28 results about "Antibiotic free" patented technology

The invention discloses a mycobacteriophage and application thereof, and relates to the technical field of bacteriophages. The mycobacteriophage is named as Mycobacter phage BZNK001, is preserved in Guangdong Microbial Culture Collection Center, has a preservation number of GDMCC No: 67907-B1, does not contain antibiotic drug-resistant genes and integrase genes, has excellent biological safety and good environmental tolerance, can maintain high biological activity in a temperature range of 37-50 DEG C under an acid-base condition of pH 5-9, and can be used for preparing the antibiotic drug-resistant gene and integrase gene-containing mycobacteriophage for preventing and treating the antibiotic drug-resistant gene and integrase gene-containing mycobacteriophage for preventing and treating the antibiotic drug-resistant gene and integrase gene-containing mycobacteriophage. The bacillus subtilis has broad-spectrum and high-efficiency non-tuberculous mycobacterium cracking activity, and can effectively crack four clinical key mycobacteria such as mycobacterium abscessus, mycobacterium avium, mycobacterium intracellularis and mycobacterium smegmatis. According to the invention, a mycobacteriophage resource library is enriched, a novel and stable biological prevention and control means is provided for efficient prevention and control of mycobacteria, and the mycobacteriophage has good clinical application and industrialization values.

The invention relates to the technical field of synthesis of natural dyes by a biological method, in particular to an antibiotic-demand-free ornamental blue high-yield strain as well as construction and application of the antibiotic-demand-free ornamental blue high-yield strain. Corynebacterium glutamicum is used as an initial strain, a tricarboxylic acid circulating gene is regulated and interfered, and the copy number of exogenous blue pigment synthetase BpsA in a genome is adjusted, so that the ornamental blue high-yield strain is obtained. The strain does not need to use antibiotics in the fermentation process, the yield is stable, the product production capacity equivalent to the level when antibiotics are used can still be achieved through fermentation under the antibiotic-free condition, and the growth performance of the strain is kept. The method for producing the ornamental blue by using the strain provided by the invention ensures that the ornamental blue product is natural and green, also improves the efficient production capacity of the strain, solves the contradiction between green environmental protection and stable high yield of the ornamental blue dye, is green and environment-friendly, and has a remarkable industrial application prospect.

The present application belongs to the field of synthetic biology and functional food technology, and provides a swallowing safety-oriented xanthan gum directional biosynthesis method. In view of the problem that existing xanthan gum is difficult to reduce the risk of swallowing residue and aspiration by relying on viscosity regulation alone, a multi-dimensional evaluation system combining interfacial tribology and extensional rheology is established to determine the side chain modification parameters suitable for swallowing disorder food. By chromosome scarless reconstruction of xanthan gum synthesis gene cluster, the gumF 、 gumG gene is knocked out and the gumL promoter is replaced, an engineering strain without antibiotic resistance gene residue and with genetic stability is constructed, and directional synthesis of xanthan gum with specific side chain structure is realized. The obtained structure customized xanthan gum has a lower mucosal interfacial friction coefficient and a more optimal extensional rheological property, and can synergistically realize interfacial lubrication and bolus cohesion, significantly reducing the wall residue in the pharynx, and keeping the consistency stable in complex food matrix. The present application can provide a safe, stable and industrialized functional xanthan gum raw material for swallowing disorder food.

The application discloses application of eugenol in preparation of a bacteriostatic agent for enhancing symbiotic nitrogen fixation efficiency of legume-rhizobium, and relates to the field of agricultural microorganisms. ‑1 The application is safe and non-inhibiting to seed germination of Medicago polymorpha and target rhizobium LL2 in a concentration range of 500-1000 muL / L ‑1 of eugenol, can significantly reshape rhizosphere microbial community structure by selectively inhibiting competitive microorganisms such as bacillus and streptomyces in soil, and is environment-friendly and free of antibiotic residue risk, thereby providing a green and efficient new approach for improving symbiotic nitrogen fixation efficiency of legume-rhizobium.

