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[Notes] Bacillus subtilis

on May 12, 2023 IN Hackable-Humans
Bacillus subtilis CRISPR/Cas 9 Dr Mikolaj Raszek (geneticist and biochemist) Dr Peter McCullough (cardiologist) Dr Robert Malone (virologist - mRNA inventor) Dr Sabine Hazan (gastroenterologist) FLCCC food-supply Genetically Modified Organisms (GMO/NWO) gut health Nattokinase New World Order food (NWO) Patents Study Synthetic Biology
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This post is to remind me to look into “Bacillus subtilis” when I get the chance. I woke up this morning thinking it was important that we understand it as the “gut microbiome” has been coming up for me a lot in my research into what “they are doing to us” (i.e. is something they are doing to kill us off disrupting or inhibiting necessary microorganisms?).

One study for example suggests “SARS COV-2 mRNA vaccine decreases levels of bifidobacteria (P = 0.0065)” (Bifidobacteria are beneficial gut microbes), and another study showed “severely symptomatic SARS-COV-2 patients had significantly less bacterial diversity (Shannon Index, p=0.0499; Simpson Index, p=0.0581), and positive patients overall had lower relative abundances of Bifidobacterium (p<0.0001), Faecalibacterium (p=0.0077) and Roseburium (p=0.0327), while having increased Bacteroides (p=0.0075). The study authors hypothesize that low bacterial diversity and depletion of Bifidobacterium genera either before or after infection led to reduced proimmune function, thereby allowing SARS-CoV-2 infection to become symptomatic. In other words – the lack of beneficial gut bacteria is ‘potentially’ the reason people “got severely sick”. The same authors in another (retracted) study, suggested a hypothesis that “Ivermectin Feeds Bifidobacteria to Boost Immunity“, and that an increase in Bifidobacterium levels can reduce inflammation levels and TNF-α function, thereby calming the cytokine storm. They said IVM has antibacterial affects against Staphylococcus aureus and other gram-positive bacteria, but not Bifidobacterium (rather IVM mechanisms of action is through feeding of Bifidobacterium), and that both IVM and Bifidobacterium act against S. auereus. Interestingly, and it looks like the same authors again, did another study that showed high-dose vitamin C supplementation improves the gut bacteria: “Patients receiving ascorbic acid supplementation had increased abundance of Bifidobacterium in their gut microbiome, which may help to explain some of the apparent health benefits and antiviral properties of vitamin C.”

I’m not sure if many scientists are even looking at this important topic because most studies are performed due to grants – and most grants are funded by pharma.

The enzymes Nattokinase (found in Nattō; a Japanese food made with soybeans fermented with Bacillus subtilis) and Serrapepetase (isolated from the bacteria Serratia E15, found in silkworms) are being recommended as “clot busters” to breakdown the calamari-like clots that are being found in those who held out their arms for a global bio-weapon experiment.

Note: GMO Soy is Poison/Toxic

I am pretty ignorant to this side of things and want to understand it. Is there credible evidence that it works with no down-sides, or is it something they want to get us to take for another reason (to do with synthetic biology and gene-editing), or what is going on in this whole area, what is all that I don’t know to do with whatever is currently going on in the world with the entire planet being weaponized and it’s connection to our gut microbes?

I trust nothing until I look into it fully and understand it completely, but I don’t have time to fully look into this at the moment but wanted to keep a post to remind me to deep dive into it later.

Maybe there are good types and bad types, maybe it is useful and also being weaponized. I don’t know, but I think we should find out before we recommend it to people who are suffering, and yet, it’s probably urgent to find out sooner rather than later because if it’s actually an antidote, delaying could mean unnecessary death. I’m not intentionally trying to confuse anyone, I’m “confused” myself, so ignore my post if you stumbled upon it for anything definitive, this is literally just notes for my own record and my own curious and skeptical nature.

There is no rhyme nor reason for the references below other than “this caught my eye”.

Patents

  • Process for preparing natto kinase and its application
  • CN1273274A
  • 2000-05-22
  • Application filed by FUMAN BIO-TECHNOLOGY INST BEIJING
  • https://patents.google.com/patent/CN1273274A/en
  • A natto kinase is prepared from (Bacillus natto) X-501 (preserved in CGMCC no.0449) through culturing in soybean culture medium, and implementing the process of solid or liquid fermentation, extraction and purification according to a defined specification. After processed, said natto kinase can be used to make capsules, health-care food or its additive, and medical injection.

