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    Strain Improvement of Microorganisms PDF

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    satriomega
    Strain improvement involves inducing genetic variation in microorganisms to improve their characteristics and productivities. Common methods include physical and chemical mutagenesis to generate random mutations, as well as targeted techniques like protoplast fusion and genetic engineering. The key steps are inducing mutations, screening mutants in fermentation processes, and analyzing their productivity to select improved strains. Optimization also involves modifying environmental and nutritional conditions to further enhance microbial performance.

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    Strain Improvement of Microorganisms PDF

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    satriomega
    445 views32 pages
    Strain improvement involves inducing genetic variation in microorganisms to improve their characteristics and productivities. Common methods include physical and chemical mutagenesis to generate random mutations, as well as targeted techniques like protoplast fusion and genetic engineering. The key steps are inducing mutations, screening mutants in fermentation processes, and analyzing their productivity to select improved strains. Optimization also involves modifying environmental and nutritional conditions to further enhance microbial performance.

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    Strain improvement involves inducing genetic variation in microorganisms to improve their characteristics and productivities. Common methods include physical and chemical mutagenesis to generate random mutations, as well as targeted techniques like protoplast fusion and genetic engineering. The key steps are inducing mutations, screening mutants in fermentation processes, and analyzing their productivity to select improved strains. Optimization also involves modifying environmental and nutritional conditions to further enhance microbial performance.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    445 views32 pages

    Strain Improvement of Microorganisms PDF

    Uploaded by

    satriomega
    Strain improvement involves inducing genetic variation in microorganisms to improve their characteristics and productivities. Common methods include physical and chemical mutagenesis to generate random mutations, as well as targeted techniques like protoplast fusion and genetic engineering. The key steps are inducing mutations, screening mutants in fermentation processes, and analyzing their productivity to select improved strains. Optimization also involves modifying environmental and nutritional conditions to further enhance microbial performance.

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    of 32

    Strain Improvement

    of Microorganisms
    Marlia Singgih Wibowo

    Definition of
    Strain Improvement
    In the field of agriculture : recovery of
    plants characters
    In the fileld of microbiology : improve the
    microbes productivities or characteristics

    Purposes of
    Strain Improvement

    Increase productivities
    To change unused co-metabolites
    To improve the use of carbon and
    nitrogen sources
    To improve morphology of cells to be a
    better cells in order to separate the cells
    and its products

    General Process

    Induction of genetic variation in some cell


    population of chosen microorganism
    Fermentation Process in lab scale for
    some cells
    Analysis (assay) to check the
    productivity of mutated strain

    How to optimize miicrobes


    productivity?

    Optimize environmental condition


    Optimize nutrition of microorganism
    Genetic Modification : Mutation,
    recombination, gene cloning

    Optimize environmental
    condition
    Modification of physical parameter
    (temperature, agitation, etc)
    Modification of chemical parameter
    (pH, O2 conc, etc)
    Modification of biological parameter
    (co-culture, enzym, etc)

    Optimize nutrition of
    microorganism

    Carbon source
    Nitrogen source
    Mineral source and other sources
    Precursor
    Enzyme

    Screening in Industry
    Stage
    1

    Process
    a.Mutation of wild type
    b.Culture on agar
    c.Selection of colonies
    d.Fermentation
    e.Selection of good strain

    Amount
    a.8 different doses
    c.25 of each dose
    d.200 strains
    e.40 strains

    a.Re test of selected strain (5 a.40 x 5 = 200 strains


    replication)
    b.Selection of best strain
    b.8 strain for primary
    metabolites, 40 strains for
    2nd metabolites
    Next screening

    MUTAGENESIS
    Mutagenesis is a process of mutation in
    strain improvement of microorganism. A
    treatment to microorganism will cause an
    improvement in their genotipic and
    fenotipic performances
    Mutation can be random or direct

    Procedure of mutagenesis
    Mutation by : physical, chemical or
    combination of physical and chemical
    method
    Protoplast Fusion
    Recombination
    Gene Cloning

    Physical Mutation
    X-ray
    Chromosomal mutation
    UV Radiation (250 270 nm)
    DNA absorps uv pada 260 nm
    Point mutation
    Dimerization of thymine
    Photo-repair : gain improvement of some
    abilities
    Radioisotop

    Stages in Physical mutation

    Wild-type of microbes is suspended in medium


    Counting number of cells in medium and
    concentration of cells is determined for
    mutation process
    Radiation is conducted in dark area with
    certain distance for certain duration.
    Exposed colonies are inoculated in growth
    medium for several days
    Check the growth and select some colonies for
    further process

    Chemical Mutation
    Base analog : substance that similar to purine or

    pirimidine, so transition will occured , example :


    5-bromouracyl : substitute C (cytosine) with T
    (thymin)
    2-aminopurine : substitute A (adenine) with G
    (guanine)
    De amine and alkylating agent : transition,
    example:
    Nitrite (NaNO2) can cause de-amined A and C so
    replication of AT will change to GC, or viceversa
    EMS (Etil metil Sulfonat), MNNG (Metil-nitro-nitrosoguanidine)

    Intercalation
    Dye acridine (proflavine) inserted between
    base pair, will cause a frame-shift and will
    misread its genetic information

    Mutation of Monascus purpureus


    using EMS

    Albino mutant of Monascus purpureus


    stable after 5 generation

    PROTOPLAST FUSION
    Protoplast fusion is a general technic to
    induce genetic recombination in some
    prokariot and eukariotic microorganisms
    Protoplast : whole cell without cell wall

