully introduced into the host strain.

Besides, we also extracted plasmids from the transformed *L. lactis* strain and confirmed the plasmid via enzyme digestion and electrophoresis, asserting the successful result of transformation.

![](https://static.igem.wiki/teams/5076/resu2.png)

**Fig. 1 Restriction digestion (w/ HindIII), and gel electrophoresis of Kit method lactis plasmid from June 24 & input plasmid used for transformation. Results align, suggesting successful transformation.**

These experimental results demonstrate that we successfully prepared competent cells and introduced the target gene into the bacteria using electroporation technology, laying the foundation for subsequent functional studies.

## PnisA Selected as the Best Promoter to Express AcGFP Protein

Nisin is an antibacterial substance secreted by lactis, which can effectively kill Gram-positive bacteria, including *Staphylococcus aureus*. To find the most effective promoter among the limited scale, firstly we construct a list of plasmids where each of them was inserted a different promoter and 6xHis and Flag-tagged AcGFP reporter gene, following Biobrick Assembly (see figure 1, a). DNA synthesis and cloning was performed by Genscript, China. For promoter PnisA, PnisR, PnisF, we did codon-optimization for *L.lactis* expression using INTEGRATED DNA TECHNOLOGIES (IDT) codon-optimization service and they were cloned into erythromycin resistant pMG36e plasmid backbone. The recombinated plasmid pMG36e-nisA (e.g.) etc. were subsequently transformed into *Lactococcus lactis* and selected by erythromycin. To check the expression status and effect of AcGFP, ELISA targeting Flag-tag and Western Blot targeting Flag-tag and His-tag were performed successively. After comprehensively consideration, we chose PnisA promoter as the final choice which was used for phase II experiment.

Based on the experimental design, plasmid pMG36e containing different promoters (P3,P5,P11,P32,P48,PTSIIC-celA,PnisA,PnisA-op,PnisR,PnisR-op,PnisF,PnisF-op) were transformed into *Lactococcus lactis*, with the empty pMG36e plasmid backbone as control group. After cultivating the transformed cells under optimal growth conditions, whole bacterial protein was extracted and analyzed using ELISA targeting Flag-tag of the reporter AcGFP gene. The data obtained were statistically analyzed, resulting in the p-value chart shown in supplementary figure 1. The results indicate that the promoters PTSIIC-celA and PnisA exhibit significantly higher expression levels compared to other tested promoters. Statistical significance is denoted by asterisks, highlighting the notable differences in protein concentration across various promoter conditions. This suggests that PTSIIC-celA and PnisA are particularly effective in driving protein expression in *Lactococcus lactis*.

We picked five promoters (P3,P11,PnisA, PnisR, PTSIIC-celA) and the negative control group (CT) to perform Western Blot to further verify the activities of these promoters. All promoters were assessed in 3 replicates, with 2 targeting the 6xHis tag and 1 targeting the Flag tag. PnisA and PTSIIC-celA showed high expression quantity and stable expression level among different replicates in both Flag-targeted and His-targeted detection (Figure 1, b&c). Considering both ELISA and Western Blot results, we eventually chose PnisA to be the target promoter for next phase.

![](https://static.igem.wiki/teams/5076/description/screenshot-2024-10-02-at-2-47-54-am.png)

**Fig. 2 Experimental Results for Promoter Screening.**

a) Overall design of the promoter testing cassette, consisting of the candidate promoter, vioA RBS, and an AcGFP reporter gene tagged with a 6xHis tag and Flag-tag.

b) Western blot results verifying promoter selection. As shown in the blot, PTSIIC-celA and PnisA showed significantly higher expression levels than the rest.

c) ELISA results of promoter screening. The box plot displays the expression levels of proteins driven by different promoters, including CT, P3, P5, P11, P48, PTSIIC-celA, nisA, nisA-op, nisR, nisR-op, and nisF. Statistical significance is indicated by asterisks (\*p-value < 0.05, \*\*p-value < 0.01), highlighting the higher expression levels achieved with the PTSIIC-celA and nisA promoters, which have a p-value of 0.0085 and 0.02 relative to the negative control, and a p-value of 0.04 and 0.0077 relative to the native nisR promoter, respectively. These p-values indicate a significant increase in expression level of the reporter gene. The complete set of ELISA data and p-values are available in Supplementary Fig. 1.

