Gim Lec Prelims - Reviewer

Chapter 6- Microbial Genomics -Separated by Gel Electrophoresis (based on
charges and size/molecular weight)

Genome- entire complement of genetic 1. PAGE- proteins
information that includes genes, genetic sequences 2. SDS-PAGE
and noncoding DNA 3. AGE- Nucleic Acids (DNA & RNA)
-reveals organisms function and Primers- short segments of DNA/RNA that

evolutionary history initiate synthesis of new strands of nucleic acids
RNA- in vitro
Genomics- discipline of mapping, sequencing, DNA- biotechnology
analyzing and comparing genomes
Automated DNA sequencing systems
New Advances in Genomics: -Bases are labeled with fluorescent dyes
- Automation if DNA sequencing -Based on Sanger concept
- Miniaturization of analytical procedures Sequencing by DNA synthesis rather than
- Development of powerful computational breakdown
methods for analysis of DNA and protein Dideoxynucleotides to block chain
sequence extension
Using labeled precursors for detection
First RNA virus 3,569 bp 1976
genome MS2 Shotgun Sequencing
sequenced -preparation of DNA for the sequencing
First DNA DNA virus 5,386 bp 1977 -much of the sequencing is redundant/repetitive
genome X174
sequenced Genomic library- molecular cloning of DNA
First Haemophilus 1,830,137 1995 fragments that cover the entire genome (7-10 fold
bacterial influenzae bp coverage)
genome (gram neg)
sequenced Second Generation DNA Sequencing
Generation- successive major changes in
technology (faster and less cost)
Largest genome sequenced (in terms of total # of
genes, more genes than humans- 25,000 coding -100x faster than Sanger
genes): - use of massively parallel methods
Black Cottonwood (45,000 genes) -very large number of samples are
Trichomonas (60,000 genes) sequenced side by side in same machine
- 2 requirement:
Linear chromosomes in several bacteria: -increased computing power
Borrelia burgdorferi- agent of Lyme disease -miniaturization
Streptomyces- antibiotic-producing
454 Life Sciences pyrosequencing
Genome Sequencing- determining the precise -DNA is broken into single stranded segments
order of nucleotides in a DNA or RNA molecule -DNA is amplified by PCR
-instead of termination (deoxyribonucleotide),
Sequence- order in which nucleotides are aligned pyrophosphate is released = energy for activity of
Luciferase (light-emitting enzyme)
-measures release of light that can handle only
First Generation DNA Sequencing short stretches of DNA
Sanger Dideoxy Method Illumina/Solexa sequencing

-invented by Fred Sanger -terminators are deoxy (rather than dideoxy)
-dideoxyribonucleotide is a specific chain ribonucleotide and can be reversibly incorporated
termination, lacks 3 OH that prevents it from -each of four deoxyribonucleotide carries its own
further elongation of DNA chain fluorescent tag that functions as a blocking group
-Bases are labeled with radioactivity for 3-OH, thus causing chain termination
SOLID/ Applied Biosystems methods

(2016:GIM Prelims) Arcilla, Cada, Misola, Montemayor, Ortega, Quilang, Sampang
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Closed Genome- entire genome sequenced
Third Generation DNA Sequencing is determined that relies on manpower
-sequencing of single molecules of DNA -more expensive
-2 main approaches: -time consuming
-based on microscopy -more information
-based on nanotechnology
Draft genome- dispensing with sequencing
the small gaps
Genome Annotation-conversion of raw sequence