The invention discloses an industrial aquaculture management system for antibiotic-free bullfrogs, and belongs to the technical field of aquaculture. The system comprises circulating water antibiotic-free treatment, antibiotic-free seedling cultivation, antibiotic-free cultivation management and a novel drainage vertical pipe system. The circulating water non-antibiotic treatment adopts a closed-loop flow, and antibiotics and ammonia nitrogen are efficiently removed through the working procedures of sewage tank microfiltration, ozone oxidation disinfection, constructed wetland biological photodegradation, aeration tank ammonia nitrogen removal, clean water tank plant bacterium adding purification reflux and the like; a staged plant nutrition and probiotic feeding mode is adopted for antibiotic-free seedling cultivation; the antibiotic-free cultivation management realizes antibiotic-free cultivation through density control, water quality and water temperature regulation and control, scientific feeding and animal health care; the novel drainage vertical pipe adopts a circular hole and vertical seam structure to prevent blocking. According to the method, whole-course antibiotic-free breeding is achieved, the survival rate in the seedling stage reaches 80% or above, the adult frog breeding period is about 120 days, the product is free of antibiotic residues, the national environmental protection and food safety specifications are met, and an efficient solution is provided for factory-like antibiotic-free bullfrog breeding.

The invention belongs to the technical field of biology, and particularly relates to a recombinant strain of high-yield riemerella anatipestifer bacteriophage lyase, a mutant strain, a construction method and application. The invention provides a coding gene of riemerella anatipestifer bacteriophage lyase, and further constructs recombinant lactococcus lactis of high-yield riemerella anatipestifer bacteriophage lyase. The mutant strain provided by the invention is obtained by taking the recombinant lactococcus lactis as a starting strain through a mutation breeding technology and a specific high-throughput screening strategy, has higher secretory expression quantity of riemerella anatipestifer bacteriophage lyase, is good in hereditary stability in an antibiotic-free culture medium, and has a good application prospect. The method is suitable for industrial efficient production of active lyase. The riemerella anatipestifer bacteriophage lyase expressed by the mutant strain has an obvious splitting effect on riemerella anatipestifer in vitro, has obvious prevention and treatment effects on riemerella anatipestifer infection in vivo, and can effectively improve the survival rate of infected chicks and remove pathogenic bacteria in vivo.

The invention discloses an imidazolyl lipid assembly as well as a preparation method and application thereof, and relates to the technical field of biomedical materials. The preparation method comprises the following steps: mixing diphenyl phosphate, alcohol and pyridine, and heating for reaction to obtain alkyl phosphate; mixing N, N-diethylethylenediamine, 5-imidazole formaldehyde and a solvent, carrying out a heating reaction in a protective atmosphere, then adding the alkyl phosphate, and continuing the heating reaction to obtain an imidazolyl lipid molecule; dissolving the imidazolyl lipid molecules in an organic solvent to obtain a mother solution; the imidazolyl lipid assembly is obtained from the mother liquor through a film hydration method. The imidazole-based lipid assembly constructed by the invention can utilize the imidazole group to destroy the normal structure of bacteria under the condition of no antibiotic load, and utilizes the catalytic action of the imidazole group to complete the destroy of a biological membrane matrix and an inflammation activation medium, so that the synergism of antibiosis and anti-inflammation is realized, and the imidazole-based lipid assembly has the effect of buffering local acidic pH.

The invention relates to a quick starting method of a biological filter bed based on fungus-bacterium coordinated regulation. The quick starting method adopts a two-step method and comprises the following steps: firstly, quickly starting a bacterium filter bed by taking benzyl alcohol as a carbon source; adjusting the pH value to 5.5-6.2, adding wheat bran, and implementing intermittent hypoxia stimulation (replacing antibiotics) until the absolute copy number ratio of fungi / bacteria is (F / B) gt; and the starting is finished when the temperature is 0.35. The whole process is free of antibiotics, the starting period is smaller than or equal to 14 days, the methylbenzene removal rate is larger than or equal to 95%, the mineralization rate is larger than or equal to 80%, and the method is remarkably superior to the prior art and suitable for large-air-volume and low-concentration hydrophobic VOCs treatment.

