After one year, we continue to feed both the Seasons Biome blend and the Spirulina Boosted on a daily basis. Elaidic acid (C18:1T) is an unsaturated trans fatty acid. a, SDSPAGE analysis of purified aa682 incubated with simulated gastric fluid supplemented with 2,000Uml1 pepsin. J. It is an omega-9 fatty acid because it has a final carbon double bond in the ninth position. PubMed Structure of smAKAP and its regulation by PKA-mediated phosphorylation. After about 8 months, June Bug's sarcoids were eradicated and she is now "healthy as a horse." Filtrated fragments were then digested in RPMI 5% FBS, 1mgml1 collagenase typeII and 40gml1 DNaseI for 40min. Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Front. 7), the amount of soluble aa682 was ~3% of total biomass. CAS Treuchtlingen is situated on the river Altmhl, 9 km southwest of Weienburg in Bayern, and 45 km northeast of Donauwrth . Rev. Specific ligand binding activity was determined on an Octet Red96e biolayer interferometry instrument (Forte Bio) using recombinant, biotinylated C. jejuni FlaA protein attached to streptavidin-coated biosensors. Groups of mice were treated with either PBS (eight mice), SP257 biomass (four mice) or SP526 biomass (four mice). adroll_currency = "USD"; High-binding, 96-well plates (Greiner Bio-one or NUNC MaxiSorp) were coated with antigen by the addition of 100l of 15gml1 recombinant protein (FlaA or RBD antigen) in carbonate-bicarbonate buffer (Sigma) to each well and incubation overnight at 4C. Spirulina Chia from Stable Feed is a powdered supplement for horses. J.X. Enteric infectious diseases are designated as high-priority antimicrobial resistance threats by the Centers for Disease Control and Prevention, adding urgency to the search for new therapeutic tools. Construction of biologically functional bacterial plasmids in vitro. Alpha-Linolenic acid (C18:3) is a polyunsaturated essential fatty acid. J. Purified aa682 binding to recombinant FlaA was used to generate a standard curve by linear regression. Biol. The phagemid library was cleaned up with QiaPrep Spin Minikit columns and transformed into electrocompetent TG1 cells (Lucigen). Food Sci. All structures generated using Pymol (Schrodinger). B.F. wrote the manuscript. Two independent mouse models were used to test the efficacy of spirulina-expressed VHHs in the treatment of C. jejuni infection. Struct. Described here are spirulina strains with constitutive VHH expression levels of 0.7, 3.4, 4.0, 4.3, 9.3 and 29% of soluble protein (Fig. Animal experiments at the University of Virginia were performed according to institutional review board (IRB) protocols. FEBS J. Spirulina strain SP79 was genotyped after continuous culture for >3years. Culture volumes ranged from 3 to 100ml.
Cyanobacteria produce high levels of ergothioneine - ScienceDirect A 1-l sample of the supernatant was used per genotyping PCR reaction. The binding activity of aa682 extracted under duodenal conditions was minimally affected by previous incubation of the biomass under the gastric environment (Fig. A 50-l sample of 1mgml1 aa682 was reduced and denatured with 6l of 0.25M DTT and 10l of 6M guanidinium HCL for 30min at 37C in the dark. Pentavalent single-domain antibodies reduce Campylobacter jejuni motility and colonization in chickens. The present study aimed to characterize the nutraceutical properties and the antimicrobial effect of Moroccan <i>Spirulina</i> (<i>Arthrospira platensis</i>). PubMed Central Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. a, Cartoons of multimeric scaffolds with sample expression data for VHHs in spirulina. Data analysis was performed using Prism (GraphPad Software). PCR from genomic DNA was performed with primers targeting the full transgene region (MP5+MP6). Stearic acid (C18:0) is a saturated long-chain fatty acid composed of an 18-carbon chain with no double bonds. Genotyping was performed once. Synthetic single domain antibodies for the conformational trapping of membrane proteins. Transition of biomass to the higher pH of a simulated duodenal environment was sufficient to extract >90% of encapsulated aa682 from spirulina within 60min (Fig. A sample of spray-dried SP1182 biomass was resuspended in PBS at 30mgml1. Total fatty acids is a better measure of the total amount of fat present in a feed sample. Cocktails comprised of ten or more therapeutic proteins are in development, whereas therapeutics comprising more than two biologics are generally impractical using conventional platforms due to the complexity of cell line development, manufacturing costs and compounding toxicities of systemic therapeutics. The following equine feeds, supplements and products contain Spirulina as an ingredient.
