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Microbial Genetics
and biotechnology
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Define Terms Genetics Genome /
Genomics Chromosomes Gene Genotype Phenotype Recombination
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Control of Gene Expression
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DNA Structure Double stranded Nucleotide
Nitrogen Bases Sugar Phosphate
Base Pairs Hydrogen Bonds
A-T C-G
Alpha helix 5’ – phosphate group 3’ – hydroxyl group
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RNA Structure Single strand Nucleotide
Nitrogen base Sugar Phosphate
Base Pairs A-U C-G
Three types mRNA rRNA tRNA
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Prokaryotic Chromosomes Location
Nucleoid region No membrane
Number Most have 1 Some species have
2, the second linear Appearance
Circular Ds Loops and coils
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E. coli genome / chromosome
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DNA Replication Semiconservative
Replication fork Single origin Bidirectional
2 Leading strands 2 Lagging strands
Enzymes Helicases DNA polymerases 5’ to 3’
I for leading strand II digest RNA primer III for lagging strand
DNA ligase DNA gyrase
Hydrogen bonds broken and reformed Methylation of adenine bases
Initiation sites for replication Turn on or Turn off gene expression Protect against viral infections DNA repair
Polymerase I & II
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DNA Replication Enzymes
DNA Helicase DNA Primase DNA Polymerase DNA Gyrase Topoisomerases DNA Ligase
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Leading/Lagging Strands
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DNA Replication Overview
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Binary Fission
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Binary Fission
Asexual reproduction DNA replicated FTs proteins
Divisome apparatus Peptidoglycan Plasma membrane
Double numbers
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Plasmids
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Plasmids 2% of genetic information (5-100 genes) ds, circular extra chromosomal DNA Independent replication Cellular Traits
F-Fertility R-Resistance : inactivate AB, toxins, heavy
metals Dissimilation: catabolism of unusual substances Bacteriocins Virulence : enzymes, toxins, attachment
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Rolling Method for DNA replication and F-Plasmid
Rolling Method One strand remains
in loop Second strand
breaks away and rolls of loop
Both strands serve as templates for daughter strand
Occurs during conjugation
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Plasmid Integration (Episome)
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Transcription DNA RNA
mRNA rRNA tRNA
Initiation Sigma factor on RNA polymerase
binds to promoter sequence on DNA
Will be release after 10 nucleotides
RNA polymerase unzips, unwinds DNA Lacks proof reading ability
Elongation 5’ to 3’, slower
Ribonucleotide sequences Base pairs :
A-U [instead of Thymine] C-G
Termination Self
Terminator sequence G-C rich area
Protein-dependant Terminator protein Separates DNA and RNA
polymerase
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Sigma Factors for RNA polymerase
Where RNA polymerase binds to DNA
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Prokaryotic RNA Transcription = RNA Polypeptides RNA
mRNA Code for several polypeptides along
strand Each code has codons: Start and
Stop tRNA
Acceptor stem Anticodon Wobble
rRNA 70S Ribosomes
50S: 23S + 5S rRNA and 33 proteins
30S: 16S rRNA and 21 proteins Binding Sites on Ribosomes
A: accepts tRNA with AA P: holds tRNA for base pairing
anticodon to mRNA codon for polypeptide
E: release [Exit] for tRNA
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Translation Steps Initiation
30S tRNA @ P site 50S GTP used
Elongation New tRNA @ A site Ribozyme in 50S
forms peptide bond GTP used
Termination Release factor
proteins Stop codon on mRNA
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Importance of rRNA structures
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Regulation of Gene Expression Constitutive
Not regulated Always “on” at fixed rate
Transcription Translation
60-80% Polypeptides need in large
amounts Regulated
Only when needed Control synthesis of genes
for enzymes Induction Repression
Control enzyme activity: feedback Noncompetitive
inhibition Competitive inhibition
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Enzyme Reguation
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Operon Parts Operator
Controls access On/off
Promoter RNA polymerase
binds All or none
Regulator Genes at distant site
control transcription Repressor binds to
operator to block Structural genes
Code for enzymes
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Operon regions on DNA
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Operation of Operon
Gene expression
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Operator
Always “on” unless switched off by repressor
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Promoter Region
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Regulation of Operator Genes Negative Control
Repressor On/Off Interacts with operon Types
Inducible Repressible
Positive Control Activator protein Determines rate Directly interacts with
genome Facilitates
transcription
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General Regulatory Control
animation
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“Negative” Genetic Control of Enzyme synthesis and formation
Operon Model Operator (O) Promoter (P) Structural genes
Regulatory genes Makes repressor
Active binds to Operator Inactive unable to bind to
Operator Types
Inducible Operons Repressor Active Operon Off Inducer needed Catabolic Pathways
Repressible Operons Repressor inactive Operon On Corepressor needed Anabolic pathways
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Repressor Proteins Regulatory Control Gene
expression Binds to operator Cues from
metabolites Active form =
blocks Inactive form =
allows transcription
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Active Repressor Proteins
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Inactive Repressor Proteins
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Inducible Operon Repressor
Active Bound to operon
Operon off Need Inducer to
inactivate repressor Inducer
metabolite that can bind to repressor
Inactivates repressor Operon “induced” on
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Lac Operon: Inducible
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Use of Lactose
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Repressible Operon Repressor inactive Operon on Need co-repressor
Metabolite Binds to repressor Activates repressor
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TRP Operon: Repressible
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“Negative” Gene Control
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Which Regulatory Gene Operon is this?