The invention belongs to the technical field of biology, and particularly relates to a method for quantitatively detecting the infection rate of lentivirus on target cells. Comprising the following steps: infecting target cells with a lentivirus-containing sample to be detected; preparing a single-cell suspension from the target cells integrated with the lentivirus, uniformly dividing the single-cell suspension into two parallel samples which are identical in number and state, and inoculating the parallel samples to obtain antibiotic treated cells and antibiotic-free treated cells; adding antibiotics into the medicine sieve group for screening, counting to obtain positive infected cells Ne, and counting the control group without adding the medicines at three time points, namely when the dichotomy operation is executed, when the antibiotics start to screen and after the antibiotics are screened, so as to obtain the total cell numbers No.2, No.1 and No.3; and calculating the infection rate result of the lentivirus on the target cells based on the total cell number. According to the method, the sampling time node of the control group is optimized, the cell number of the control group is selected as the denominator of the infection rate calculation when the medicine sieve group samples start to be added, and the accuracy of the experimental result is further improved.

The invention discloses a stable and antibiotic-free plasmid expression system and application thereof in preparation of antitumor drugs. The plasmid expression system comprises a replicon, a regulatable promoter, a ribosome binding site RBS and an axe / txe toxin-antitoxin stabilization module, and the plasmid expression system can realize long-term stable expression of exogenous genes under the antibiotic-free condition. The replicon is selected from one or a combination of more of pUC, pMB1, p15A or pSC101; the promoter is selected from one or a combination of more of J23100, J23150 or J23112, and the RBS is selected from one or a combination of more of B0032, B0033 or B0034. According to the invention, an antibiotic-free and lasting plasmid platform is established for salmonella-based therapy, and a framework, synthetic biology and cancer treatment opportunity are provided for reliable gene delivery in vivo.

The application discloses a traditional Chinese medicine feed additive for preventing and treating Simmental respiratory disease syndrome and a preparation method and application thereof, and belongs to the technical field of livestock feed additives. The additive is prepared from traditional Chinese medicine raw materials, such as ephedra, honeysuckle, forsythia, radix scrophulariae, radix houttuyniae, radix isatidis, platycodon, bitter apricot kernel, radix glycyrrhizae, radix astragali, rhizoma atractylodis, mint, loquat leaf and radix codonopsis, through decocting, extracting, low-temperature extracting after mint, concentrating and spray drying. The application adopts a three-step prevention and treatment strategy of 'dispelling lung-evil, clearing heat and resolving toxin and supporting healthy body', can effectively prevent and adjuvantly treat Simmental respiratory disease syndrome caused by mixed infection of multiple pathogens, significantly reduces the morbidity of calf BRDC, enhances the immune function of the body, promotes growth and weight gain, has the characteristics of no antibiotic residue, no drug resistance, good palatability and the like, and meets the requirements of green and healthy breeding.

The invention provides an antibiotic-free fermented feed for sows and a preparation method thereof, and relates to the technical field of feeds for sows, the antibiotic-free fermented feed for sows and the preparation method thereof comprise 30-35% of corn, 26-28% of wheat, 10-15% of bran and 12-15% of soybean meal. The feed is prepared from the following raw materials in percentage by weight: 0.5-1% of calcium phosphate, 0.5-1% of calcium hydrophosphate, 0.2-0.5% of sodium chloride, 1-2% of soybean oil, 0.2-0.5% of lysine, 0.1-0.3% of threonine, 1-2% of premix and 0.6-1.2% of compound Chinese herbal medicine through a fermentation process. The fermented feed is free of antibiotics, avoids the situation that the balance of animal intestinal microorganisms is damaged by abuse of antibiotics, is good in palatability and high in nutrient substance utilization rate, can improve the intestinal microflora, can increase the feed intake of lactating sows and piglets, reduces the number of escherichia coli in fecal microorganisms, increases the number of probiotics, and further has the effects of improving the yield of the lactating sows and the piglets. And the added composite Chinese herbal medicine agent can enhance the immunity of the sows and improve the defense resistance of the body when the sows eat the feed, which means that the sows have better disease resistance.

