BIOTECHNOLOGY APPLICATIONS PHENOTYPIC GENOTYPIC CHARACTERIZATION OF FOODBORNE BACTERIA
BIOTECHNOLOGY APPLICATIONS: PHENOTYPIC & GENOTYPIC CHARACTERIZATION OF FOOD-BORNE BACTERIA Presented by Dr. Stalis Norma Ethica M. Si. Universitas Muhammadiyah Semarang In Euro Global Conference on Food, Agronomy & Technology FAT’ 18 with: Prof. Agus Sabdono, M. Sc. Dr. Ana Hidayati Mukaromah, M. Si. Ayu Rahmawati Sulistyaningtyas, M. Si Tara Puri Ducha, M. Sc. Indonesia Forestry Institute
PHENOTYPIC & GENOTYPIC CHARACTERIZATION OF FOODBORNE BACTERIA Genotypic characteristics Bacterial Genome composition Environmental factors: • Temperature & light • Nutrition • Drugs & chemicals • Toxins • Weathers Species-specific molecular Identification, following steps: • Bacterial genomic DNA isolation • Amplification and sequence analysis of bacterial 16 S r. RNA gene using PCR method (Polymerase Chain reaction) Case: Alcaligenes javaensis JG 3 (Ethica et al. , 2018) Phenotypic characteristics Functional gene characterization following step: • Bacterial genomic DNA isolation • Primer design and in silico amplification • Amplification and sequence analysis of functional genes Case: glp. D, glp. K, mo. AC, ben. E (Ethica et al. , 2018; Ethica 2017) Metabolism characteristics Bioochemical reaction tests: • Catalase, hydrolase tests • Automated Microbiology System (BD Phoenix, Vitek, API, etc. ) Result example: 44 biochemical parameters of Alcaligenes sp. JG 3 (Ethica, et al. , 2014; 2018) (varied) Observable traits Genes on Genes off Our object: Strain JG 3 isolated from root of Zea Mays in Central Java Availability levels of factors supporting gene expression to proteins: • Enzymes, • Cofactors • Hormones • etc. Recombination & Mutations Bacterial morphology www. biologyjuntion. com Morphological characteristics tests: • Scanning Electron microscopy (SEM) observation • Transmission Electron Microscopy (TEM) observation Result example: Short rod shape of Alcaligenes sp. JG 3 (Ethica, et al. , 2014; 2018) Examples: Unique cell colors, shapes, sizes, different secondary metabolites, Case: Unique ability to metabolize glycerol (Ethica, et al. , 2014; 2018)
PHENOTYPIC & GENOTYPIC CHARACTERIZATION OF FOODBORNE BACTERIA Genotypic characteristics Phenotypic characteristics Gene expression to proteins DNA replication & transcription RNA DNA Bacterial Genome composition PROTEIN www. biologyjuntion. com Observable traits
In silico PCR product from designed primers METHODS & RESULTS (/) • In silico PCR Product Tool: Free software http: //insilico. ehu. es PCR product: Electroforegram of isolated gene fragments using designed primers In silico PCR Functional gene isolation PCR product: Electroforegram of isolated 16 S r. RNA gene fragments using universal primers Novel species: Alcaligenes javaensis JG 3 -96% similarity to other bacteria’s 16 S r. RNA genes according to BLASTn (Ethica et al. , 2018) BD Phoenix result glp. D & glp. K (gene fragment) Genbank Acc. no: AB 826685, & AB 894421 (Ethica et al. , 2013; 2014) Proof of glycerol degradation by strain JG 3 Species-specific gene isolation Trait characterization
SIMPLE STRATEGY FOR POLYPHASIC IDENTIFICATION Biochemical test -BD Phoenix Microbiological System Morphological test - SEM & TEM Molecular test - Analysis on 16 S r. RNA gene sequence
OTHER RESULTS RESEARCH ON STRAIN JG 3 Characterization of moa. C and a nontarget gene fragments of food-borne pathogen Alcaligenes sp. JG 3 using degenerate colony and arbitrarly-primed PCRs METHODS: Arbitrarily-primed PCR: Two rounds of amplification (Espinosa-Urgel, Salido, & Ramos, 2000) 1 st round of PCR was performed using the genomic DNA of strain JG 3 as a template, with an arbitrary primer ( ARB 1; 50 -GGCACGCGTCGACTAGTACNNNNNGATAT-30) and an internal primer of glp. D internal fragment (GSP 3’). The themocycling condition was: 3 min at 958 C; 6 cycles of 30 min at 958 C, 30 min at 308 C, and 1 min at 728 C; 30 cycles of 30 min at 958 C, 30 min at 508 C, and 1 min 728 C; and an extension period of 7 min at 728 C. 2 nd round of PCR was done with 5 ml of the 1 st round PCR product as the template as follows: 3 min at 958 C; 30 cycles of 30 min at 958 C, 30 min at 578 C, and 1 min at 728 C; and 7 min at 728 C. Primers used for the 2 nd round were those corresponding to the conserved region of ARB 1 (ARB 2; 5’GGCACGCGTCGACTAGTAC-3’) and similar GSP 3’ primer. Similar condition was applied for amplification of 5’ end using similar arbitrary primers ARB 1 and ARB 2, but different gene specific primer, GSP 5’. RESULTS: The developed degenerate colony PCR resulted in 421 -bp DNA, while the set arbitrary PCR resulted in non-targeted 436 -bp DNA. Both sequences shared 94% amino acid similarity with molybdenum cofactor biosynthesis (Moa. C)of Pseudomonas stutzeri and benzoate membrane transporter (Ben. E) of Alcaligenes faecalis
Bibliography Ethica, Stalis Norma, Mohammad Kasfu Hammi, Puji Lestari, Endang Semiarti, Jaka Widada, and Tri Joko Raharjo. "Amplification of Azospirillum sp. JG 3 glp. D gene fragment using degenerate primers generated by web-based tools. " The Journal of Microbiology, Biotechnology and Food Sciences 3, no. 3 (2013): 231. Ethica, Stalis Norma, Endang Semiarti, Jaka Widada, Oedjijono, and Tri Joko Raharjo. "Characterization of moa. C and a nontarget gene fragments of food‐borne pathogen Alcaligenes sp. JG 3 Ethica, Stalis Norma, and Tri Joko Raharjo. "Detection of genes involved in glycerol metabolism of Alcaligenes sp. JG 3. " Ph. D diss. , Universitas Gadjah Mada, 2014. using degenerate colony and arbitrary PCRs. " Journal of Food Safety 37, no. 4 (2017): e 12345. Ethica, Stalis Norma, Oedjijono, Endang Semiarti, Jaka Widada, and Tri Joko Raharjo. "Genotypic And Phenotypic Characterization of Alcaligenes Javaensis Jg 3 Potential as an Effective Biodegrader. " BIOTROPIA-The Southeast Asian Journal of Tropical Biology 25, no. 1 (2018): 1 -10.
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