PubMed

  • Nattokinase: An Oral Antithrombotic Agent for the Prevention of Cardiovascular Disease
  • Weng Y, Yao J, Sparks S, Wang KY. Nattokinase: An Oral Antithrombotic Agent for the Prevention of Cardiovascular Disease. Int J Mol Sci. 2017 Feb 28;18(3):523. doi: 10.3390/ijms18030523. PMID: 28264497; PMCID: PMC5372539.
  • https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5372539/

  • Nattokinase decreases plasma levels of fibrinogen, factor VII, and factor VIII in human subjects
  • Hsia CH, Shen MC, Lin JS, Wen YK, Hwang KL, Cham TM, Yang NC. Nattokinase decreases plasma levels of fibrinogen, factor VII, and factor VIII in human subjects. Nutr Res. 2009 Mar;29(3):190-6. doi: 10.1016/j.nutres.2009.01.009. PMID: 19358933.
  • https://pubmed.ncbi.nlm.nih.gov/19358933/

Slideshows

  • Spore-Forming Gram-Positive Bacilli
  • https://slideplayer.com/slide/10847723/
  • Bacillus Species
    1. B anthracis, which causes anthrax, is the principal pathogen of the genus. It’s a major agent of bioterrorism and biologic warfare.
    2. Bacillus cereus causes food poisoning and occasionally eye or other localized infections.
    3. Most members of this genus are saprophytic organisms prevalent in soil, water, and air and on vegetation, such as Bacillus cereus and Bacillus subtilis. Such organisms may occasionally produce disease in immuno-compromised humans (e.g, meningitis, endocarditis).

Nattō Articles & Testimonials

  • What is B. subtilis Used For?
  • https://www.powerofprobiotics.com/Bacillus-subtilis.html
  • Bacillus subtilis strains, many of them genetically-engineered, are used in various applications. Some of these are:
    1. As a bacteriocide and fungicide sprayed on plants or seeds or mixed in the soil
    2. As an important source of industrial enzymes and polymers
    3. In the production of natto, a traditional Japanese dish of fermented soy beans
    4. In the production of medically important enzymes such as nattokinase or Douchi fibrinolytic enzyme (DFE) used to reduce blood clotting
    5. In animal feed, predominantly as a fattening agent
    6. As part of a genetically modified corn, Maize MON 87460, to increase the corn’s drought resistance
    7. As a probiotic
  • B. subtilis, a species with probiotic strains, food industry usage (including natto), GMO usage, and industrial applications as well as harmful toxin-producing strains and opportunistic-infection strains
  • https://www.powerofprobiotics.com/Bacillus.html

  • Nattokinase detoxifies covid spike proteins from the body
  • Thursday, February 23, 2023 by: Ethan Huff
  • https://www.naturalnews.com/2023-02-23-nattokinase-detoxifies-covid-spike-proteins-from-body.html
  • https://t.me/NaturalNewsMedia/3855
  • Dr. Peter McCullough @P_McCulloughMD, MD, MPH, has dropped another bombshell revelation, this time about how nattokinase, a natural enzyme found in fermented soybeans, is a powerful remedy against Wuhan coronavirus (Covid-19) spike proteins.
  • McCullough says he is asked all the time by people who got “vaccinated” for covid what they can do to “get this out of my body.” This inspired him to write a piece about nattokinase that calls the substance the “holy grail of covid-19 vaccine detoxification.”
  • McCullough highlights a paper published in the journal Molecules that unpacks the effects of nattokinase on the spike protein of SARS-CoV-2. In a cell lysate preparation that he says could be analogous to a jab recipient, researchers successfully demonstrated that spike proteins degraded in a time- and dose-dependent manner when exposed to nattokinase.
  • In a second experiment as part of the same study, researchers replicated the findings of a similar study published in 2021 in which nattokinase successfully degraded covid jab spike proteins in cells infected with SARS-CoV-2.
  • “Kurosawa and colleagues have shown in humans that after a single oral dose of 2000 FU D-dimer concentrations at 6, and 8?hours, and blood fibrin / fibrinogen degradation products at 4?hours after administration elevated significantly (p?<?0.05, respectively). Thus an empiric starting dose could be 2000 FU twice a day.”
  • Destroying spike protein! – COVID-19 update 60
  • https://youtu.be/3tqwqEqOBe4
  • Dr. Mikolaj Raszek
  • We introduce one potential natural way of destroying spike protein that has been proposed in a published paper: a suggestion that nattokinase which can be found in Japanese natto food can destroy the spike protein found on the surface of cells in a laboratory setting. Another added benefit is that nattokinase is apparently the best-known natural compound that prevents fibrin aggregation of clots.
  • Nattokinase + spike Study: https://www.mdpi.com/1420-3049/27/17/5405/htm