Development of spirulina for the manufacture and oral delivery of 2b). PubMed Plates were washed three times with 300l of PBS supplemented with 0.05% Tween-20 (PBS-T). J.R. directed overall research and wrote the manuscript. 2 were here. This supplement is intended to promote the healing of skin issues in horses. were measured for triplicate samples at either 40h (81176) or 66h (CG8421) after plating. Bars indicate meanSD. SP1313 lysate was run in reducing conditions with DTT for 6 independent experiments with 3 lysates assessed in each run, and in non-reducing conditions for 5 independent experiments with 2 lysates assessed in each run. e, Strategy of markerless transgene integration (see main text for details). Koromyslova, A. D. & Hansman, G. S. Nanobodies targeting norovirus capsid reveal functional epitopes and potential mechanisms of neutralization. In brief, dried biomass samples were diluted to a concentration of 0.2mgml1 using water and a 5master mix prepared from an EZ Standard Pack1, in either reducing or nonreducing format (Bio-Techne). The single polypeptide also contains the solubility enhancer MBP and C-terminal 6X-his affinity tag. How. Dried spirulina biomass was resuspended in PBS containing Pierce Protease Inhibitor minitablets and 1mM PMSF at a biomass concentration of 1040mgml1 in 1.7-ml Eppendorf tubes. Our Spirulina Chia supports and promotes the healing of: Roughly 30% of horseswon't eatthe Spirulina Biscuit, so we recommend starting with the loose variety. Pellets were resuspended in 1ml of 50mM bicarbonate buffer to a final biomass concentration of 30mgml1 and incubated in a water bath at 37C for 30min. Plasmid DNA was purified from E. coli by the QIAprep Spin Miniprep Kit (Qiagen) and transformed into spirulina strain SP205. Syndr. Global burden of diarrheal diseases among children in developing countries: incidence, etiology, and insights from new molecular diagnostic techniques. 3 Expression of representative transgenic proteins in spirulina. This resuspension was diluted 1:30 with prechilled SGF (50mM citrate-phosphate buffer pH3.0, 94mM NaCl, 13mM KCl pH 3.5 with 2,000Uml1 pepsin (MP Biomedicals)) and incubated in a water bath at 37C. The resuspension was passed through a French pressure cell twice to lyse the cells. Adverse events were grade1, were deemed unlikely to be related to the therapeutic and were similar in frequency between experimental and placebo groups (Supplementary Table 3). Phycocyanin, a natural blue pigment protein present in wild type spirulina, was overexpressed with a transgenic copy of the native gene. Whole biomass samples were denatured and analyzed on a Jess system. Present address: Merck & Co., West Point, PA, USA, Present address: Sandia National Laboratories, Livermore, CA, USA, Present address: TScan Therapeutics, Waltham, MA, USA, Benjamin W. Jester,Hui Zhao,Mesfin Gewe,Thomas Adame,Jan Agosti,Nhi Khuong,Rolf Kuestner,Caitlin Gamble,Kendra Cruickshank,Jeremy Ferrara,Rachelle Lim,Troy Paddock,Colin Brady,Stacey Ertel,Miaohua Zhang,Alex Pollock,Jamie Lee,Michael Tasch,Tracy Saveria,David Doughty,Jacob Marshall,Damian Carrieri,Lauren Goetsch,Jason Dang,Nathaniel Sanjaya,David Fletcher,Anissa Martinez,Bryce Kadis,Kristjan Sigmar,Esha Afreen,Tammy Nguyen,Amanda Randolph,Alexandria Taber,Ashley Krzeszowski,Brittney Robinett,Ryo Takeuchi,Brian Finrow,Craig Behnke&James Roberts, Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Universit della Svizzera Italiana, Bellinzona, Switzerland, Division of Infectious Diseases & International Health, University of Virginia School of Medicine, Charlottesville, VA, USA, Department of Pharmaceutical Chemistry, Vaccine Analytics and Formulation Center, University of Kansas, Lawrence, KS, USA, You can also search for this author in A sliding window of 30amino acid fragments, with a two-amino-acid interval along the length of FlaA, was designed as oligos for cloning into the phagemid pADL-23c (Antibody Design Labs). Your input is very much appreciated. Rev. Both horses received one pound of Season feed per day and one scoop of the Spirulina Boosted. The study was performed in accordance with ICH guidelines and in compliance with all local and international requirements. 1c). Curve fits were performed using a global fit across all concentrations of aa682 assuming a 1:1 binding model.
