Nisin solution

Promoter Screening

Through literature review and promoter optimization, we finally selected the following 14 promoters to be tested for subsequent genome editing to increase nisRK expression and thus nisin production. 

Some of promoter sequences were optimized using Integrated DNA Technologies' codon optimization tool [1], a process carried out by Sam Shen, PhD, Product Support Specialist at IDT, and Hans Packer, PhD, former Scientific Writer at IDT. This optimization involved selecting the DNA bases, specifying the product type (a gene), and indicating the organism (Lactobacillus acidophilus). The sequence was adjusted to enhance transcriptional efficiency and stability, aiming to strengthen the promoter's capability to reliably regulate gene expression. This modification is particularly beneficial in applications that demand continuous production of antimicrobial peptides and other valuable proteins. 

PnisA: a nisin-inducible regulatory DNA sequence widely used in the Nisin-Controlled Expression (NICE) system [2]. Derived from Lactococcus lactis, this promoter controls the transcription of the nisin biosynthetic genes [3].

PnisA-op: the optimized nisA promoter sequence.

PnisR: The nisR promoter is part of a two-component regulatory system in Lactococcus lactis, controlling gene expression in response to nisin. It regulates the expression of the nisR and nisK gene, which encodes a response regulator protein essential for activating transcription of nisin-responsive genes [2]. 

PnisR-op: the optimized nisR promoter sequence.

PnisF: The nisF promoter is a nisin-inducible regulatory DNA sequence from Lactococcus lactis associated with the nisin operon. PnisF controls the expression of the nisFEG genes [2].

PnisF-op: the optimized nisF promoter sequence. P3, P5 and P8: constitutive promoters derived from L. lactis N8, known for the efficient and constant gene expression in lactic acid bacteria. These promoters are characterized by the ability to continuously drive high levels of transcription without the need for inducers or additional regulatory elements [4].

P11 and P48: synthetic promoters from the Synthetic promoter library (SPL), containing consensus sequence that derived from rRNA promoters extracted from the L. plantarum WCFS1 genome [5].

PTS-IIC promoter: an endogenous constitutive promoter from L. lactis., mediates protein expression in B. subtilis and E coli Nissle 1917. The activity of PTS-IIC promoter can be enhanced by Cellobiose [6].

Plasmid Design

Our test plasmids for promoter screening were designed in the following structure: Candidate Promoter + AcGFP +Reporter Tag. We inserted a modified version AcGFP reporter CDS codon optimized for expression in Lactococcus (Gensmart, Genscript, China) into the Lactic Acid Bacteria expression vector pMG36e backbone following Biobricks Assembly, the CDS attached with 6x His-tag and Flag-tag for ease of detection. Candidate promoters screened in the previous step was then inserted into pMG36e backbone before the CDS to complete the expression cassette.

GFP expression test

We performed two tests for GFP expression, Enzyme-Linked Immunosorbent Assay (ELISA) and Western blot (WB). ELISA is an immunoassay method that binds an antigen or antibody to the surface of a solid phase carrier and uses the specific binding of the antigen or antibody and the enzyme labeled on the antibody or antigen to catalyze the color reaction of a specific substrate to achieve the detection of the target object. In our experiment, ELISA detects the Flag tag attached to AcGFP.

WB is a method in which protein samples are separated according to molecular weight by polyacrylamide electrophoresis, then transferred to a hybrid membrane (blot), and then the target protein is specifically detected by the primary antibody/secondary antibody complex. It has the advantages of large analytical capacity, high sensitivity and strong specificity, and is one of the most commonly used methods to detect protein characteristics, expression and distribution. In this experiment, the antigen used by WB is the 6xHis tag.

Compared with WB, the advantage of ELISA is that the experimental procedure is simple, the antigen can be quantitatively detected without purification, and the use scenario is more flexible. When the antibody is highly specific to the antigen, it is easy to produce highly sensitive results. But in contrast, Although WB can only achieve semi-quantitative detection, the probability of false positive caused by non-specific binding in WB is lower than WB, which makes the qualitative detection results are more accurate.

NisRK Overexpression

In order to enhance the production of nisin, whose expression is regulated by an inducible promoter PnisA, we overexpress the PnisA regulating genes nisRK in L. lactis using a plasmid vector to achieve the effect of increasing nisin production with the NICE system. It also provides validation of the correlation between nisRK expression and nisin expression, which work as proof-of-concept for the genome editing aspect of the project.

Fig. 1 Organization of genes involved in nisin production, regulation and immunity. P* and Pc indicate nisin-inducible and constitutive promoters, respectively. Hairpins indicate putative transcription terminator loops. nisA, nisin structural gene; nisB and nisC, post-translational modification; nisT, translocation; nisP, serine protease involved in precursor processing; nisR and nisK, regulation; nisI and nisFEG, immunity [7][8].

The Nisin expression system relies on self-regulation [9]. nisA promoter activity was dependent on the proteins NisR and NisK, which constitute a two-component signal transduction system that responds to the extracellular inducer nisin [10]. 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 [11].

We used pMG36e with PnisA-GFP to express nisRK, and detected GFP expression by ELISA and WB after transfer to L. lactis. The aim of this method is to indirectly judge the promotion effect of overexpressed nisRK on nisA promoter in L. lactis genome by measuring its promotion effect on nisA promoter in plasmid, so as to provide theoretical support for Pnis protocol. At the same time, this regimen alone also increased nisin expression by introducing regulatory factors. Although plasmids introduce additional erythromycin resistance compared to CRISPR, which has a certain risk if applied outside the laboratory, but it can be used as a relatively complete and simple replacement solution.