## NisRK Overexpression as an Amplifier to PnisA-Guided Genes

In this part, we introduced Myc-tagged nisR CDS and HA-tagged nisK CDS (sequences obtained from Genbank HM219853.1, codon optimized by Genscript’s Gensmart codon optimization service for expression in *Lactococcus casei* and to comply with biobricks assembly), guided by nisA promoter screened from previous experiments, into the pMG36e plasmid with PnisA-GFP reporter cassette, aiming to detect the effect of nisRK on nisA promoter activity through changes in GFP expression level, and verifying nisRK expression through detection of respective tags. Western Blot (WB) experiment included nisRK overexpression strain containing PnisA-AcGFP reporter cassette (nisRK), PnisA-AcGFP reporter cassette standalone (nisA), and a control group (CT) containing empty pMG36e plasmid. 3 biological replicates were performed for nisRK and nisA groups, with 2 technical replicates of the WB experiment performed on each. WB experiment followed standard protocols, using primary antibodies targeting Flag-tag and HA-tag to detect expression of AcGFP reporter gene and nisK protein, respectively. The results demonstrated successful nisK expression in nisRK group, as well as a significant increase in AcGFP expression in nisRK group, indicating that introduction of nisRK cassette was successful in enhancing gene expression guided by nisA promoter. However, fluctuations in expression level can still be observed among samples in the same experimental group, indicating a minor instability in the activity of this nisRK amplification system of nisA promoter.

![](https://static.igem.wiki/teams/5076/resu4.gif)

**Fig. 3 Target protein expression in nisA and nisRK.**

a) construct of nisRK group plasmid, which includes a PnisA-GFP reporter cassette and a nisRK expression cassette.

b) construct of nisA group plasmid, which includes a PnisA-GFP reporter cassette tagged with Flag tag and His tag.

c) western blot result. Strong nisK expression has been detected and GFP expression was significantly stronger in nisRK group than in nisA group.

![](https://static.igem.wiki/teams/5076/description/screenshot-2024-10-02-at-2-48-26-am.png)

**Supplementary Fig. 1. Supplementary data for ELISA results.**

a) This table displays the p-values obtained from ELISA data analysis after calibration. Each row represents a specific promoter, with columns labeled I1, I2, II1, II2, II3, III1, III2, and III3 indicating different biological or technical replicates. The last column, "AVR," shows the average p-value across all conditions for each promoter.

b) This table presents the p-values comparing each promoter to the control (CT). The values indicate the statistical significance of differences between each promoter and the control condition.

c) This table provides p-values for comparisons between each promoter and the nisR condition. These values reflect the statistical significance of differences between each promoter and nisR.

## Construction of Lactococcus compatible CRISPR plasmid

We constructed the chimeric plasmid named pMG-Cas, combining Cas9 gene and Red/ET recombinase system from pCas, and pWV01 origin of replication and Erythromycin resistance gene from pMG36e. The pWV01 origin of replication and Erythromycin resistance gene was cloned from pMG36e with extension PCR which introduced ApaI and XhoI restriction sites to the fragment. pCas fragment was prepared by digesting pCas with ApaI and XhoI (EasyCut restriction kit, Adamas Life, China), electrophoresing and cutting the gel to retrieve the larger fragment of 9120 bp. The amplified pMG36e fragment was then digested with ApaI and XhoI (EasyCut restriction kit, Adamas Life, China) and cloned into ApaI and XhoI digested pCas fragment using T4 DNA ligase (Adamas Life, China). Ligation product was transformed into E. coli DH5-alpha competent cell and spreaded on Erythromycin selective LB plates for culture. A colony with Erythromycin resistance was selected and amplified, and its plasmid was extracted for verification of plasmid construction. The extracted pMG-Cas plasmid was enzyme digested by PciI which introduced a single cut on the plasmid, and a PCR targeting ligation junction with a product size of around 300bp was also performed. As shown in figure 4, both enzyme digestion and PCR results demonstrated successful construction of the pMG-Cas plasmid.

![](https://static.igem.wiki/teams/5076/description/screenshot-2024-10-02-at-9-47-43-am.png)

**Fig. 4 Electrophoresis result of PCR and enzyme digestion of pMG-Cas. The Lanes, from left to right, are: Ladder, PCR replicate 1, PCR replicate 2, PciI digestion replicate 1, PciI digestion replicate 2, PciI digestion replicate 3. PCR results had a size of around 300bp while PciI digestion result is around 12kbp, which aligns with the size of pMG-Cas plasmid, which is 11939 bp.**

## H2O2 Solution

## Standard Curve for Phenol Red Assay

Before testing the H2O2 concentration, we preformed the phenol red assay successfully produced a standard curve linking known H2O2 concentrations to absorbance at 620 nm (Fig. 5a). This curve enables the estimation of H2O2 levels in bacterial cultures by comparing their absorbance values. This is critical for quantifying the production of H2O2 by the bacterial strains in subsequent assays, ensuring that absorbance data can be reliably converted to actual concentration values for analysis.

## Bacterial Growth over Time

Four groups were tested with different strains and was done under different conditions (Fig. 5b). 3 replicates were done for each group and average values were taken for all the data. Following with the bacterial growth curve (OD600) demonstrates that all strains entered exponential growth during incubation (Fig. 5c). We can clearly find that the concentration of successfully transfected lactobacillus (pMG36E\_LJ0548-0549) is higher than that of the control group during the whole growth process. However, both aerobic strains exhibited significant cell death after approximately 800 minutes of culture. The cessation of data collection at the final time point, due to observed dead cells, suggests that the high level of bacterial death may have distorted the OD600 readings. This information is essential for interpreting any related results, such as H2O2 production, which may be impacted by bacterial viability.