Physico-chemical and nutritional properties of meat - Springer The relative copy number of the integrated transgene was calculated as the fold difference between transgene and endogenous genes using the Ct method. Improving containment strategies in biopharming. These are often reasons for clinical failure and a major driver of the high cost of new drug development48. 2a and Extended Data Fig. Spirulina strain SP526 expressed a monovalent fusion of FlagV6 VHH and MBP, which bound to recombinant FlaA with a KD of 53nM and was constitutively expressed in the cell cytoplasm at 3% of dry biomass (equivalent to 8% of soluble protein) (Fig. d. CEIA of spirulina expressing a single-domain antibody, an anti-SARS-CoV-2 VHH fused to a self-dimerizing scaffold (SP1741); VHH represents 9.3% of cell protein. The promoterterminator pair consisted of a constitutively active, native A. platensis promoter (600bp upstream of the cpcB gene, named Pcpc600) and the terminator of the E. coli ribosomal RNA gene rrnB (named TrrnB). 3e). Uninfected mice were treated with vehicle (n=4). Sign up for the Nature Briefing: Translational Research newsletter top stories in biotechnology, drug discovery and pharma. VHHs and other biologics in dry spirulina powder are shelf stable without refrigeration, facilitating distribution, especially into regions lacking high-quality infrastructure. Super Horses Need SuperFood! Summary Spirulina (Athrospira sp.) Supplementary Table 2: Characterized spirulina strains. It is calculated by subtracting plant pigments, esters, and aldehydes from crude fat. Cell 181, 14361441 (2020). Development of spirulina for the manufacture and oral delivery of protein therapeutics. B., Ewing, C., Poly, F., Wang, F. & Egelman, E. H. Atomic structure of the Campylobacter jejuni flagellar filament reveals how Proteobacteria escaped Toll-like receptor 5 surveillance. K.C., R.L., D.C., N.S., E.A., T.N., A.R., A.T., A.K. We demonstrated stable intracellular expression of diverse exogenous proteins, including bioactive peptides, antigen-binding domains (VHHs), protein pigments and enzymes (Extended Data Fig. Spirulina is a dietary supplement made from blue-green algae (cyanobacteria: Arthrospira platensis) that is used both as a food and as a medicinal supplement to counteract unintentional weight loss and to ameliorate a variety of medical conditions including attention-deficit hyperactivity disorder, allergic rhinitis, hypertension, diabetes, stress, fatigue, anxiety and depression. Sci. Diabetes Metab. J. Gastroenterol. Gastroenterology 75, 10831089 (1978). There were no major adverse events or laboratory abnormalities reported during or following the trial. Biochem. Article 21, 570578 (2003). Genomic DNA from spirulina strains SP205 (parental), SP985 (Pcpc600-driven expression of anti-C. difficile VHH 5D), and SP1182 (Pcpc600-driven expression of aa682) were used as templates. One insertion site corresponded to NCBI reference sequence NIES39_RS07765, which codes for a protein homologous to kanamycin aminoglycoside acetyltransferases (hereafter referred to as KmR). In contrast, prophylaxis using the spirulina platform could be affordable, even in the developing world. Samples B and C yielded approximate EC50 binding values of 85.8 and 29.2gml1 biomass, respectively. b, CEIA quantification of aa682 expressed in SP1182. Sequencing reads were filtered for quality and analyzed to minimize false positives. Annu. Yang, B., Liu, J., Jiang, Y. A. et al. J. Biotinylated MBPFlaA protein was prepared using an EZ-Link NGS-PEG4-Biotin kit (Thermo Scientific) following the manufacturers guidelines. 