Bacillus subtilis Articles

  • Bacillus subtilis
  • by Rob Dunn
  • https://robdunnlab.com/projects/invisible-life/bacillus-subtilis/
  • Your body is covered with the species you can read about elsewhere in this book, species of Staphylococcus, Corynebacteria and many more. An inch of your skin might house hundreds of species of life. The total surface of your skin, or really anyone’s skin—from ears to anus and back out—might house thousands of species. A great number of these species eat the substances oozing out of our bodies. For example, several of the bacteria species that lives on our feet, including Bacillus subtilis, eat leucine, an amino acid common in the sweat on our feet. When these species eat leucine, they fart isoflavic acid which smells like stinky feet. The farts of B. subtilis are more awful smelling than those of other skin species, but Bacillus species, Staphylococcus species and a handful of others, when eating leucine, all stink. It is an unavoidable consequence and requires no special circumstances. All of this is known and contains, in and of itself, a hint of the idea I am about to offer. You now have the flint stones.
  • What if our hands and feet sweat more so as to feed their bacteria even more. What if those bacteria in turn help to ward off fungal infections in the very places—hands and feet—where we are most likely to be exposed to fungal or bacterial pathogens. What if our body has intentionally fortified us with microscopic forces precisely where such forces are most needed? One can find hints (or at least an excited idea maker can…) suggesting this could be right. Bacillus subtilis, the stinkiest species on our feet is known to produce antibiotics capable of killing foot fungus.
  • Meanwhile I can’t help wondering, if Bacillus subtilis, that common common bug, is not using its antibiotics to ward of fungi, who is it warding off? On each of us there are wars.
  • Thurston County Review Summary
  • Bacillus subtilis is a bacterium that can be found in water, air, soil. and decomposing plants. As well as its use for pesticides, it is also fermented for the production of proteases, amylases, antibiotics, and specialty chemicals. There are currently four strains of Bacillus subtilis that are registered by the EPA for pesticidal use. These strains include; MBI 600 – used on crops or seeds, QST 713 – used on crops and in residential gardens, FZB24 and GB03 – used on turf, ornamentals, shrubs, nursery stock, crops and other agricultural sites). Bacillus subtilis does not contain traits that cause disease and is not considered pathogenic to humans, animals, or plants, and is rated low in hazard by Thurston County’s pesticide review criteria
  • Bioaccumulation
  • Bacillus subtilis is a living bacteria that has no known pathological effects in animals and is eliminated within 14 days of administration.
  • Although bioaccumulation may not be the correct measure for Bacillus subtilis, accumulation from oral, inhalation, or dermal exposures is not likely to occur and cause toxic or pathogenic effects.
  • The EPA concluded that Bacillus subtilis may inhabit the skin and/or gastrointestinal tract for a short period of time, but is unlikely to colonize other places in the human body.
  • Acute Wildlife Toxicity Values and Risk Assessment
  • The EPA reports that there have been 17 cases of bovine mastisis where Bacillus subtilis was thought to be the causal agent, although, it was not noted if the exposure to Bacillus subtilis was from soil or introduced.
  • Acute Human Toxicity Risk Assessment
  • Bacillus subtilis is known to produce the toxic chemical “subtilisin” that is capable of causing allergic reactions. This potential allergic response is only expected from high concentration exposures within fermentation facilities and exposure to it is regulated by the Occupational Safety and Health Administration.
  • Risk to humans from potential exposures to Bacillus subtilis from pesticidal use is expected to be minimal due to its lack of acute oral toxicity/pathogenicity.
  • Potential worst-case inhalation exposures to workers within a fermentation facility are calculated to range from 650 to 1,200 cfu/day.
  • Rats were dosed up to 113,000,000 cfu/day without any adverse effects.
  • Chronic Human Toxicity Hazards
  • Due to lack of toxicity in acute toxicity testing, the EPA waived the requirements for carcinogenicity, immunotoxicity, reproductive fertility effects, and infectivity/pathogenicity testing.
  • There have been cases in which Bacillus subtilis was attributed to human infections, although the infections only occurred in people with compromised immune systems.
  • So, even in a fermentation facility, the potential for infection due to potential exposures to Bacillus subtilis is considered low by the EPA.

