seleksi dan penyaringan

Dina Rahma Fadlilah

Commonly, first step in development of biotechnology is find the compatible organism.

PRODUCT TO INDUSTRY

Steps culture from natural habitat -> industry:1. Research culture: What is the utility? -> SELECTING2. Culture development: Exhibit the utility -> CULTIVATION3. Culture production: used for industrial production

Characteristic of selecting Industrial organisms are expected:-Pure cultures-Stable genetic-Easily propagated-Rapid growth-There is no toxic byproducts-Geneticly manipulated

Microorganisms are organism that frequently used

E. coli Klebsiella pneumoniae

Lactobasillus bulgaricus

Acinetobacter sp.

Streptomyces sp. Saccharomyces sp.

a. Choosing habitatb. Isolate

microorganismc. Choosing screening

methode

Enviromental Condition:

pH

Temperature

0o - 30o Physchrophile

25o - 37o Mesophile

40o- 75o Thermophile

65o - 114o

Hyperthermophile

Oxygen Partial

Pressure

Acidophil Neutrophil Alcaliphil

2.0 - 5.0 5.5 – 8.0 8.4 – 9.5

Osmotic

Pressure

Osmophile

Halophile

Haloduric

Xerophile

High sugar

Salinity: 3.5%

Salinity:15%-30%

Very dryenvironment

Nutrition

Elemental requirements

MacronutrientCarbon ( C ) Nitrogen (N) Phosphorus (P)Sulfur (S) Potassium (K)Magnesium (Mg)Calcium (Ca)Sodium (Na)

Iron (Fe)

Specific nutrient

Amino acid

Vitamin

Energy Requireme

ntsLight Energy(photoautotro

f)Chemical Energy

(chemotrof)

Organic (Organotroph

)

Inorganic (Lithotroph)

MicronutrientCobalt ( Co)Zinc (Zn) Molybdenum (Mo)Copper (Cu)Manganese (Mn) Nickel (Ni)Tungsten (Tu)

Seleneum (Se)

Screening is selective procedure used in population to detect and isolate desirable microorganism or metabolite.

Purpose: get the most potential microorganism to be used in industry.

Screening is divided by 2 prototype:a.Random screening: calibration all isolateb.Rational screening: praselection first, by

biochemistry knowledgeex: Cephalosporium acrenomium -> antibiotik

enzim proteolitik

Purpose: to prevent contamination and genetic changes also maintain activity level, viability and genetic quality of cell.

a. Vanili kering & segar; b. hasil pengeringan beku

Medium Agar

Selecting culture cultivation/ preservation method affected by:

1. Viability level required (nutrition)2. Possibility of genetic changes3. The number of isolates4. Culture storage duration5. Cost

is the genetic change in a cell by the alterations in the cell's genetic material (usually deoxyribonucleic acid [DNA]) to stable state -> mutation

Purpose: improve organism productivity to produce a product

Agent that causing mutagenesis is called mutagen, whereas the product is called mutant.

Mutation 1. Spontaneous

2. Induction

Physical treatment Chemical treatment Recombinant DNA technology

Random mutagenesisSite-directed mutagenesisKunkel's methodCassette mutagenesisPCR site-directed mutagenesisWhole plasmid mutagenesisIn vivo site-directed mutagenesis methodsCombinatorial mutagenesisInsertional mutagenesis

The target enzymes were subjected to random mutagenesis (step 1). The mutated genes were cloned into a pET expression vector and transformed to E. coli carrying the pGro7 plasmid, which conditionally overexpressed GroEL/GroES (step 2). In each round, 360 randomly picked colonies were individually re-grown in liquid media within 96-well plates (step 3). The plates were duplicated. To one set of plates, arabinose was added to induce GroEL/GroES overexpression. Glucose was added to the parallel set to suppress GroEL/GroES expression (step 4). Overexpression of the mutated enzyme variants was subsequently induced by IPTG addition (step 5). The cells were lysed, enzyme activity with and without GroEL/GroES overexpression was recorded (step 6), and representative variants were analysed (green box). To continue the drift, clones showing activity above the chosen threshold under arabinose induction were taken for the next round of mutagenesis and screening (step 7).

Smith, John E. 1985. Prinsip Bioteknologi. Jakarta: Gramedia.

Rahayu, Umi. 2007. Skrining Bakteri Termofilik Penghasil Protease. Bul. Tek. Lit. akuakultur 6 (2): 145 - 149

Yulneriwarni. 2008. Mikroba, dari Habitat ke Industri. Vis Vitalis 01 (2): 13-18

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