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? -> SELECTING 2. Culture development: Exhibit the utility -> CULTIVATION 3. 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 Acinetobacter sp.

Lactobasillus bulgaricus Streptomyces sp. Saccharomyces sp.

Choosing habitat b. Isolate microorganism c. Choosing screening methode

a.

 Enviromental Condition:
Acidophil Neutrophil 5.5 8.0 0o - 30o 25o - 37o 40o- 75o 65o - 114o Alcaliphil 8.4 9.5 Physchrophile Mesophile Thermophile Hyperthermophile

pH

2.0 - 5.0

Temperature

Oxygen Partial Pressure Osmotic Pressure

Osmophile Halophile High sugar Salinity: 3.5% Haloduric Xerophile Salinity: 15%-30% Very dry environment

Nutrition Elemental requirements

Macronutrient Carbon ( C ) Nitrogen (N) Phosphorus (P) Sulfur (S) Potassium (K) Magnesium (Mg) Calcium (Ca) Sodium (Na) Iron (Fe)

Specific nutrient
Amino acid

Vitamin

Micronutrient Cobalt ( Co) Zinc (Zn) Molybdenum (Mo) Copper (Cu) Manganese (Mn) Nickel (Ni) Tungsten (Tu) Seleneum (Se)

Energy Requirements
Light Energy (photoautotrof) Chemical Energy (chemotrof) Organic (Organotroph) Inorganic (Lithotroph)

 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:

Random screening: calibration all isolate b. Rational screening: praselection first, by biochemistry knowledge ex: Cephalosporium acrenomium -> antibiotik enzim proteolitik
a.

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

Agar medium Reduce metabolism: Mineral oil, liquid paraffin Drying: sterile soil, silika gel Culture Cultivation Freeze drying

Short-term

Long-term
cryopreservation

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 changes 3. The number of isolates 4. Culture storage duration 5. 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 mutagenesis

Site-directed mutagenesis
Kunkel's method Cassette mutagenesis

PCR site-directed mutagenesis

Whole plasmid mutagenesis In vivo site-directed mutagenesis methods

Combinatorial mutagenesis
Insertional 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 http://ditjenbun.deptan.go.id/bbp2tpsur/index.php?option=com_content&vi ew=article&id=268:teknik-penyimpanan-mikroba-dengan-menggunakanaquadest-dan-tanah-steril&catid=14:proteksi&Itemid=25