Britannica

  • Bacillus subtilis
  • https://www.britannica.com/science/bacillus-bacteria
  • In 1877 German botanist Ferdinand Cohn provided an authoritative description of two different forms of hay bacillus (now known as Bacillus subtilis): one that could be killed upon exposure to heat and one that was resistant to heat. He called the heat-resistant forms “spores” (endospores) and discovered that these dormant forms could be converted to a vegetative, or actively growing, state. Today it is known that all Bacillus species can form dormant spores under adverse environmental conditions. These endospores may remain viable for long periods of time. Endospores are resistant to heat, chemicals, and sunlight and are widely distributed in nature, primarily in soil, from which they invade dust particles.
  • Some types of Bacillus bacteria are harmful to humans, plants, or other organisms. For example, B. cereus sometimes causes spoilage in canned foods and food poisoning of short duration. B. subtilis is a common contaminant of laboratory cultures (it plagued Louis Pasteur in many of his experiments) and is often found on human skin. Most strains of Bacillus are not pathogenic for humans but may, as soil organisms, infect humans incidentally. A notable exception is B. anthracis, which causes anthrax in humans and domestic animals. B. thuringiensis produces a toxin (Bt toxin) that causes disease in insects.
  • Medically useful antibiotics are produced by B. subtilis (bacitracin). In addition, strains of B. amyloliquefaciens bacteria, which occur in association with certain plants, are known to synthesize several different antibiotic substances, including bacillaene, macrolactin, and difficidin. These substances serve to protect the host plant from infection by fungi or other bacteria and have been studied for their usefulness as biological pest-control agents.

Browsing ResearchGate

  • Bacillus subtilis, a Swiss Army Knife in Science and Biotechnology
  • May 2023
  • Journal of Bacteriology 205:e00102-23
  • DOI:10.1128/jb.00102-23
  • https://www.researchgate.net/publication/370516306_Bacillus_subtilis_a_Swiss_Army_Knife_in_Science_and_Biotechnology
  • Next to Escherichia coli, Bacillus subtilis is the most studied and best understood organism that also serves as a model for many important pathogens. Due to its ability to form heat-resistant spores that can germinate even after very long periods of time, B. subtilis has attracted much scientific interest. Another feature of B. subtilis is its genetic competence, a developmental state in which B. subtilis actively takes up exoge-nous DNA. This makes B. subtilis amenable to genetic manipulation and investigation. The bacterium was one of the first with a fully sequenced genome, and it has been subject to a wide variety of genome-and proteome-wide studies that give important insights into many aspects of the biology of B. subtilis. Due to its ability to secrete large amounts of proteins and to produce a wide range of commercially interesting compounds , B. subtilis has become a major workhorse in biotechnology. Here, we review the development of important aspects of the research on B. subtilis with a specific focus on its cell biology and biotechnological and practical applications from vitamin production to concrete healing. The intriguing complexity of the developmental programs of B. sub-tilis, paired with the availability of sophisticated tools for genetic manipulation, positions it at the leading edge for discovering new biological concepts and deepening our understanding of the organization of bacterial cells.

  • New CRISPR-Cas9 vectors for genetic modifications of Bacillus species
  • January 2019
  • FEMS Microbiology Letters 366(1)
  • DOI:10.1093/femsle/fny284
  • https://www.researchgate.net/publication/330655937_New_CRISPR-Cas9_vectors_for_genetic_modifications_of_Bacillus_species
  • Genetic manipulation is a fundamental procedure for the study of gene and operon functions and new characteristics acquisition. Modern CRISPR-Cas technology allows genome editing more precisely and increases the efficiency of transferring mutations in a variety of hard to manipulate organisms. Here, we describe new CRISPR-Cas vectors for genetic modifications in bacillary species. Our plasmids are single CRISPR-Cas plasmids comprising all components for genome editing and should be functional in a broad host range. They are highly efficient (up to 97%) and precise. The employment and delivery of these plasmids to bacillary strains can be easily achieved by conjugation from Escherichia coli. During our research we also demonstrated the absence of compatibility between CRISPR-Cas system and non-homologous end joining in Bacillus subtilis.

