MB 206 Microbial Biotechnology

MB 206 Microbial Biotechnology

MB206 May-Aug 09 Angelia Teo

CHAPTER 1.1 NUCLEIC ACID

2

A Comparison of DNA and RNA

Ribosome: containsribosomal RNA(rRNA)

Transfer RNA (tRNA)

largesubunit

1 2

tRNA/amino acidbinding sites

catalytic site

attachedamino acid

anticodon

smallsubunit

Messenger RNA (mRNA)

Central Dogma of Molecular BiologyCentral Dogma of Molecular Biology

How does the sequence of How does the sequence of a strand of DNA correspond a strand of DNA correspond to the amino acid sequence to the amino acid sequence of a protein?of a protein?

• DNA codes for RNA production.• RNA codes for protein production.• Protein does not code protein, RNA or DNA production. The end.

Or in the words of Francis Crick: Once information has passed into protein, it cannot get out again!

Revision of the "Central Dogma"Revision of the "Central Dogma"

CAN go back from RNA to DNA (reverse transcriptase)CAN go back from RNA to DNA (reverse transcriptase) RNA can also make copies of itself (RNA polymerase) RNA can also make copies of itself (RNA polymerase)

Still NOT possible from Proteins back to RNA or DNAStill NOT possible from Proteins back to RNA or DNA

Not known mechanisms for proteins making copies of themselves. Not known mechanisms for proteins making copies of themselves.

Gene ExpressionGene Expression

Expression of genetic determinants in bacteria Expression of genetic determinants in bacteria involves the involves the unidirectional flow of information from DNA to RNA unidirectional flow of information from DNA to RNA to to protein. protein. Two processes involved are transcription and Two processes involved are transcription and translation.translation.

Transcription & TranslationTranscription & Translation Prokaryotic vs Eukaryotic cellsProkaryotic vs Eukaryotic cells

In a prokaryotic cell, which does not contain a nucleus, this In a prokaryotic cell, which does not contain a nucleus, this process happens at the same time. process happens at the same time. In Eukaryotic cells, occur at different In Eukaryotic cells, occur at different cell compartments.cell compartments.

Prokaryotic cellProkaryotic cell Eukaryotic cellEukaryotic cell

TranscriptionTranscription The The DNA-directed synthesis of RNA is called transcription. is called transcription. Transcription produces Transcription produces RNA molecules that are complimentary copies of one strand of DNA. . Only one of the dsDNA strands can serve as template for Only one of the dsDNA strands can serve as template for synthesis of a specific mRNA molecule.synthesis of a specific mRNA molecule. mRNAs transmit information from DNA, and each mRNA in mRNAs transmit information from DNA, and each mRNA in bacteria function as a template for synthesis of one or more bacteria function as a template for synthesis of one or more specific proteins.specific proteins.

Translation Translation

Initiated at an AUG codon for methionine.Initiated at an AUG codon for methionine. Codons are translated sequentially in mRNA from 5' to 3'. Codons are translated sequentially in mRNA from 5' to 3'. The corresponding polypeptide chain / protein is assembled The corresponding polypeptide chain / protein is assembled from the amino terminus to carboxy terminus. from the amino terminus to carboxy terminus. The sequence of amino acids in the polypeptide is, therefore, The sequence of amino acids in the polypeptide is, therefore, co-linear with the sequence of nucleotides in the mRNA and the co-linear with the sequence of nucleotides in the mRNA and the corresponding gene. corresponding gene.

The Genetic codeThe Genetic codeThe "universal" genetic code employed by most organisms is a triplet code and it The "universal" genetic code employed by most organisms is a triplet code and it determines how the nucleotides in mRNA specify the amino acids in the determines how the nucleotides in mRNA specify the amino acids in the polypeptide. polypeptide.

• 61 of 64 possible trinucleotides 61 of 64 possible trinucleotides (codons) encode specific amino (codons) encode specific amino acids.acids.

• 3 remaining codons (UAG, UAA or 3 remaining codons (UAG, UAA or UGA) code for termination of UGA) code for termination of translation (nonsense codons = do translation (nonsense codons = do not specify any amino acids) not specify any amino acids)

Exceptions:Exceptions:1)1) UGA as a tryptophan codon in some UGA as a tryptophan codon in some

species of Mycoplasma and in species of Mycoplasma and in mitochondrial DNA.mitochondrial DNA.

2)2) Few codon differences in Few codon differences in mitochondrial DNAs from yeasts, mitochondrial DNAs from yeasts, Drosophila, and mammals. Drosophila, and mammals.

Gene expression occurs in 2 steps:

Transcription of the information encoded in DNA into a molecule of RNAof the information encoded in DNA into a molecule of RNATranslation of the information encoded in mRNA into a defined sequence of of the information encoded in mRNA into a defined sequence of amino acids in a protein.amino acids in a protein.

