Microbial Genetics

• Definition

  Virtually all the microbial traits are controlled or influenced by heredity

  Heredity → heretable variation

  The inherited traits of microbes include their shape and structural features, their metabolism, their ability to move, and their ability to interact with other organisms – perhaps causing disease.Individual organism transmit these characteristics to their offspring through genes, the units of the heredity material that contain the information that determines these characteristics

• Structure and Function

  • The genetic information in a cell is called the genome.
  • The base pairs always occur in a specific way : A – T and C – G

• Replication / Replikasi DNA

  • In DNA replication,one “parental” double-stranded DNA molecule is converted to two identical “daughter” molecules
  • Because the bases along the two strands of double-helical DNA are complementary, one strand can act as a template for the production of the other strand
  • The point at which replication occurs is called the replication fork
  • Enzymes
  • The process of replication is reffered to as semiconservative replication

• RNA and PROTEIN SYNTHESIS

  • Transcription
  • Translation

• term

  • GENOM
  • GENOTYPE
  • PHENOTYPE
  • PROTOTROPH
  • AUXOTROPH

• Operon model

  Jacob and Jacques Monod (1961) formulated this general model

  • Structural genes
  • Operator genes
  • Regulatory gene

Mutation

• overview

  • Is a change in the base sequence of DNA
  • Natural selection
  • Can occur spontaneously
  • Can be inherited to the offsprings
  • Back mutation

• Mutation rate :

  • relatively constant

  • spontaneous rate of mutation is about once in 106 replicated genes

  • can be increased 10 – 1000 times by mutagens

• Mutagens

  • chemicals : nitrate, acridine, N-mustard
  • physicals : x-ray, gamma-ray, UV-ray

• Morphological Mutant

  

• Biochemical Mutant

  

• Mutation Effect

  

• Type of mutation

  • Base Substitution (or point mutation) :

    • missense mutation

    • nonsense mutation

  • Addition / Deletion :

    • frameshift mutations

  • Inversion

• Intercelullar Mechansm- Genetic Transfer

  INVOLVE DONOR CELLS AND RESIPIENT CELLS

  • 1.TRANSFORMATION
  • 2.TRANSDUCTION
  • 3. CONJUGATION

• Plasmid

  • Extrachromosomal genetic elements

  • Replicate independently of chromosomal DNA

  • Usually contain from 5 – 100 genes

  • May carry genes for such activities as antibiotic resistance, tolerance to toxic metals, the production of toxin, and the synthesis of enzymes

  • Can be transferred from one bacterium to another

  • Is used for gene manipulation in biotechnology

  • Examples :

    F plasmids

    Penicillinase plasmid

    R factors : RTF + r-determinant

• Transposon

  • Small segments of DNA that can move (be ‘transposed’) from one region of DNA molecule to another
  • Also called as ‘jumping genes’
  • These pieces of DNA are 700 – 400,000 bp
  • The frequency of transposition 10-5 – 10-7 per generation
  • The simplest transposon, also called insertion sequences (IS), contain only a gene that codes for an enzyme (transposase) and recognition sites
  • Complex transposons also carry other genes not connected with the transposition process, for example → may contain genes for enterotoxin or for antibiotic resistance
  • Because they may be carried between cells on plasmids or viruses, they can also spread from one organism – or even species – to another

BIOTECHNOLOGY and RECOMBINANT DNA

• Overview

  Biotechnology, the use of microorganisms, cells, or cell components to make a produc

  Genetic engineering, manufacturing and manipulating genetic material in vitro; also called recombinant DNA technology

  Genetic recombination, the process of joining pieces of DNA from different source

• Gene Cloning

  PROCEDURE :

  1. ISOLATION OF DNA CONTAINING GENE OF INTEREST

  • DNA IS CLEAVED BY AN ENZYME → RESTRICTION ENZYME

    produced by bacteria (> 2000 enzymes)- molecular scalpel → cut DNA on the

    particular sites- recognize particular nucleotide base sequences(4,6,8 bp)

    • cutting point :

      • blunt-ended fragments

      • sticky ends

  • GENE IS INSERTED INTO PLASMID (VECTOR) → RECOMBINANT DNA (PLASMID)

    • CLONING VECTOR

      • must has origin of replication

      • has ≥ 1 cloning site

      • has gene that code for product to differentiate :

      transformed cells and untransformed cells

    • Types

      • PLASMID

        circular DNA molecules → extrachromosomal

        small size (± 5000 bp)

        replicate autonomously

      COSMID

      BACTERIOPHAGE λ

      PHAGE M 13

    • By using the same restriction enzyme DNA vector

    and DNA that contain gene of interest are cutted

    • Gene of interest inserted into pd DNA vector + ligase
    • → Recombinant DNA
  1. TRANSFORMATION → PLASMID IS TAKEN UP BY A SUCH CELL SUCH AS BACTERIUM (RECOMBINANT BACTERIUM)
  • SELECTING A CLONE

• Application OF GENETIC ENGINEERING

  • THERAPEUTIC APPLICATIONS :

    1. Synthetic genes → to make human insulin

    2. Subunit vaccines → Hepatitis B

    3. Producing microbial product → antibiotic

    4. etc

  • BASIC RESEARCH & MEDICAL APPLICATIONS

    1. DNA fingerprinting → kedokteran forensik
    2. Gene therapy
    3. PCR (polymerase chain reaction)
    4. DNA sequencing
    5. DNA probes for pathogen identification