211, 13341341 (2015). Competent spirulina (UTEX LB1926 and NIES-39) were exposed in liquid culture to an integrating DNA vector containing a selectable marker and a gene of interest, flanked on both sides by sequences homologous to the spirulina chromosome. Natural competence and the evolution of DNA uptake specificity. Its great for managing fungus and helping their coat truly develop in a natural way! CAS 308, 116 (2005). Cost per unit biomass includes labor, capitalized cost of operating lighting system (varies by light intensity), and capitalized costs of other upstream components (independent of light intensity). The flash-freeze procedure was repeated an additional two times. Adoption of new expression platforms depends on the availability of methods for genetic manipulation of the organism to achieve stable, high expression of exogenous proteins, and on whether the organism possesses biological traits compatible with large-scale manufacturing and commercialization. Source data for the findings of this study will be made available on FigShare. With minor optimization of throughput, inlet and outlet temperatures and air flow rates, equivalent dryer performance at a ~20, single-step scale-up was achieved. Cohen, S. N., Chang, A. C., Boyer, H. W. & Helling, R. B. The library was transformed into strain SP3 in triplicate, following the transformation method described above, and cultured with streptomycin. To prepare for spray-drying, the harvested biomass was rinsed with a dilute (0.1%) trehalose solution to remove excess media salts, concentrated again by filtration and then spray-dried in a centrifugal nozzle spray-dryer. Burgers, P. P. et al. Some sensitivity of aa682 to chymotrypsin, but not trypsin, was evident when it was exposed to the higher protease levels that are present immediately after a meal33 (Fig. CAS
Alfred Haase | Historica Wiki | Fandom Spirulina: Dosage, eye health, oral health, and more - Medical News Today Spirulina - LiverTox - NCBI Bookshelf - National Center for After loading, probes were allowed to reach a baseline equilibrium in kinetics buffer (PBS with 1% bovine serum albumin and 0.05% Tween-20) for 2min. Hyperimmune bovine colostrum as a novel therapy to combat Clostridium difficile infection. A single 400-l dose of spray-dried spirulina resuspended in PBS was administered by oral gavage to mice 1.5h before infection with C. jejuni 81176 (108CFU200l1 PBS). Genomic DNA was extracted from bacterial samples following the extraction method described above. The amino sequence of aa682 was confirmed by proteolyzed peptide fragment analysis, with 98% coverage of the expected protein sequence (Extended Data Fig. Taton, A. et al. The difference in mass would be consistent with loss of the N-terminal methionine. Peroxidase activity was quenched after 510min with 50l of either 1M HCl or 2M sulfuric acid. Binding activity of the extracts to FlaA was measured by ELISA. PCR product libraries were subcloned and sequenced. 3b,c). She is constantly battling scratches, even on stall rest. 217, 111 (2016). Kreutzberger, M. A. To obtain Each point represents a different batch of biomass, presented as the mean of two technical replicates. "To say my horse has been doing and looking incredible on StableFeed's Spirulina Chia is an absolute understatement! b, VHH binding activity of SP526 stored at a range of temperatures over 6months. We introduced a variety of exogenous DNA vectors into the spirulina genome, including single genes, tandem genes, operons and sequential engineering of different insertion sites (Supplementary Table 2). R.T. contributed foundational research. This destination vector included integrating homology arms for the kmR locus flanking an ORF for the native kmR gene and a terminator. Monomeric (MBP and thioredoxin (TRX)), dimeric (5HVZ), trimeric (cTRP) and heptameric (4B0F) scaffolding proteins were used to multimerize VHHs expressed in spirulina. Nucleic Acids Res. Wash was discarded, 100l of primary antibody