Organization of bacterial chromosome Prokaryotic DNA replicate, transcription

& translation

by Angelia Teo (Jan 09) 13

Prokaryote the genome of prokaryotes is not in a separate

compartment, haploid. Single chromosome: it is located in the cytoplasm (although sometimes confined to a particular region called a “nucleoid”). Prokaryotes contain no membrane-bound organelles; their only membrane is the membrane that separates the cell form the outside world. Nearly all prokaryotes are unicellular.


Eukaryotes are defined as having their genetic material enclosed in a membrane-bound nucleus,

separate from the cytoplasm. In addition, eukaryotes have other membrane-bound organelles such as mitochondria, lysosomes, and endoplasmic reticulum. almost all multicellular organisms are

eukaryotes.

Prokaryote cond..

Prokaryotes are haploid, and they contain a single circular chromosome. In addition, prokaryotes often contain small circular DNA molecules called “plasmids”, that confer useful properties such as drug resistance. Only circular DNA molecules in prokaryotes can replicate.


Eukaryotes are often diploid, and eukaryotes have linear chromosomes, usually more than 1.

Prokaryote cond..

In prokaryotes, translation is coupled to transcription: translation of the new RNA molecule starts before transcription is finished.


In eukaryotes, transcription of genes in RNA occurs in the nucleus, and translation of that RNA into protein occurs in the cytoplasm. The two processes are separated from each other.

2005-2006

Bacteria Bacteria review

one-celled organisms prokaryotes reproduce by mitosis

binary fission rapid growth

generation every ~20 minutes 108 (100 million) colony overnight!

dominant form of life on Earth incredibly diverse

2005-2006

Bacterial genome

Single circular chromosome haploid naked DNA

no histone proteins ~4 million base pairs

~4300 genes 1/1000 DNA in eukaryote

2005-2006

No nucleus! No nuclear membrane

chromosome in cytoplasm transcription & translation are coupled

together no processing of mRNA

no introns but Central Dogma

still applies use same

genetic code

Bacterial Chromosome

Molecules of double-stranded DNA Usually circular Tend to be shorter Contains a few thousand unique

genes Mostly structural genes Single origin of replication

20

Bacterial Chromosome cond.. The bacterial chromosome is found

in region called the nucleoid (not membrane-bounded- so the DNA is in direct contact with the cytoplasm)


Bacterial Chromosome cond..

The circularity of the bacterial chromosome was elegantly demonstrated by electron microscopy in both Gram negative bacteria (such as Escherichia coli) and Gram positive bacteria (such as Bacillus subtilis).

Bacterial plasmids were also shown to be circular.

Linear chromosomes found in Gram-positive Borrelia & Streptomyces.


Bacterial Genome is haploid, single chromosome

Not all bacteria have a single circular chromosome: some bacteria have multiple circular chromosomes, and many bacteria have linear chromosomes and linear plasmids.

The Operon Model


The operon model of prokaryotic gene regulation was proposed by Fancois Jacob and Jacques Monod. Groups of genes coding for related

proteins are arranged in units known as operons. An operon consists of an operator, promoter, regulator, and structural genes. The regulator

gene codes for a repressor protein that binds to the operator, obstructing the promoter (thus, transcription) of the structural genes.

The regulator does not have to be adjacent to other genes in the operon. If the repressor protein is removed, transcription may occur.

The Operon Model


The Operon Model


Extra-chromosomal Elements

DNA molecules that replicate as discrete genetic units in bacteria are called replicons.

Extrachromosomal replicons: - bacteriophages - plasmids (non-essential replicons)

These determine resistance to antimicrobial agents or production of virulence factors.


Angelia Teo Jan 09

Regulation of Gene Expression in Bacteria

Regulation of Gene Expression A cell contains the entire genome of an

organism– ALL the DNA. Gene expression = transcribing and

translating the gene Regulation allows an organism to

selectively transcribe (and then translate) only the genes it needs to.

Genes expressed depend on the type of cell the particular needs of the cell at that time.

How Are Genes Regulated?

Genes located in coherent packages called operons

operons has 4 parts regulatory gene - controls timing or rate of

transcription promoter - starting point operator - controls access to the promoter

by RNA polymerase structural genes

NOTE = operons regulated as units

Angelia Teo Jan 09

Most genes are not expressed at a particular time Not all of the genes in a bacteria will be

expressed at the same time. Even in some of the smallest bacteria,

about 500 different genes exists Of the 4279 genes in E. coli , only about

2600 (~60%) are expressed in standard laboratory conditions.

Only about 350 genes are expressed at more than 100 copies (i.e. molecules!) per cell, making up 90% of the total protein.

Angelia Teo Jan 09

Possible target in control of gene expression

How Do Mutations in DNA Affect the Function of Genes?

