Microbial Genetics. Dr. Umi Fatmawati, S.Pd.,M.Si

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Microbial Genetics

Dr. Umi Fatmawati, S.Pd.,M.Si

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Structure and Function of Genetic Material

DNA & RNA

DNA=deoxyribonucleic acid

RNA=ribonucleic acid

Basic building blocks:

Nucleotides

Phosphate group

Pentose sugar

Nitrogenous base

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DNA Structure

Phosphate-P Sugar-blue Bases-ATGC

Arah pemanjangan

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Bacterial DNA

• Bacteria have closed, circular DNA

• Genome: a whole

genetic material in an organism

• E. coli

– 4 million base pairs – 1 mm long (over 1000

times larger that actual bacterial cell)

– DNA takes up around 10% of cell volume

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AKTIVITAS MATERI GENETIK (DNA)

1. Replikasi DNA : Perbanyakan DNA (bisa terjadi secara in vivo maupun in vitro)

In Vivo : Sebelum pembelahan sel (interfase)

In vitro : menggunakan primer dan mesin PCR

2. Sintesis protein

• Transkripsi : Pengkopian DNA menjadi mRNA

• Translasi: penerjemaahan mRNA menjadi asam amino

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1. DNA Replication

• Arah pemanjangan 5’ to 3 ‘

• DNA helikas membuka double stranded DNA + DNA template (cetakan) membentuk garpu replikasi

– Leading stand (5’ to 3’-continuous)

*DNA polymerase-joins growing DNA strand after nucleotides are aligned (complimentary)

– Lagging strand (5’ to 3’-not continuous)

*RNA polymerase (makes short RNA primer)

*DNA polymerase (extends RNA primer then digests RNA primer and replaces it with DNA)

*DNA ligase (seals Okazaki fragments-the newly formed DNA fragments)

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Garpu Replikasi

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2. Sintesis Protein

• DNA--- mRNA--- protein

transcription translation

Central Dogma

of Molecular Genetics

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a. Transcription

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b. Translation

• Three parts:

– Diawali start codon (AUG)

– Ribosom berjalan sepanjang mRNA

– Terminasi: sampai di stop codon polipeptida protein lepas dari ribosom

• rRNA=subunits that form the 70 S ribosomes (protein synthesis occurs here)

• tRNA=transfers amino acids to ribosomes for protein synthesis)

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Types of RNA

• Three types:

– mRNA: messenger RNA

• Contains 3 bases ( codon)

– rRNA: ribosomal RNA

• Comprises the 70 S ribosome

– tRNA: transfer RNA

• Transfers amino acids to ribosomes for protein synthesis

• Contains the anticodon (3 base sequence that is complimentary to codon on mRNA)

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Genetic Code

• Codons: code for the production of a specific amino acid

• 20 amino acids

• 3 base code

• Degenerative: more than 1 codon codes for an amino acid

• Universal: in all living organisms

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Genetic Code

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Mutations

• Changes in base sequence of DNA/lethal and inheritable

• Can be:

– Harmful – Lethal – Helpful – Silent

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Some fundamentals of bacterial genetics

Bacterial DNA (like any DNA) can be altered by mutations

Mutations can result in changes in proteins

diversity acquisition of resistance

new traits can be transmitted to other microbes

GENE MUTATION

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(a) Normal DNA dan (b) Missense Mutation

Normal

Terjadi subtitusi asam amino

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(c) Nonsense Mutation dan (d) Frameshift Mutation

Menyebabkan perubahan mjd STOP

KODON

Pergeseran

penerjemahan kodon

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Mutation can caused by

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Genetic Transfer in Bacteria

• Genetic transfer-results in genetic variation

• Genetic variation-needed for evolution

• Three ways:

– Transformation: genes transferred from one bacterium to another as “naked” DNA

– Conjugation: plasmids transferred 1 bacteria to another via a pilus

– Transduction: DNA transferred from 1 bacteria to another by a virus

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• Sel yang belum kompeten dapat

dibuat menjadi kompeten melalui 2 teknik, yaitu :

– Heat shock : dengan mendinginkan- memanaskan-mendinginkan lagi sel bakteri

– Elektroforasi : dengan mengejutkan sel bakteri dengan medan listrik

berkekuatan tinggi (10-20 kV/cm)

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Phage DNA itself is the new genetic element.

Bacteriophages – 2 Types of life cycle

• Lytic or virulent cycle – progeny viruses build up inside host bacterium, which rupture to release them.

• Temperate or nonlytic or lysogenic cycle – host bacterium is unharmed.

Transduction by a Bacteriophage

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Transformation

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Conjugation in E. coli

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What is different in prokaryotes from eukaryotes?

The prokaryotic genome is circular, so it is replicated differently than linear

eukaryotic DNA

What is gene expression?

transcription: synthesis of RNA from a DNA template

translation: formation of a protein (amino acid sequence) from RNA sequence

How does this process differ in prokaryotes?