The invention discloses an antibiotic-free screening gene expression system, plasmid, engineering bacteria and application thereof, and relates to the technical field of bioengineering. The expression system provided by the invention comprises a first strain, a second strain, a third strain and plasmids of antibiotic-free screening genes, the first strain, the second strain and the third strain are escherichia coli strains with a thyA gene knocked out, the first strain is used for molecular cloning, the second strain is used for expression of target protein, and the third strain is used for biosynthesis of natural products such as polyketone and non-cellulose peptide; a thyA complementary gene expression cassette is inserted into the plasmid of the antibiotic-free screening gene, and a resistance gene is knocked out; the thyA complementary gene expression box contains a thyA gene.

The application relates to a traditional Chinese medicine and additive compound preparation for treating gizzarditis of laying hens and application, and relates to the technical field of veterinary drugs. The compound preparation is prepared by mixing a traditional Chinese medicine extract and a functional feed additive. The raw material composition of the traditional Chinese medicine extract comprises torrefied malt, torrefied medicated leaven, radix codonopsis, rhizoma atractylodis macrocephalae, rhizoma coptidis, andrographis, dried tangerine or orange peel, white arillus, and liquorice. The additive comprises neutral protease, a mould inhibitor and probiotics. The mould inhibitor is selected from aspergillus flavus B1 degrading enzyme, corn strobukenone degrading enzyme, fumonisin degrading enzyme and vomitoxin degrading enzyme. The probiotics are selected from lactobacillus plantarum, bacillus subtilis and fructo-oligosaccharide. The application is based on the traditional Chinese veterinary theory of food digestion and spleen health, dampness expelling and qi regulation, heat clearing and toxin resolving and mucosa repairing. In combination with the effects of the additive on digestion, mould expelling and intestinal micro-ecological regulation, the application has a remarkable treatment effect on gizzarditis of laying hens, can improve the feed intake and weight gain, reduce the gizzard index, promote the repair of gastric mucosa, and has no antibiotic residue and no toxic side effect.

The invention provides a resistance-free selection marker plasmid, a preparation method, recombinant bacteria and application, and belongs to the technical field of gene engineering. The plasmid comprises an EM7 promoter, a ccdA41 antitoxin gene and an R6K gamma replication origin, the length of the plasmid molecule is less than 600bp, and the plasmid molecule does not contain antibiotic resistance genes. The plasmid is small in molecular weight and free of antibiotic resistance genes, effectively avoids safety risks caused by use of antibiotics, strictly depends on designated host replication of chromosome integration ccdB, meets biological safety requirements and is suitable for safe production of biological products such as vaccines and recombinant proteins. According to the invention, the recombinant bacterium is used for producing the plasmid without the resistance selection marker simply, conveniently and quickly, the success rate is high, and the recombinant bacterium utilizes the GT115-ccdb strain and the plasmid containing the ccdA41 antitoxin gene to realize stable maintenance and expression of the plasmid in a host through a toxin-antitoxin composite regulation mechanism.

The invention relates to the technical field of feed processing and bioactive substance extraction, and particularly discloses a combined preparation method of antibacterial peptide based on fresh shrimp shells and an antibiotic-free feed premix. According to the method, fresh shrimp shell waste serves as a raw material and is prepared into slurry through pretreatment, and a culture medium containing energy substances and composite strains is prepared and used for breeding housefly maggots; and then, extracting the high-purity antibacterial peptide from the harvested fly maggots through the processes of homogenization, ultrasonic-assisted extraction, salting-out, dialysis and the like. Meanwhile, culture medium residues obtained after breeding and fly maggot extraction residues are mixed, dried and smashed, and then auxiliary materials such as probiotics, mineral substances and vitamins are added, so that the antibiotic-free feed premix is prepared. According to the method, full-chain and high-value utilization of the fresh shrimp shell waste is achieved, the high-value-added antibacterial peptide product and the high-nutrition antibiotic-free feed are synchronously produced, the process is compact, environmentally friendly and economical, and an innovative technical scheme and core raw material support are provided for antibiotic-free breeding.