diluted with PBS-TM was added to each well and plates were incubated at room temperature for 1h. A 1:10,000 dilution of either a mouse anti-His-tag antibody (GenScript) or rabbit anti-camelid VHH antibody cocktail (GenScript) was used as the primary antibody. While these studies established that orally delivered antibodies can be effective, commercial development has suffered from reliance on production systemssuch as the milk of hyperimmunized cowsthat have proven difficult to scale cost effectively while maintaining FDA-grade lot-to-lot consistency. Uniform expression of GFP in a population of spirulina transformed with a transgene (SP699). 5 Effect of MBP fusion on VHH expression in spirulina. On day0, mice were given an inoculum of 106live C. jejuni cells (strain 81176, resuspended in PBS) by oral gavage. The pellet was resuspended in a total volume of 35ml with lysis buffer (50mM sodium phosphate buffer pH8.0, 500mM NaCl, 20mM imidazole) supplemented with Pierce Protease Inhibitor Minitablets (Thermo Scientific) and 1mM phenylmethylsulfonyl fluoride (PMSF). Unless noted otherwise, experiments were performed once. J. Virol. Signup today to be sentnutrition tips, new product alerts, and special discounts! All the CEIA analysis was performed performed once for each strain. 6a). Moreover, Spirulina shows promising antioxidant and anti-inflammatory properties supporting general equine health as well as increased muscle recovery. Manuka Honey also supports proper immune responses but is best known for its antimicrobial and antibacterial properties. Glycerol stocks of C. jejuni were first streaked on Campy Blood Agar Blaser plates (Thermo Scientific) and grown for 48h. Bacteria were then used to inoculate 0.4% soft agar MuellerHinton (MH) plates by stab and incubated for 48h. A slice of agar from the leading edge of motility halos was used to inoculate 10ml of MH broth. To exclude the possibility of nonspecific integration of the vector backbone DNA, PCR was performed with primer pairs targeting the ampicillin resistance gene and E. coli origin of replication. Cells were pelleted from 5-ml cultures by centrifugation for 10min at 1,600RCF. Use of quantitative molecular diagnostic methods to identify causes of diarrhoea in children: a reanalysis of the GEMS case-control study. Food and water were available ad libitum and mice were monitored daily. A phase1 clinical trial in healthy volunteers aged 1850years assessed the safety and tolerability of LMN-101. Multimers routinely demonstrated subnanomolar apparent KD levels (Fig. PubMed Therefore, the subset of therapeutic proteins requiring these for activity would be excluded. d. Ribbon representation of heptameric VHH (orange) with the heptamerization motif (blue; PDB ID: 4B0F)58. Single-cell suspensions from cecal lamina propria were stained with labeled antibodies diluted in PBS with 2% FBS for 20min on ice. A chronic cough is no joke. Expression of VHHs as high-avidity multimers could bypass a need for affinity maturation and may accelerate product development. Huang, H., Liao, D., Pu, R. & Cui, Y. Quantifying the effects of spirulina supplementation on plasma lipid and glucose concentrations, body weight, and blood pressure. Biotechnol. E. coli-expressed and purified monomeric (PP622) and dimeric (PP661) forms of RBD-binding VHH were assayed with RBD and compared with the binding activity of identical proteins present in spirulina extracts (SP1464 and SP1477, respectively). Conditions were identified (Extended Data Fig. 4a,b). Weight measurements were made daily for 7days. The biomass was first incubated (A), brought to intestinal pH (pH>5.0) (B) then compared with aa682 extraction by direct biomass resuspension in bicarbonate buffer (pH>7.0) (C). Animals were fed a regional basic diet for 10days, followed by 3days of antibiotic treatment. Information on 30spirulina strains referenced in this study is provided, including numbers of ORFs, introduced protein constructs, integration sites and antibiotic markers. Mice were given three doses of spirulina before and after challenge. Extended Data Fig. Savarino, S. J. et al. Development of spirulina for the manufacture and oral delivery of protein therapeutics, https://doi.org/10.1038/s41587-022-01249-7. Result is representative of dozens of independent experiments. 