## H2O2 Production by Strain

H2O2 production level was measured using phenol red assay and then calculated by the standard curve from Fig. 5a. The data indicate that anaerobic conditions lead to significantly higher hydrogen peroxide (H2O2) production in *L. johnsonii*. In the anaerobic control group (Group C), H2O2 levels began to increase around 500 minutes and peaked at 700 minutes before declining, possibly due to feedback inhibition or depletion of necessary substrates. The anaerobic experimental group (Group D), which is the overexpressing strain for gene LJ\_0548 and LJ\_0549, showed higher H2O2 production after 800 minutes, peaking at 41.2μM, suggesting that the overexpression of these genes enhances H2O2 output under anaerobic conditions.

In contrast, aerobic conditions appear to inhibit significant H2O2 production. Both the aerobic control and experimental groups (Groups A and B) showed consistently low H2O2 levels with no major fluctuations throughout the observation period. This suggests that the aerobic environment suppresses the accumulation of hydrogen peroxide in *L. johnsonii*.

Overall, the data suggest that anaerobic conditions favor higher H2O2 production, and the overexpression of LJ\_0548 and LJ\_0549 amplifies this effect. Aerobic conditions, on the other hand, limit H2O2 production, regardless of gene overexpression.

## H2O2 Production in Transformed Lactobacillus Johnsonii

After successfully transforming *L. johnsonii* with genes regulating H₂O₂ production, we observed significant H₂O₂ production under both aerobic and anaerobic conditions. This was evident in the transformed strains compared to controls, with notable increases in H₂O₂ levels in both conditions.

![](https://static.igem.wiki/teams/5076/screenshot-2024-10-01-at-6-09-24-am.png)

**Fig. 5 H2O2 expression test and protein expression level test of *L. johnsonii*.**

a) Phenol red assay was performed with H2O2 standard solution to generate a standard absorbance curve. This curve can be used to estimate H2O2 concentrations in the growth medium based on their relative absorbance at 620nm.

b) Description of the four different groups about their strain chosen and culturing environment. pMG36E stands for the strain with the plasmid backbone and pMG36E\_LJ0548-0549 stands for the overexpression strain.

c) Value of OD600 of the growing bacteria was measured at different time points during the strains’ incubations. Each strain was observed to have exponential growth. Notably, both aerobic strains experienced significant bacterial death after 800 minutes of culture. (\*last data point not collected due to observation of dead cells, which may have affected OD600).

d) H2O2 production measured using phenol red assay for four groups.

f) Western Blot result for Flag Tag (up) and 6xHis tag (down). For each group, 2 replicates were done.

g) Result of ELISA test for Flag tag which is attached to the gene LJ\_0548 after culturing for 16h. Group B2 was ignored because of too low protein concentration.

## Protein Expression of Inserted Genes

To examine the expression of the inserted genes, we tagged the genes LJ\_0548 and LJ\_0549 with 6xHis Tag and Flag Tag, respectively, to facilitate subsequent protein analysis. We conducted Western blot (WB) analysis under both aerobic and anaerobic conditions to compare the protein expression in the insertion groups and control groups. For the WB result for the protein FRedA encoded by gene LJ\_0548, no significant differences were observed for anaerobic group and the result was not trustworthy due to too much unspecific binding of proteins whose length was not our target (46.29kD). (Fig. 5f) Under aerobic conditions, the expression levels of the protein FRedB encoded by gene LJ\_0549 were moderately increased. Although this result means gene LJ\_0549 in the plasmid successfully expressed, it is not significantly elevated. Under anaerobic conditions, protein expression levels were minimal. (Fig. 5f) Adjustments to experimental conditions, such as changing developing reagents and normalization controls, did not result in more substantial findings.

## Quantification of Protein Expression via ELISA

To further validate protein expression, we employed ELISA to test the expression level of gene LJ\_0548 attached with Flag Tag under both aerobic and anaerobic conditions. The results revealed a significant downregulation of protein levels in the insertion groups under both conditions, aligning with the WB observations for the 6xHis tag. (Fig. 4g)

## Negative Correlation Between H₂O₂ Production and Protein Expression

Upon analyzing the relationship between H2O2 production and protein expression, we observed a negative correlation. While H2O2 levels increased following gene insertion, the corresponding protein levels for LJ\_0548 and LJ\_0549 decreased. We hypothesize that this reduction in protein levels may be due to the consumption or degradation of these enzymes after H2O2 production. Additionally, this may represent a regulatory mechanism by *L. johnsonii* to prevent excessive H2O2 accumulation, protecting the bacteria and its environment from oxidative damage.

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