The invention belongs to the technical field of aquaculture, and discloses an immunopotentiator for aquaculture and application of the immunopotentiator. The immunopotentiator for aquaculture is prepared from the following components in parts by weight: 50 to 60 parts of ginsenoside, 15 to 20 parts of organic saccharicterpenin, 6 to 10 parts of a nutritional supplement, 1 to 5 parts of an immunopotentiating aid, 0.5 to 2 parts of an anti-stress aid and 0.2 to 1 part of an antioxidant. The ginsenoside and the organic saccharicterpenin which are naturally extracted are used as core active ingredients, safe auxiliaries are used as auxiliary materials, harmful ingredients such as antibiotics and hormones do not exist, medicine residues cannot be generated after the composition is used, and safety is high; meanwhile, through scientific compounding, all the components play a synergistic role, the non-specific immunity and anti-stress ability of aquatic animals can be improved, intestinal microecological balance can be improved, growth can be promoted, the culture water environment can be indirectly improved, and multiple effects are achieved.

The invention discloses a method for biosynthesizing gamma-polyglutamic acid without antibiotics, which particularly comprises the following steps: knocking out an alr gene for coding alanine racemase on a chassis strain corynebacterium glutamicum genome to construct alanine auxotroph cells, transferring alr to an expression plasmid pZM1 to construct a complementary plasmid, and successfully constructing a plasmid expression system without antibiotic addition; on the basis, gamma-polyglutamic acid degrading enzyme genes pgdS and pgsBCA derived from bacillus subtilis are subjected to tandem expression, and a dual-module formed by gamma-polyglutamic acid synthetase and degrading enzyme is further subjected to metabolism regulation by screening a promoter, RBS and a protein degradation tag at three levels of transcription, translation and post-translation modification. Under the regulation and control of a PtacM-UTR2-DAS + 8 element combination, the efficient synthesis of the gamma-polyglutamic acid is realized, the yield can reach 24.2 g.L <-1 >, and the production intensity is 0.55 g.L <-1 >. H <-1 >. And a gamma-polyglutamic acid production process without antibiotic addition is successfully constructed.

The application belongs to the technical field of biology, and particularly relates to a recombinant strain of high-yield duck Riemerella anatipestifer phage lytic enzyme, a mutant strain, a construction method and application. The application provides a coding gene of duck Riemerella anatipestifer phage lytic enzyme, and further constructs a recombinant Lactococcus lactis of high-yield duck Riemerella anatipestifer phage lytic enzyme. The mutant strain provided by the application is obtained by using the recombinant Lactococcus lactis as a starting strain, through mutagenesis breeding technology and specific high-throughput screening strategies, has higher secretion expression of duck Riemerella anatipestifer phage lytic enzyme, and has good genetic stability in an antibiotic-free culture medium, and is suitable for industrialized and efficient production of active lytic enzyme. The duck Riemerella anatipestifer phage lytic enzyme expressed by the mutant strain has obvious lytic effect on duck Riemerella anatipestifer in vitro, has significant prevention and treatment effect on duck Riemerella anatipestifer infection in vivo, can effectively improve the survival rate of infected chicks, and can remove pathogenic bacteria in the body.