  • Bacillus subtilis Cell Factory
  • January 2023
  • DOI:10.1007/978-981-19-7911-8_8
  • https://www.researchgate.net/publication/367456184_Bacillus_subtilis_Cell_Factory
  • In book: Biomanufacturing for Sustainable Production of Biomolecules (pp.165-173)
  • Increasing demands for natural products from microbial origin have given much attention to researchers to exploit these microorganisms to produce a variety of natural products due to its versatile nature and ease in optimization processes at industrial scale. Presently, Bacillus subtilis has attracted lots of recognition by researchers for producing enzymes, terpenoids and other biomolecules due to its generally recognized as safe status. Extensive research work was carried out in Bacillus subtilis for more than the past five decades for the production of active biomolecules, which is considered as a representative cell factory in the Gram-positive bacterium. B. subtilis has the ability to secrete high-level of enzyme and production of other biomolecules. Therefore, Bacillus subtilis has emerged as an important platform for researchers into synthetic biology. A number of SSF and SmF approaches have been optimized with Bacillus subtilis for the bioprocessing of important biomolecules of industrial importance by using a variety of agro-industrial residues.

  • Susceptibility of Bacillus subtilis to Zinc Oxide Nanoparticles Treatment
  • January 2023
  • La Clinica terapeutica 174(1):61-66
  • DOI:10.7417/CT.2023.2498
  • https://www.researchgate.net/publication/367281730_Susceptibility_of_Bacillus_subtilis_to_Zinc_Oxide_Nanoparticles_Treatment
  • Aim: With the characteristics such as low toxicity, high total surface, ability to inhibit the growth of pathogenic microorganisms, zinc oxide nanoparticles (ZnO NPs), as one of the metallic nanoparticles, have been chosen as an antibacterial agent to treat various skin infections. The present study was aimed to determine the antibacterial potential of ZnO NPs on Bacillus subtilis, the Gram-positive bacterium that can cause skin and wound infections. Methods: B. subtilis was exposed to 5 to 150 μg/mL of ZnO NPs for 24 h. The parameters employed to evaluate the antimicrobial potential of ZnO NPs were the growth inhibitory effect on B. subtilis, the surface interaction of ZnO NPs on the bacterial cell wall, and also the morphological alterations in B. subtilis induced by ZnO NPs. Results: The results demonstrated a significant (p <0.05) inhibition of ZnO NPs on B. subtilis growth and it was in a dose-dependent manner for all the tested concentrations of ZnO NPs from 5 to 150 μg/mL at 24 h. Fourier transformed infrared (FTIR) spectrum confirmed the involvement of polysaccharides and polypeptides of bacterial cell wall in surface binding of ZnO NPs on bacteria. The scanning electron microscopy (SEM) was used to visualize the morphological changes B. subtilis illustrated several surface alterations such as distortion of cell membrane, roughening of cell surface, aggregation and bending of cells, as well as, the cell rupture upon interacting with ZnO NPs for 24 h. Conclusion: The results indicated the potential of ZnO NPs to be used as an antibacterial agent against B. subtilis. The findings of the present study might bring insights to incorporate ZnO NPs as an antibacterial agent in the topical applications against the infections caused by B. subtilis.

  • Genome Editing Methods for Bacillus subtilis
  • January 2022
  • Methods in molecular biology (Clifton, N.J.) 2479:159-174
  • DOI:10.1007/978-1-0716-2233-9_11
  • https://www.researchgate.net/publication/360679050_Genome_Editing_Methods_for_Bacillus_subtilis
  • In book: Recombineering (pp.159-174)
  • Bacillus subtilis is a widely studied Gram-positive bacterium that serves as an important model for understanding processes critical for several areas of biology including biotechnology and human health. B. subtilis has several advantages as a model organism: it is easily grown under laboratory conditions, it has a rapid doubling time, it is relatively inexpensive to maintain, and it is nonpathogenic. Over the last 50 years, advancements in genetic engineering have continued to make B. subtilis a genetic workhorse in scientific discovery. In this chapter, we describe methods for traditional gene disruptions, use of gene deletion libraries from the Bacillus Genetic Stock Center, allelic exchange, CRISPRi, and CRISPR/Cas9. Additionally, we provide general materials and equipment needed, strengths and limitations, time considerations, and troubleshooting notes to perform each method. Use of the methods outlined in this chapter will allow researchers to create gene insertions, deletions, substitutions, and RNA interference strains through a variety of methods custom to each application.

