A plasmid vector is used to control the expression of the Holin gene, and a method for producing the ghost bacteria Lactobacillus plantarum is also developed using this plasmid vector.

Abstract

OCR 10KL (02 / 02 / 2569) This invention involves the development of a plasmid vector containing a modified holin gene. The promoter that controls the expression of the holin gene (PsppA), the signaling peptide (Lp_3050), the holin gene, and... A method for producing ghost bacteria from Lactobacillus plantarum using holin, focusing on the use of bacterial strains. Safe varieties with reduced ghost cell initiation time were created by modifying the holin gene in the L. plantarum genome. The nucleotide sequence is optimized for expression in E. coli to create a modified plasmid vector. pLp_3050LpHo is used to control holin expression within cells. When cells containing this plasmid are Using this developed cultivation method, living cells can be transformed into ghost cells or ghost bacteria within 14 hours. The resulting ghost bacteria can be developed into molecular delivery systems for applications in biotechnology. Moving forward to the food and medical industries.

Claims

OCR 10KL (02 / 02 / 2569) 1. The plasmid vector for controlling the expression of the Holin gene consists of: Promoter that controls the expression of the holin gene (PsppA) Signaling peptide (Lp_3050) Holin gene 2. A plasmid vector for controlling the expression of the holin gene, according to claim 1, where the holin gene has the following sequence. Nucleotide sequence number 1 (SEQ ID NO.1) and / or amino acid sequence number 2 (SEQ ID NO.2), respectively, as follows: ATGGGCTTCT GGAAGCGTAT CCTGATTAAC ACCATTCTGT TTATCGCGAT TGCGGGTTTC 60 TTTCAGAGCA GCTTCCACGT GGCGAGCGTT TGGATGGCGC TGATCGCGAG CTTTGTGCTG 120 GCGATTCTGA ACGCGGCGAT CAAACCGTTC CTGCTGCTGC TGAGCCTGCC GATTACCCTG 180 CTGACCCTGG GCCTGTTCAG CATCGTTATT AACGGTTTTA TGCTGCAAAT GACCAGCTAC 240 GTGGTTGGCA AGGCGAACTT CGGCTTTAGC AGCTTTGGTA TGGCGATGGT GGTTAGCATC 300 CTGATGAGCA TTGCGAACGT GATCGTTAGC AACTTCTTTG CGAAAGACGG CGTGGATGGC 360 GAGGGCAGCC ACCACCACCA CCACCACTAA 390 Nucleotide sequence number 1 (SEQ ID NO:1) Amino acid sequence number 2 (SEQ ID NO: 2) 3. Plasmid vectors for controlling the expression of the Holin gene, pursuant to claim 1 or 2, where the Holin gene... It is further composed of a glycine-serin linker and a histidine label, with the histidine label bound to the holin gene by... It uses a glycine-serin crosslinker.

4. Modified microbial cells for producing ghost bacteria with gene introduced into the cells using a plasmid vector for control. (MGFWKRILINTILFIAIAGFFQSSFHVASVWMALIASFVLAILNAAIKPFLLLLSLPITLLTLGL FSIVINGFMLQMTSYVVGKANFGFSSFGMAMVVSILMSIANVIVSNFFAKDGVDGEGSHHHHHH*) The expression of the Holin gene consists of: A. Host cells of Lactobacillus plantarum and B. Plasmid vector for controlling holin gene expression introduced into Lactobacillus plantarum. Where the plasmid vector for controlling the expression of the Holin gene is included. The promoter that controls the expression of the holin gene (PsppA), the signaling peptide (Lp_3050), and the holin gene.

5. Modified microbial cells for generating ghost bacteria, with genes introduced into the cells using a plasmid vector for control. The expression of the holin gene, according to claim 4, where the host cell is Lactobacillus plantarum species. Variety TBRC2-4 6. Method for producing Lactobacillus plantarum ghost bacteria by introducing genes into cells using a plasmid vector. The following steps are involved in controlling the expression of the Holin gene: a. Culturing colonies of modified microbial cells Lactobacillus plantarum containing a plasmid vector. For controlling the expression of the olin gene (pLp_3050LpHo) in liquid culture medium containing antibiotics. The bacterial solution containing modified microbial cells was then incubated at 30 degrees Celsius for a certain period of time. 16-18 hours Where the plasmid vector for controlling the expression of the Holin gene includes a promoter. Controlling the expression of the holin gene (PsppA), signaling peptide (Lp_3050), holin gene. B. Dilute the bacterial solution into fresh liquid culture medium containing antibiotics until cell turbidity is achieved. (OD600) 0.025-0.04, then incubate at 30°C until the cell turbidity (OD600) is in the range of 0.3, plus or minus 0.

05. and C. Add the inducing agent and incubate at 30 degrees Celsius for 14-16 hours.

7. Method for producing Lactobacillus plantarum ghost bacteria by introducing genes into cells using a plasmid vector. For controlling the expression of the holin gene, according to claim 6, which concerns the concentration of antibiotics in the food. The culture medium should be in the range of 3-5 micrograms per milliliter.

8. Method for producing Lactobacillus plantarum ghost bacteria by introducing genes into cells using a plasmid vector. For controlling the expression of the Holin gene, according to claim 6, where the concentration of the inducing agent... It is in the range of 100-200 micrograms per milliliter.

9. A method for producing Lactobacillus plantarum ghost bacteria by introducing genes into cells using a plasmid vector. For controlling the expression of the Holin gene, under claim 6, where the inducing agent is IP-673.

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