3a). The standard curve was used to calculate the concentration of aa682 in SP1182 lysates. Mathers, C. et al. Thank you, Natalie! Spirulina ameliorates aspirin-induced gastric ulcer in albino mice by alleviating oxidative stress and inflammation, Antibacterial phenolic components of New Zealand manuka honey, Antioxidant, Immunomodulating, and Microbial-Modulating Activities of the Sustainable and Ecofriendly Spirulina, Antioxidant, Immunomodulating, and Microbial-Modulating Activities of the Sustainable and Ecofriendly, Potential health benefits of spirulina microalgae*. Spirulina hold FDA Generally Recognized as Safe status44. DNA fragments were gel extracted and characterized by Sanger sequencing. High levels of ET (up to 0.8 mg per g dry mass) were measured in cyanobacteria preparations sold as dietary supplements for humans and in fresh Scytonema and Oscillatoria cultures. Eligible, healthy volunteers aged 1850years were enrolled following informed consent. A phase 1 clinical trial was designed and conducted to assess the safety and tolerability of LMN-101. To establish axenic spirulina strains, cells were washed with SOT medium on 10-m filters to exclude small, unicellular bacteria, and single filaments were isolated from cells captured on the filter. Nat. VHH volumetric productivity in spirulina is approximately 2550 versus 500mg antibodyl1d1 in CHO cells42. Dis. Phage from an overnight culture in 2YT medium was precipitated and washed following the manufacturers protocol. 58, 822830 (2017). We thank C. Shoemaker, B. Stoddard, G. McDonald, L. Tonkovich, M. Spigarelli, M. Heinnickel, C. Sherr, F. Cross, B. Kerwin, K. Stein, W. Chen, D. Wattendorf and O. Vandal for help, discussions and advice. Methylglyoxal (MGO), the major antibacterial component of manuka honey, has also been identified as efficacious in protecting the mucosal lining of the intestinal wall. f. CEIA of spirulina expressing an enzyme, the catalytic domain from a C. difficile-specific, bacteriophage-derived endolysin (SP1287); enzyme represents 0.54% of cell protein. adroll_pix_id = "MSO5WTCZ6BHRXIKYE7AF4Z"; Bunnak, P., Allmendinger, R., Ramasamy, S. V., Lettieri, P. & Titchener-Hooker, N. J. Life-cycle and cost of goods assessment of fed-batch and perfusion-based manufacturing processes for mAbs. a. Prophylactic efficacy of hyperimmune bovine colostral antiadhesin antibodies against enterotoxigenic Escherichia coli diarrhea: a randomized, double-blind, placebo-controlled, Phase 1 trial. A Correction to this paper has been published: https://doi.org/10.1038/s41587-022-01323-0. At no point were these fragments observed in spirulina, suggesting that there is no integration of the vector outside of the homology arms. Untreated water was provided for 1day before C. jejuni challenge. Absorbance spectra for wild-type (black) and transgenic (red) spirulina are shown. The phage library was diluted with PBS-TM to a concentration of 1012phagesml1 and incubated at room temperature for 30min. This process was repeated until the supernatant was clear. Silver, Lukas Ker, Maximilian Rotter, Peter Czermak, Neil D. Butler, Sabyasachi Sen, Aditya M. Kunjapur, Joseph Kreitz, Mirco J. Friedrich, Feng Zhang, Jae Sung Cho, Dongsoo Yang, Sang Yup Lee, Young Hun Chung, Derek Church, Nicole F. Steinmetz, Joachim J.