The invention belongs to the technical field of piglet feeding, and particularly relates to a method for preventing and treating piglet diarrhea. The method comprises the step of feeding piglets with a functional feed prepared by a specific strain and a specific fermentation process. The preparation method of the functional feed comprises the following steps: adding the bacillus licheniformis and the bacillus subtilis into a solid-state fermentation culture medium containing overground stem and leaf powder of the galangal and wheat bran powder, and carrying out staged fermentation on the bacillus licheniformis and the bacillus subtilis in a solid-state fermentation culture medium containing overground stem and leaf powder of the galangal; and drying and granulating the fermentation product. According to the invention, agricultural waste, namely overground stems and leaves of galangal, is combined and co-fermented with specific probiotics for the first time and is applied to the piglet feed, so that the diarrhea rate of piglets is effectively reduced, the growth of the piglets is promoted, the utilization rate of the feed is improved, and a safe, efficient and antibiotic-residue-free green solution is provided for solving the problem of piglet diarrhea; and meanwhile, the high-value utilization of the galangal byproduct is realized.

Disclosed arc vectors including i) a first region of nucleic acid sequence comprising a gene of interest, a telomerase occupancy site, and 5' and 3' ends; and ii) a spacer region that is less than 1000 bp links the 5' and 3' ends of the first region of nucleic acid sequence. Vectors may also include a bacterial replication origin or a nucleic acid sequence encoding a RNA selectable marker. Vectors may be double-tranded, linear, close-ended nucleic acid vector. Also disclosed are methods for producing double-stranded, linear, close-ended nucleic acid vectors.

The invention belongs to the field of additives, and provides an animal feed additive and a preparation method thereof, and the animal feed additive is prepared by mixing a Chinese herbal medicine fermentation liquid and black gadfly pulp according to a mass ratio of 1: (200-500), the Chinese herbal medicine fermentation liquor is prepared by fermenting the following raw materials: 10-15 parts of astragalus membranaceus, 6-10 parts of liquorice, 8-10 parts of salvia miltiorrhiza, 10-12 parts of white paeony root, 10-15 parts of scutellaria baicalensis, 6-12 parts of rheum officinale, 8-12 parts of golden cypress, 10-15 parts of sapium sebiferum, 8-12 parts of tobacco and 4-6 parts of medicated leaven, and the raw materials are crushed and fermented with a compound microbial strain to obtain the Chinese herbal medicine fermentation liquor; the compound microbial strain comprises bacillus subtilis, lactic acid bacteria and bacillus licheniformis, and the viable count ratio of the bacillus subtilis to the lactic acid bacteria to the bacillus licheniformis is (2-3): (1-2): 1; the fermented Chinese herbal medicine is taken as a core, Chinese herbal medicine macromolecules are decomposed into micromolecular active substances (such as fermented astragalus polysaccharide) through microbial conversion of bacillus subtilis and the like, the drug effect is improved by 4-28 times, antibiotic residues are avoided, the problems of drug resistance and drug accumulation are avoided, and the food safety requirement is met.

Antibiotic-free systems for maintaining plasmids in host cells and methods of using same. The plasmids can comprise one or more plasmid-maintenance genes and a gene of interest configured to express in the host cell. The one or more plasmid-maintenance genes can comprise one or more of a prophage repressor gene, a sucrose phosphorylase gene, and an alcohol / aldehyde dehydrogenase gene. The methods can comprise growing the host cell with the plasmid comprised within the host cell, wherein the plasmid can be maintained during growth in the host cell without the use of antibiotics. In systems with a plasmid comprising a sucrose phosphorylase gene, the methods can comprise growing the host cell in the presence of sucrose as a sole carbon source. In systems with a plasmid comprising an alcohol / aldehyde dehydrogenase gene, the methods can comprise growing the host cell in the presence of glucose as a sole carbon source.

The invention relates to an antibiotic-free biological fermentation compound feed for poultry and a preparation method thereof, and belongs to the technical field of feeds, the feed takes a fermentation basic raw material as a carrier, plant extracts and chemical addition components are added, and the synergistic effects of resisting bacteria, diminishing inflammation, regulating intestinal flora, promoting nutrient absorption and improving the immunity of poultry are achieved in a biological fermentation synergistic manner; and antibiotic residues are avoided, so that the safety of poultry products is guaranteed.