In prokaryotes, an mRNA

molecule can carry information for several genes

e.g., all of the genes in a biosynthetic pathway

(this doesn’t happen in eukaryotes)

The RNA polymerase in constructed differently in prokaryotes

Process consists of initiation, elongation, termination

Same steps are seen in translation

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Plasmid Replication

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The difference of Prokaryotic and

Eucaryotic genome

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The difference of Prokaryotic and

Eucaryotic genome

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The entire genomes (DNA sequences) of many bacteria are known

>1000 times smaller than human genome

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GENE EXPRESSION

• Gene expression is carefully controlled.

• Bacteria must be able to adapt to environment and divide quickly

• Some genes are constitutive (always expressed)

• Some are inducible (only expressed when needed) example: enzymes that metabolize lactose

• Some are repressible ( expressed most of the time,but can be turned off)

• example: enzymes involved in biosynthesis

• These genes are organized into operons

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Operons: a group of genes expressed together

• Repressor: binds to operator (a region of DNA adjacent to genes)

• If something is bound to operator, trasncription cannot occur

• Inducer: binds to repressor so it can’t bind to operator

• Therefore transcription can take place

• Example: lactose operon.

genes are expressed only if lactose (inducer) is present

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Jumping gen (Transposon)

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Transposon

(Jumping Genes, Barbara McClintock)

DNA segment that can move between chromosome & plasmids

Transposons are not self replicative, they depend on chromosomal or plasmid DNA for replication

Insertion of transposon into a functional gene would destroy the function of the gene (internal mutagenic agents)

Plasmid

Chromosome

Transposon

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Bacterial Transposon

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Transposon organisms

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REKAYASA GENETIKA MIKROBA

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KARAKTERISTIK PLASMID

• Ukuran kecil, dapat

dipisahkan secara mudah dari DNA kromosom inang

• Gen ORI, gen awal tempat replikasi plasmid

• Multiple Clonning Sites (MCS) atau polylinker,

daerah pemotongan oleh enzim restricdi pd

sequence DNA

• Selectabel marker genes berupa gen resisten

antibiotik

• Promotor gen, gen awal transkripsi

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3 KEMUNGKINAN HASIL TRANSFORMASI GEN REKOMBINAN KE DALAM SEL BAKTERI

1. Sel bakteri tidak menerima/dimasuki oleh plasmid

2. Sel bakteri menerima/dimasuki oleh plasmid tetapi buka yang rekombinan

3. Sel bakteri menerima/dimasuki oleh plasmid

rekombinan

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SELEKSI DNA REKOMBINAN PADA BAKTERI

Terdapat 2 mekanisme:

1. Antibiotik selection

2. Blue white selection

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BLUE WHITE SELECTION

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BLUE WHITE SELECTION

• Adalah teknik yang biasa digunakan untuk membedakan antara koloni / bakteri yang mengandung vektor kloning rekombinan maupun non rekombinan.

• Teknik ini didasarkan pada vektor/plasmid seperti pUC dan seri M13mp yang membawa fragmen gen β-galaktosidase menkode α-β- fragmen dari galaktosidase.

• Teknik ini membutuhkan penggunaan strain bakteri yang kompeten untuk membawa

fragmen gen rekombinan.

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BLUE WHITE SELECTION

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Insersi gen asing pada gen lac Z

• Gen lac Z adalah gen yang mengkode ß-galatosidase (enzim yang mendegradasi laktosa menjadi glukosa dan galaktosa)

• Ketika lac Z diinsersi dengan gen asing, maka lac Z tidak mampu memproduksi ß-gal.

• Koloni bakteri ditumbuhkan pada media dengan penambahan substrat untuk ß-gal yg disebut X-gal (5-bromo-4-chloro-3-

indolyl ß-(5)D-galactopyranoside, selain itu ditambahkan inducer berupa IPTG.

• Hasil:

- non rekombinan bakteri yg mengandung lac Z fungsional menghasilkan enzim ß-gal (biru)

- rekombinan bakteri mengandung Lac Z non fungsional tidak menghasilkan enzim ß-gal (putih)

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SELEKSI ANTIBIOTIK

• Teknik ini pertama kali ditemukan oleh Cohen dan Boyer

• Menggunakan plasmid yang memiliki dua gen resisten

antibiotik, resisten ampicilin (amp_R) dan resisten tetracyclin (tetR)

• DNA asing diinsersikan pada salah satu gen resisten antibiotik, misal gen tet_R akan mencegah gen menghasilkan protein

resisten antibiotik.

• Sel bakteri ditumbuhkan pada media yang mengandung ampicilin, dimana bakteri non transformed tidak akan tumbuh.

• Dicopy dengan menggunakan replica platting kemudian ditumbuhkan pada media tetracilin, hanya bakteri non rekombinan yang tumbuh.

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ANTIBIOTIC SELECTION

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ANTIBIOTIC SELECTION

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