    Fusion has some benefits to improve


    productivity of industrial microorganisms, e.g.
    Actinomycetes.
    The Method is easy, no transduction phage
    needed, plasmid or competention development,
    the only things is how to get the stable protoplast
    In protoplast fusion will produce hybrid cells

    Method of Protoplast Fusion


    Protoplast is prepared by removing the cell wall
    using lytic enzyme in a osmotic solution.
    With the help of PEG (polyetilenglicol) at certain
    conc. (fusogenic agent), the fusion can be done
    and hybrid cells will produced (diploid)
    During this stage , genome (or chromosom)
    from both cells will be fused and mixed . The
    genetic recombination will occured

    Protoplast Fusion for microorganism


    For prokariot Bacilli and Streptomycetes ,
    recombination will occur in a very high frequency
    because its chromosomes is free in the
    cytoplasm
    For eukariot microorganism, protoplast fusion
    should be followed by fusion of nucleus to
    obtain the real recombination
    The important step in protoplast fusion is
    REGENERATION of the cells after fusion
    Protoplast fusion mostly done for the same
    species to obtain the best strain with high
    productivity

    Exampe of Protoplast Fusion :


    Aspergillus terreus

    Strain 1

    Strain 2

    Fusant after fusion

    Genetic Engineering

    Genetic Engineering
    Rekayasa genetik adalah salah satu cara pembuatan
    DNA baru, biasanya melalui rekombinasi DNA dari
    organisme yang berbeda dan meproduksi banyak
    kopi dari DNA rekombinan tersebut melalui proses
    yang disebut KLONING
    Kloning adalah suatu proses dimana suatu urutan
    DNA tertentu disisipkan ke dalam suatu vektor
    (berupa plasmid atau kromosom faga) dan
    selanjutnya direplikasi sebanyak mungkin. Replikasi
    terjadi di dalam suatu inang (host) yang
    memungkinkan replikasi itu terjadi

    Prinsip mutasi dengan


    rekayasa genetik
    Informasi genetik yang dituju, diisolasi dari
    organisme donor dan dipotong menjadi bagian
    tunggal menggunakan enzim restriksi
    Potongan ini digabungkan di dalam suatu DNA
    pembawa (vektor) dan selanjutnya bersama
    vektor tersebut ditransfer ke dalam suatu sel
    inang (host)
    Sel inang akan bereplikasi dan mensekresi
    metabolit yang sesuai dengan informasi genetik
    yang ditransfer sebelumnya.

    Syarat untuk vektor

    Memiliki Selectable markers


    (penanda selektif)
    Memiliki Restriction sites
    (situs restriksi)
    Berat molekul serendah mungkin

    Plasmid
    Plasmid adalah suatu molekul DNA sirkular
    (BM 106 2 x 108) dari bakteri, yang
    membawa 1-3% genom sel dan mengkode
    suatu sifat genetik penting yang tidak dikode
    secara normal oleh kromosom bakteri
    tersebut
    Sifat yang banyak digunakan : resistensi
    antibiotik, produksi antibiotik, degradasi
    senyawa aromatik, dll.

    Faga (bakteriofaga)
    suatu sub-kelompok virus yang menginfeksi
    bakteri dengan cara menyisipkan asam
    nukleat nya ke dalam bakteri host nya.
    Faga terkecil hanya mengandung 3 gen
    pengkode ss RNA
    T4, suatu faga bakteri yang mengandung lebih
    kurang 60 gen pengkode ds genom DNA faga
    tersebut

    Cosmid
    Cosmid adalah partikel sintetik yang dapat bereplikasi
    sendiri
    Berasal dari plasmid yang mengandung fragmen
    lambda-DNA (DNA-) yang mengkode urutan cos site
    (situs pengenalan) untuk sistem yang akan
    membentuk struktur sirkular.
    DNA faga ini bereplikasi melalui suatu mekanisme
    khusus yaitu rolling circle yang yang selanjutnya
    membentuk struktur concatemer (berulang secara linear)
    setelah disisipkan DNA asing.
    Bentuk linear ini merupakan substrat untuk reaksi
    packaging secara in-vitro membentuk partikel.
    Selanjutnya partikel tersebut digunakan untuk
    menginfeksi sel inang, dan bentuk linear cosmid akan
    membentuk sirkular kembali pada sisi cos site

    Prinsip kloning gen menggunakan


    plasmid E.coli pBR 322
    Plasmid yang mengandung gen resistensi
    thp ampisilin dan tetrasiklin dipotong pada
    sisi tetrasiklin dengan enzim restriksi
    DNA asing di sisipkan pada plasmid dengan
    enzim ligase
    Plasmid yang telah mengandung DNA
    asing tsb ditransfer ke dalam sel bakteri

    Pembiakan

    Seleksi sel yang hanya


    mengandung gen
    resistensi ampisilin
    dan DNA asing

    Jenis Mutan

    Mutan Aukosotrof (Auxotrophic mutan)


    Mutan Resisten (Resistant)
    Mutan yang sensitif terhadap temperatur
    Mutan Konstitutif katabolik
    Mutan Konstitutif anabolik

    Mutan aukosotrof adalah mutan yang tidak dapat


    mensintesis suatu molekul organik yang sebenarnya
    sangat diperlukan untuk salah satu jalur biosintesis
    metabolit nya. Misalnya : nitrat, antibiotik

    Mutan resisten adalah mutan dari organisme yang


    sebelumnya sensitif terhadap suatu senyawa

    Mutan yang sensitif terhadap temperatur

    adalah mutan dari oraganisme yang semula tidak sensitif


    terhadap temperatur

    Mutan yang memiliki enzim konstitutif


    katabolik : antiinduksi, limiting factor, dll.
    Mutan yang memiliki enzim konstitutif
    anabolik : antimetabolit

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