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.

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.

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.

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.

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.

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.

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.