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The “other” one Positive Control of
Gene Expression Catabolite
Activator Protein (CAP) Binds to promoter
region Enhance affinity for
RNA polymerase Stimulate gene
expression
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CAP (Catabolite Activator Protein)
cAMP binds and activates CAP (crp)
cAMP-CAP bind to promoter
Increase RNA polymerase affinity
Allow efficient transcription
Determines rateNo cAMP, no CAP binding, rate slows
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Mutations Define Types
Silent Point
Mis-sense Non-sense Sense [aka silent]
Substitution Transition: purine for purine Transversion: purine for
pyrimadine Frameshift
Insertions Deletions
Causes Spontaneous Induced
Chemical Physical
Conditional Adaptive Transposons
Thymine dimer (radiation)
Inversion(transposons)
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Repair of Mutations
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Transposons (Transposable elements)
DNA fragments within chromosomal DNA
Gene producing enzymes for insertion
Types Simple
Insertion only Complex
Additional genes Jumping genes Move
Within one chromosome
From one chromosome to another
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Transposon Jumping Patterns Conservative
Simple Not replicated Move pre-existing
Replicative Complex Copies Original in tact New site with new
genes (jump)
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Transposon copies
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Genetic Transfer & Recombination Vertical
Parent to offspring Horizontal
Lateral transfer to same generation
Donor to recipient DNA transfer Plasmid transfer Types
Transformation Transduction Conjugation
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Transformation Occurance
1% Random Naturally in certain species
Haemophilus Neisseria Pseudomonas Streptococcus Staphylococcus
Griffith experiment Genetic transfer
Environmental surroundings Naked DNA assimilated Competent cells
Cell wall Plasma membrane
Bacterial lysis DNA Plasmids
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Griffith Experiments
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Transduction Transfer of
bacterial genes via viruses Donor to recipient Virus:
Bacteriophages Types
Generalized Specialized
Replication Cycle Lytic Lysogenic
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Transduction
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Generalized Lytic Cycle Random pieces of
host cell DNA (any genes)
Packaged with phage during lytic cycle
Donor DNA combines with recipient
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Lytic Cycle Summary
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Specialized Transduction Cycle
Only certain specific bacterial genes are transferred
Example: Toxins Corynebacterium
Diphtheria toxin Streptococcus
pyogenes Erythrogenic toxin
E. coli Shiga-like toxin
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Randomgenestransferred
SpecificGenestransferred
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Lysogenic Cycle Summary
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Conjugation
Sex
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Conjugation Define Bacteria
Gram Neg : F.pilus Gram Pos: sticky
surface molecules Types
F+ [plasmid] R [plasmid] Hfr [DNA]
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Conjugative Plasmid
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Plasmid F-factor F = fertility
F+ = male F- = female
~25 genes Sex Pilus Replicates in
synchrony with host DNA
Rolling method DNA replicates from
parent If integrated with host
DNA becomes Hfr
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Hfr Interrupted Stages
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Other Plasmids R factors
Resistance to AB ~10 genes Different bacterial
species share Bacteriocin factors
Specific protein toxins Kill other cells of same
or similar species Virulence
Pathogenicity Structures Enzymes Toxins
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Conjugation: R Plasmid transfer
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Genetic Recombination General
Homologous chromosomes Any location DNA breakage and repair
Site Specific Non-homologous Viral genomes in bacterial chromosomes
Replicative Health and Food industries
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Recombinant DNA
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Genetic Engineering Use
Plasmids Recombinant DNA
Applications Therapeutic
Hormones Enzymes Vaccines Gene therapy
Agricultural Scientific
Southern Blot ELISA tests
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Biotechnology
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Questions?