Mutations result from Nucleotide Substitutions, Insertions, or Deletions

Mutations may have a variety of effects on protein structure and function

Mutations provide the raw material for evolution

Chapter 10.

MUTATION Process of mutation may result in a

gene coding for a new protein Mutations are not good or bad, they do

provide the raw material of change. There are many different types of

mutations.

Chapter 10.

Nucleotide substitution can result in either a change or no change in amino acid of the codon.

MUTATION

Point Mutation - change in one nucleotide.

Insertion - new nucleotide pairs are inserted into DNA molecule

Deletion - nucleotide pairs are removed from DNA molecule

Chapter 10.

Different Types of Mutations

Plasmid

First plasmid described was discovered in Japan in Shigella species during an outbreak of dysentery in the early 1940‘s

3 main components:

• Origin of replication• Selectable marker• Restriction enzyme site(s)

• Enzymes that cut at specific sequence on DNA

Plasmid – small, circular, extrachromosomal DNA which replicates independently of host chromosomal DNA

Plasmids Content

Replication factors

Genes

Ori Region

Ori, actual site of replication

Proteins that assist in replication (varies)

Recognition sequences for control factors

The ori determines the Range

Plasmids

Discrete, extrachromosomal genetic elements in bacteria

Usually much smaller than bacterial chromosome Size varies from < 5kb to > 100 kbp Mostly supercoiled, circular, ds DNA molecules Replicate independently of the chromosome Exist in multiple copies in bacterial (the average

number of plasmid per bacterial is called copy number).

Usually encode traits that are non-essential for bacterial viability.

Plasmid is an ideal structure for genetic engineering because Simple in structure

Easy to extract & isolate in the lab

Easy for genetic manipulation & transformed back into bacteria

Contains genetic information which can be used by the bacteria

Most plasmid present in high copy number

Plasmid codes for antibiotic resistant gene eg. Ampicillin, Apr or Tetracyclin Tcr - selection of bacteria with transformed plasmid.

Non-essential for bacteria’s growth, thus possible to manipulate plasmid

DNA without affecting bacteria growth.

Exchange of Genetic Information in bacteria

Medically important - rapid emergence and dissemination of antibiotic resistance

plasmids - flagellar phase variation (eg. Salmonella) - antigenic variation of surface antigens (eg. Neisseria & Borrelia)

Sexual processes in bacteria involve transfer of genetic information from a donor to a recipient, results in:

- substitution of donor alleles for recipient alleles - addition of donor genetic elements to the recipient genome.

3 major types of genetic transfer found in bacteria: a) Transformation b) Transduction c) Conjugation In all three cases, recombination between donor and recipient DNA

result in formation of stable recombinant genomes

Types of transfers:

Non-transmissible = cannot initiate contact with recipient or transfer DNAConjugative = can initiate contact with recipient bacteriumMobilizable = can prepare its DNA for transferSelf-transmissible = is both conjugative & mobilizable

4 stages of plasmid transfer: a) Effective contact

b) Mobilization - preparation for DNA transfer c) DNA transfer d) Formation of F in recipient

Donation - a conjugative plasmid (F) can provide conjugative function to a mobilizable plasmid (eg. ColE1) such that both plasmids can be transferred.

Plasmid conduction - a self-transmissible plasmid (F) can recombine with a non-mobilizable plasmid and transfer the co-integrate.

Fin genes & plasmid transfer

fin genes - fertility inhibition

- codes for repressor that prevents transcription of genes

required for transfer.

F plasmid has 1 fin gene so transfer system is always ‘ON’

R plasmid has 2 fin genes so cannot always transfer. - in new recipients (repressor is absent) so transfer

can occur soon after receiving the R plasmid but after

time (when repressor is made) transfer can't occur

introducing DNA from donor to recipient result in uptake and integration of fragments of donor

DNA into recipient genome. produce stable hybrid progeny. is most likely to occur when the donor and recipient

bacteria the same or closely related species.

(a) Bacterial Transformation

"Transformation" is simply the process where bacteria manage to "uptake" a piece of external DNA. Usually, this process is used in the laboratory to introduce a small piece of PLASMID DNA into a bacterial cell.

Bacteria transformation in the lab

Bacteriophage infect donor bacterium form rare abnormal bacteriophage particles contain DNA

from donor bacteria. abnormal bacteriophage infect recipient bacteria & inject

DNA into recipient donor DNA integrated / recombined into recipient DNA

resulting in transduced bacterium.

Bacteria Transduction

Bacterial Conjugation

Transfer of DNA between 2 bacteria in contact with each other

Contact between donor and recipient (initiated by sex pili)

DNA transfer through a conjugation bridge Mediated by a plasmid

Called an F-factor (fertility factor) or conjugative plasmid

Several important properties of F

F is a self-replicating plasmid and is maintained in a dividing cellular population.