The invention discloses a large-scale laying hen farm double-antibiotic-free breeding technology, and relates to the technical field of laying hen breeding, and the method mainly comprises two technical systems of harmful positive virus antigen purification for chicken flocks and complete-cycle antibiotic-free health management. Wherein a method for purifying harmful positive virus antigens comprises the following steps: strictly executing a full-in full-out feeding management mode, introducing chicken flocks with negative ALV, CIAV and REV antigens, and regularly monitoring and eliminating cages in which the chickens with positive ALV, CIAV and REV antigens are located and adjacent chicken flocks with high-risk cages by utilizing a PCR technology. The whole-cycle antibiotic-free health management method comprises the following steps: controlling drinking water pollution from the day when chicks enter a farm, and using cold boiled water as drinking water within a week when the chicks enter the farm; a feed beneficial microorganism colonization promoting system is constructed, precise immunization is implemented, harmful microorganism invasion is prevented, the resistance of chicken flocks is enhanced, and no antibiotic is added in the whole breeding process.

This invention proposes a compound plant composition for inhibiting intestinal inflammatory response in pigs, its preparation method, and its application, relating to the field of veterinary technology. The compound plant composition contains the following active ingredients in parts by weight: 50-70 parts carvacrol, 10-20 parts thymol, 30-50 parts cinnamaldehyde, 5-10 parts baicalein, 5-10 parts apigenin, and 3-8 parts chlorogenic acid. The preparation method involves mixing the active ingredients in proportion, adding water and Tween-80, and stirring until homogeneous. This invention can significantly reduce the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in the serum and small intestinal tissue of pigs infected with enterotoxigenic Escherichia coli, and increase the level of the anti-inflammatory cytokine IL-10. It also exerts its anti-inflammatory effect by inhibiting the activation of the TLR4-MyD88-NF-κB signaling pathway. This invention uses a compound of natural plant extracts, eliminating the risk of antibiotic resistance and veterinary drug residues, and is simple to prepare.

The invention relates to the technical field of microbiological detection, and particularly discloses a culture medium for selectively separating and culturing Burkholderia cepacia, which comprises the following components: 8-12 g / L of tryptone, 8-12 g / L of sucrose, 1-2 g / L of yeast extract, 1-2 g / L of monopotassium phosphate, 5-10 g / L of sodium pyruvate, 4-7 g / L of sodium chloride, 15-23 mg / L of phenol red, 10-18 g / L of agar, 0.8-1.3 mg / L of crystal violet, 0.5-5 g / L of selective inhibitor, and the balance of water. 10-30 g / L of a specific accelerant and the balance of water; the selective inhibitor is selected from one or more of sodium deoxycholate, sodium taurocholate, sodium glycocholate and propyl gallate; the specific accelerant is lactose, cane sugar and vanillic acid; and the culture medium does not contain any antibiotic component. The culture medium disclosed by the invention is free of antibiotic components, good in selectivity and high in growth promoting ability, and has a very good market application prospect.

This invention provides a recombinant plasmid for expressing the Bacillus subtilis resistance marker gene without antibiotic resistance, its construction method, and its application. This invention utilizes Bacillus subtilis SCK6 supercompetent cells to directly construct a Bsggt recombinant expression plasmid that does not contain the Ori replication origin site of Escherichia coli or the ampicillin resistance gene. This results in a smaller free plasmid expression system, providing a stable expression tool for heterologous proteins in production applications. The use of Cas9n-AID base editing technology in this invention simplifies the strain screening process. The constructed resistance marker-free expression plasmid is smaller than traditional shuttle plasmids, and no inducing agent is needed during expression. Because the expression plasmid is free of antibiotic resistance markers, the growth rate is significantly increased during culture due to the absence of antibiotic pressure. High expression without antibiotic introduction is more suitable for food safety-grade production applications.