Genome editing

In order to enhance expression of the PnisA regulators nisRK, thus enhancing nisA production, we employed a Red/ET recombinase-assisted CRISPR/Cas9 system to modify the native nisR promoter of the Lactococcus lactis plasmidome [12], substituting it with the stronger promoter identified through the screening process to improve nisin production.

To achieve this, we constructed a chimeric plasmid named pMG-Cas, combining elements from both the pCas and pMG36e plasmids [13]. This plasmid includes the following key components: Cas9 gene, which facilitates targeted DNA cleavage; a Red/ET recombinase system sourced from pCas, mediating homologous recombination, which is essential for the precise replacement of the native nisR promoter. It also contains a gRNA scaffold and homology arms of the target site, which is responsible for guiding specific cleavage and homology-directed repair. The pWV01 origin of replication (ori) is sourced from pMG36e, and ensures efficient plasmid replication in Lactococcus lactis. In addition, the Erythromycin resistance gene is carried in replacing the kanamycin resistance gene in pCas, which enables the selection of successful transformants in Lactococcus lactis and avoids interference to the selection process due to L. lactis’ native resistance to Kanamycin. 

 For the homology arm design, we used Blast tool and identified the nisin coding and regulating cluster in ATCC11454 genomic scaffold 23, 7088-22102. we selected the 1000-bp regions immediately upstream and downstream of the nisR promoter for homology, and introduced our identified strong promoter, PnisA, to complete the template. 

We plan to transform the plasmid into L. lactis through electroporation, select successful transformants, and continue to pass on the culture for a few generations until the plasmids are lost. Then genomic DNA will be extracted and PCR targeting the modified PnisA-nisRK cassette will be performed to verify successful editing. Successful strains will be preserved for later testing of nisin expression level and bacteriostatic effects of the strain.

H2O2 Solution

Literature Review

In L. johnsonii NCC 533 strain, studies have found that two adjacent and highly similar genes (LJ_0548 and LJ_0549) can influence the production of hydrogen peroxide. (Hertzberger et al., 2014) Later studies found that these two genes encode FRedA and FRedB. These two proteins are the building blocks of a protein dimer, FMN reductase. (Valladares et al., 2015) FMN (flavin mononucleotide) reductase is an enzyme that catalyzes the reduction of FMN to FMNH2 using NADH or NADPH as electron donors, playing a crucial role in various biological redox reactions. The deletion of the two genes led to a 40-fold reduction of the H2O2 production ability of L. johnsonii. They also constructed a strain using a strong promoter which overexpressed these two genes. The result showed even higher H2O2 production ability in vivo than the wild strain (Fig. 1).

Fig. 2 H2O2 production in L. johnsonii NCC 533 and NCC 9359, with or without complementation of LJ_0548 and LJ_0549 under aerobic conditions. Strain or plasmid called pDP1019 has pDP794 with LJ_0548 and LJ_0549 expression plasmid.

Vector Construction

Based on these findings from the predecessors, we enhanced the expression levels of the two genes by cloning them into the pMG36E plasmid backbone and subsequently transforming the plasmid into the L. johnsonii strain (ATCC 33200) (Fig. 2). The 2 genes were optimized using service from GenScript. The expressing host selected was L. lactis and five enzyme cleavage sites were avoided to meet the standards of BioBricks™: EcoRI, Xbal, Spel, Pstl, and Notl. Then, they were added following the promoter P32. To test the expression level of the 2 proteins, Flag Tag was added after LJ_0548 and 6xHis Tag was added after LJ_0549.

Fig. 3 Construction of plasmid pMG36E_LJ0548-LJ0549.

Hydrogen Peroxide Production Test

In order to properly quantify the H2O2 production of our transformed L. johnsonii species, we began testing with Phenol Red Assay. This method was chosen based on its simple mechanism, ease of performing, and affordability. The oxidation of phenol red by horseradish peroxidase is H2O2 dependent and creates a linear relationship with H2O2 production and spectrophotometer absorbance at 610nm (Pick & Keisari, 1980).

In total, we cultured and tested two strains representing two different environmental conditions. Since the vaginal environment is neither fully aerobic nor anaerobic, two strains were cultured in both environments to test the effect that available oxygen may have on H2O2 production. Therefore, we designed 4 different groups for our experiment (Table 1). As transformed bacteria were being cultured, small samples were periodically taken out and aliquoted into microplate wells. Phenol red and horseradish peroxidase solution was then added to each of the wells and incubated for 10 minutes, after which stop solution was added.

Table. 1 Description of the four different groups about their strain chosen and culturing environment. pMG36E is the empty plasmid and pMG36E_LJ0548-0549 is the plasmid used for overexpression of the two genes.

Unfortunately, after performing the Phenol Red method, we realized that the MRS broth we used to culture L. johnsonii had a similar absorption spectrum to 610nm, which may have interfered with the sensitivity and accuracy of the assay. So, we switched to another assay that did not rely on visible light absorbance, but fluorescence: the Amplex Red assay. Amplex Red is also oxidized by horseradish peroxidase in the presence of H2O2, however, unlike phenol red, it emits a fluorescent signal when oxidized (Fig. 3). This signal can be detected by a fluorescence plate reader. After building a standard curve, H2O2 can be quantified in concentrations of less than 0.1uM (Towne et al., 2004).

Fig. 4 Mechanism for Amplex Red Assay: Oxidation of nonfluorescent Amplex red into highly fluorescent resorufin using hydrogen peroxide as a coreactant. (Towne et al., 2004)

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