  • Bacillus subtilis WB800N alleviates diabetic wounds in mice by regulating gut microbiota homeostasis and TLR2
  • March 2022
  • Journal of Applied Microbiology 133(2)
  • DOI:10.1111/jam.15547
  • https://www.researchgate.net/publication/359456966_Bacillus_subtilis_WB800N_alleviates_diabetic_wounds_in_mice_by_regulating_gut_microbiota_homeostasis_and_TLR2
  • Objective: This study aims to investigate the effect of Bacillus subtilis WB800N on diabetic wounds. Methods: Hematoxylin & eosin (H&E) staining was used to observe the healing of skin wounds. Collagen deposition was assessed by Masson staining. Western blotting and qRT-PCR were used to detect vascular endothelial-related factors (VWF), CD31, TLR2, NLRP3, ASC, and Caspase-1 expression. 16S rDNA sequencing detected microbiota distribution. The concentrations of IL-1β and IL-37 were measured by ELISA. Apoptosis was measured by the TUNEL assay.
  • Results: Compared with the control group, the wound healing was delayed in diabetic mice. The wound area in the Bacillus subtilis group decreased more significantly than the diabetic wounds group. H&E staining showed that Bacillus subtilis WB800N promoted wound healing and increased re-epithelialization. Masson staining showed that Bacillus subtilis WB800N increased collagen deposition in diabetic wounds mice. Bacillus subtilis WB800N upregulated VWF and CD31 protein expression in diabetic wounds mice. The 16S rDNA results showed that Bacillus subtilis WB800N reduced the diversity of the gut microbiota of diabetic wounds mice and regulated the microbial composition. At the genus level, Bacillus subtilis WB800N reduced the relative abundance of Muribaculaceae and CG-005 in diabetic wounds mice, while increasing the relative abundance of Lactobacillus. Bacillus subtilis WB800N increased the expression of TLR2, NLRP3, ASC, and Caspase-1. Bacillus subtilis WB800N increased the concentrations of IL-1β and IL-37 in serum. Bacillus subtilis WB800N upregulated cell apoptosis. The TLR2 antagonist Sparstolonin B (SsnB) reduced the expression of TLR2, NLRP3, ASC, Caspase-1, IL-1β, and IL-37 and the apoptosis in diabetic wounds mice, while the combined intervention of Bacillus subtilis and SsnB reversed the effect of SsnB treatment alone.
  • Conclusion: Bacillus subtilis WB800N alleviated diabetic wounds healing by regulating gut microbiota homeostasis and TLR2. Significance and impact of research: Our findings might provide potential therapeutic targets for diabetic wounds.

  • Enhance endogenous Subtilisin Secretion in Bacillus subtilis by CRISPR-mediated engineering of Promoter and Signal Peptide
  • March 2023
  • Conference: 5th international and 17th Iranian Genetic Congress
  • https://www.researchgate.net/publication/370060560_Enhance_endogenous_Subtilisin_Secretion_in_Bacillus_subtilis_by_CRISPR-mediated_engineering_of_Promoter_and_Signal_Peptide
  • Proteases have widespread use in various industries, such as food, pharmaceutical, chemical, and agriculture. The ability to continuously produce and secretion of large and stable quantities of the desired recombinant products has made Bacillus subtilis a suitable and inexpensive source of industrial enzyme production. The major extracellular protease produced by B. subtilis is serine alkaline protease or subtilisin, AprE (39 kDa), a 381 amino acid residue containing a 29 amino acid signal peptide. Among the family of proteases, subtilisin (EC 3.4.21.62) has been widely used in detergent industries due to its stability in alkaline and high-temperature environments. The signal peptide is an essential element in the translocation of the AprE across the membrane through the Sec pathway and is removed by a type I signal peptidase. The mature protein then secretes to the culture medium, considerably simplifying product recovery and significantly reducing the subsequent purification and downstream processing steps. In this project, we aimed to enhance the production and secretion of endogenous AprE in Bacillus subtilis strain 168 by replacing its native promoter and signal peptide, respectively. Therefore, a single plasmid CRISPR-Cas9 system was constructed to edit the genome of B. subtilis. To build the editing template fragment, two PCR products from flanking region of AprE promoter fused to the amplified promoter of YlbP via overlap extension PCR. The primers were designed so that the final editing template included the new signal peptide of DacB, a new, improved ribosome binding site (RBS) fused to the YlbP promoter. DacB has a 27 amino acid residues signal peptide with an optimized start codon (ATG) that demonstrated the best heterologous subtilisin production performance in B. subtilis in previous studies. We expect that the transformation of the constructed plasmid by replacing the endogenous promoter and signal peptide of AprE will enhance the production of subtilisin.

Posts tagged: Bacillus subtilis | Synthetic Biology | Nattokinase

Penny (PennyButler.com)
Penny (PennyButler.com)

Truth-seeker, ever-questioning, ever-learning, ever-researching, ever delving further and deeper, ever trying to 'figure it out'. This site is a legacy of sorts, a place to collect thoughts, notes, book summaries, & random points of interests.

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