Cells carrying F produce pili, minute proteinaceous tubules that allow the F+ cells to attach to other cells maintaining contact.

F+ cells can transfer its F plasmid to a F- cell , turning the recipient cell into an F+ cell. F+ cells are usually inhibited from making contact with each other.

Occasionally, F can integrate into the host bacterial chromosome and transfer the host chromosomal markers to the recipient cell.

Bacterial CellsOr tissue culture cellsOr bloodOr flies………..

HOW?

Extract

Cells

Pure DNA

Organic extraction

Work with Plasmid DNAs

Isolation and Purification

After 10 hrs centrifugation at 100,000 rpm (450,000 xg), two distinct bands, corresponding to linear nuclear DNA above and circular mitochondrial DNA below, are visible under ultraviolet light.

Banding of plasmids and chromosomal DNAs in CsCl-EtBr and in iodixanol-DAPI gradients.

CsCl Gradient centrifugation or CsCl dye-bouyant density method

(d) collecting plasmid DNA by centrifugation (after ethanol precipitation or through filters - positively charged silicon beads),

(e) check plasmid DNA yield and quality (using spectrophotometer and gel electrophoresis).

Plasmid DNA Isolation continued

Midi Prep Mini PrepTranditional Ways

spectrophotometer and gel electrophoresis

DNA Quantification

The absorption spectra of DNA and RNA are maximal at 260 nm.

It is often a good practice to measure the absorption of nuclei acid solution at multiple wavelengths.

Most spectrophotometers used in research laboratories at the HKUST are equipped to measure absorbance at various wavelengths simultaneously.

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DNA, RNA and proteins carry negative charges, and migrate into gel matrix under electro-fields.

The rate of migration for small linear fragments is directly proportional to the voltage applied at low voltages.

At low voltage, the migration rate of small linear DNA fragments is a function of their length.

DNA Electrophoresis

At higher voltages, larger fragments (over 20kb) migrate at continually increasing yet different rates. Large linear fragments migrate at a certain fixed rate regardless of length.

In all cases, molecular weight markers are very useful to monitor the DNA migration during electrophoresis.

The process using electro-field to separate macromolecules in a gel matrix is called electrophoresis.

http://en.wikipedia.org/wiki/Electrophoresis

Conformations of Plasmid DNAs

Plasmid DNA may appear in the following five conformations:

Super Coiled

Linear DNA

SC

Relaxed region

Nicked DNAs

1) "Supercoiled" (or "Covalently Closed-Circular") DNA is fully intact with both strands uncut.

2) "Relaxed Circular" DNA is fully intact, but "relaxed" (supercoils removed).

3) "Supercoiled Denatured" DNA. small quantities occur following excessive alkaline lysis; both strands are uncut but are not correctly paired, resulting in a compacted plasmid form. 4) "Nicked Open-Circular" DNA has one strand cut.

5) "Linearized" DNA has both strands cut at only one site.

Conformation of Plasmid DNAs

The relative electrophoretic mobility (speed) of these DNA conformations in a gel is as follows:

Nicked Open Circular (slowest)

Linear

Relaxed Circular

Supercoiled Denatured

Supercoiled (fastest)

MB 206 : Module 2-C

Enzymes used in Gene Manipulation

Prepared by Angelia Teo 09

Enzymes used in gene manipulation

Aid in recombinant DNA technology. Originally identified and isolated from different bacteria

strains. Commercially available as highly purified recombinant

enzymes.


Enzymes used in gene manipulation

Enzymes used in gene manipulation, based on their functions – 5 classes:

1) Nucleases – cut or degrades DNA molecules

2) Polymerases – copy or make new strands of DNA

3) Ligases – joins pieces of DNA fragments together

4) Modifying enzymes – modify the DNA by adding or

removing chemical groups

5) Topoisomerase – remove or introduce supercoils from

covalently closed- circular DNA


Degrade DNA within DNA

a) Cleave DNA at nonspecific cleavage sites: - DNAase I (isolated from bovine pancrease) – digest dsDNA - mung bean nuclease (from sprouts of mung bean) – digest ssDNA b) Cleave DNA at sites specify by specific DNA sequences - Restriction endonucleases (RE)

Degrade DNA at either ends

Exonuclease III (Exo III) - cleave dsDNA

(Exo VII) – cleave ssDNA

Nucleases



blunt end sticky end

What is PCR History of PCR

How PCR works Optimizing PCR

Fidelity, errors & cloning PCR primer design Application of PCR

It’s a means of selectively amplifying a particular segment of DNA.

The segment may represent a small part of a large and complex mixture of DNAs: e.g. a specific exon of a human gene.

It can be thought of as a molecular photocopier. Now used for:

CloningAnalysis of gene expression

SNP detectionMutagenesis

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MB 206 Microbial Biotechnology