Spirulina Chia (Stable Feed) - Equine Nutrition Analysis | Feed Bank Steele, J., Sponseller, J., Schmidt, D., Cohen, O. C. Behnke designed experiments and wrote the manuscript. The elution buffer was neutralized with 40l of 2M Tris pH7.5 and used to reinfect phage-competent TG1 cells (Antibody Design Labs).
PDF Inclusion of Spirulina in Floating Fish Feed Production: Protein The supernatant was transferred to fresh tubes and stored at 4C. Samples were kept on ice throughout. A week later I was able to soften the scabs with Vaseline and gently wash the area. Resuspensions were transferred to a water bath at 37C for 210min. Cultures were inoculated at a concentration of 0.10.5gl1 biomass by ash-free dry weight, and harvested by filtration at 24gl1. 6 CEIA analysis of the oxidation state of disulfides in dimeric proteins expressed in spirulina strain SP1313. Low productivity and potency in plants not only amplify production costs but may also necessitate expensive downstream processing, which is reported to be a weakness of plant-based production systems11. Crypto Aero Whole Food Horse Feed. J. Pharm. 11, 729742 (2018). Well-separated single filaments were picked with a 1-ml pipette tip and transferred in 3ml of SOT with appropriate antibiotics in a round-bottom tube. b, CEIA of pepsin-digested spirulina biomass resuspension. Engineering and functionalization of large circular tandem repeat protein nanoparticles. Stool samples were collected on days1, 3 and 7 post challenge. Llama-derived single-chain antibody fragments directed to rotavirus VP6 protein possess broad neutralizing activity in vitro and confer protection against diarrhea in mice. 3e) and was therefore predicted to inhibit pathogenesis in vivo. Once collected from the hydrocyclone, the powder was sealed and stored in airtight, opaque mylar bags to prevent exposure to moisture or light. It conferred kanamycin resistance in E. coli, and its deletion rendered spirulina kanamycin sensitive. Obes. Segregation of the chromosome was assessed using a primer pair annealing to regions within the RHA and LHA. The vector backbone sequences outside of the homology arms should not integrate into the genome and thus should be absent from spirulina genomic DNA. Recombinant protein expression in spirulina was measured by capillary electrophoresis immunoassay (CEIA) using a Jess system (ProteinSimple), which was run as recommended by the manufacturer. In brief, a 10-ml pellet of spirulina cells from strain SP1182 was collected from 2l of culture by centrifugation. We were also struggling to keep weight on him through the winter, even though we were feeding him copious amounts of senior feed with a fat supplement (in addition to hay). Oleic acid (C18:1C) is a long-chain mono-unsaturated fatty acid. It is an omega-6 fatty acid because it has a final carbon double bond in the sixth position. Klindworth, A. et al. The VHH Nano26 was expressed with no fusion (SP1772) or MBP fused to the C-terminus (SP1773). The VHH FlagV6 binds to flagellin (FlaA), a subunit of C. jejuni flagella26. However, when delivered within dry spirulina biomass, >70% of aa682 remained intact after 2h of incubation under gastric conditions (Fig. Especially for oral delivery, the simplification of both equipment and upstream and downstream processes can more than offset the benefit apparent from a comparison of volumetric productivities. Markers for antibiotic resistance in spirulina were cloned between the two homology arms of the plasmid. Supplementary Table 4: Oligos used in this study. Culturing, induction and lysis of E. coli expressing VHHs followed the same protocol as for FlaA expression. Cell 11, 223232 (1977). Sasou, A. et al. It has a chemical structure of 14 carbon atoms with no double bonds. Using a standard curve of purified protein (Extended Data Fig.
My upper level jumper is basically allergic to life. Then, the microbiological analysis . J.M. The theoretical mass was 54913Da, with a measured mass of 54783Da.
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