data into a list of genes present in the genome
-bottleneck in genomics
HeliScope Single Molecule Sequencer
-fragments of single stranded DNA is attached in an Bioinformatics-use of computers to store
array on a glass slide and analyze DNA and protein sequences for
-complementary strands are synthesized comparative purposes
-Fluorescent tags are monitored on a microscope
-can monitor billion DNA fragments simultaneously
-computer assembles the fragments into a
complete sequence
functional Open Reading Frame (ORF)
Pacific Biosciences SMRT (single-molecule real- -in bacterial genomes
time) -yields a polypeptide/ encodes a protein
-uses zero mode waveguides technique -computer search for ORFs
-reactions are carried out inside nanocontainers -look for start/stop codons and Shine
-fluorescent tags are attached to the Dalgarno sequences
pyrophosphate group -functional if ORF sequence is similar to ORFs in the
-computer assembles the fragments into a genomes of other organisms and ORF includes a
complete sequence sequence known to encode protein functional
domain
Fourth Generation DNA Sequencing -the number of genes whose role can be clearly
-post light sequencing identified is approximately 70% of the total
-optical detection is no longer used number of ORFs detected
Ion torrent sequencing method Codon bias/ codon usage-codons that are
-measures release of protons whenever a new used more frequently than the others and differs
deoxyribonucleotide is added to a growing strand greatly between organisms
of DNA
-capable of sequencing an entire human genome in Hypothetical proteins- encoded by
less than a day uncharacterized or unknown ORFs (unknown
function)
Silicon chip- worlds smallest pH meter
Uncharacterized or unknown ORFs
Oxford Nanopore Technologies System -have uninterrupted reading frames
-Nanopore detectors are extremely narrow pores -proteins they encode lack sufficient amino
that permit single stranded DNA to pass through acid sequence homology with known protein
one at a time -likely to encode nonessential genes
-passes DNA through nanoscale biological pores -In E.coli, predicted to encode regulatory or
-a detector records the change in electrical current redundant proteins
Noncoding RNA
Genome Assembly- connecting DNA fragments in -untranslated RNA molecules = doesnt code for
the correct order and eliminating overlaps protein
-lack start codons or have multiple stop codons
-lack codon bias
-eg. tRNA, rRNA, noncoding regulatory RNA
molecules
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-Archaea and Bacteria have relatively large
numbers of genes whose functions are unknown or
that encode only hypothetical proteins
Microbial Genomes
Genomes of Organelles
Genome Size -Mitochondria and chloroplasts are organelles
-size and gene number are directly proportional derived from endosymbiotic bacteria found in
-prokaryotic gene (1,000 bp long) eukaryotic cytoplasm that contains a small genome
-share many fundamental traits related with
Autotrophs need only a few genes than Bacteria
Heterotrophs
Chloroplast Genome
Small Prokaryotic Genomes -all are circular DNA molecules
-belong to prokaryotes that are parasitic or -typically 120-160 kbp
endosymbiotic -contains 2 inverted repeats of 6-76 kbp
-obligate parasites range from 490 kbp -largest sequenced: Floydiella terrestris
(Nanoarchaeum equitans) to 4,400 kbp -some proteins in the chloroplast are encoded by
(Mycobacterium tuberculosis) nuclear genes
-endosymbionts can be smaller (eg. Carsonella -introns are common and are primarily of the self-
ruddii- 160 kbp) splicing type
-minimum number of genes for a viable cell is 250- -many genes encode proteins for photosynthesis
300 genes and autotrophy (C02 fixation)
Large Prokaryotic Genomes RubisCO- enzyme that catalyzes the key

-can be as large as eukaryotes = more genes than step in CO2 fixation in Calvin cycle
eukaryotes
-largest bacterial genome: Sorangium Cellulosum rbcL- large subunit that is always
(12.3 Mbp) present on the chloroplast
-largest archaeal tend to be smaller (5Mbp)
rbcS- small subunit that resides in
the nucleus
Gene Content
-complement of genes in a particular organism Mitochondrial Genomes
reveals its capabilities -can be linear or circular
-genomes are molded by adaptation to an -smaller than chloroplast genomes
organisms lifestyle -primarily encode proteins for oxidative
-metabolic genes are typically the most abundant phosphorylation
class in prokaryotic genomes -larger in plants than in mammals
-the smaller the genome, the greater the -may contain plasmids (in fungi)
percentage of genes that encode translational Variability in Genetic Code
processes -Genetic code is not universal
-the percentage of an organisms genes devoted to -animal mitochondria uses stop codons as sense
a particular function is to some degree a function codons
of genome size -Mycoplasma (Bacteria) and Paramecium (Eukarya)
-many genes can be identified by sequence use certain stop codons to encode amino acids
similarity to genes found in other organisms
Comparative analysis- allow for prediction Symbionts and Organelles
of metabolic pathways and transport systems -many insects and other invertebrates contain
-Archaea devote a higher percentage of their symbiotic bacteria
genomes to energy and coenzyme than Bacteria -no longer capable of independent existence
-Archaea appear to contain fewer genes for -genomes of some symbionts contain fewer genes
carbohydrate metabolism or membrane functions -host cannot survive without symbiont
than Bacteria
-fewer genome sequences are available from Eukaryotic Microbial Genomes
species of Archaea -largest eukaryotic genome (parasite)- Trichomonas
Most important eukaryotic parasite:
Plasmodium- causes malaria
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-smallest eukaryotic genome- Encephalitozoon 3. Scanned and Analyzed by a computer
cuniculi (causes lung infections) Hybridization between a specific RNA and a
-as for prokaryotes, smallest eukaryotic genome- DNA segment on the chip indicates that the
nucleomorph (degenerate remains of a eukaryotic
gene is transcribed
endosymbiont)
Yeast Genome *mRNA must be measured when studying

-about 2/3 of yeast ORFs encode proteins whose protein- encoding gene (if few mRNA, it
functions are known must be amplified)
-about 900 ORFs are essential known by knockout - Photolithography process used to
mutations produce computer chips and microarray
chips (1-2 cm and can hold thousands of
Knockout mutations- mutations that completely
inactivates genes different DNA fragments)
-by generating knockout
mutations in diploid cells and then investigating APPLICATIONS OF GENE CHIPS: GENE
whether they can also exist in haploid cells = EXPRESSION
determine whether a particular gene is essential Global gene expression is monitored by
for cell viability assembling an array of oligonucleotides
complementary to each gene in the genome
FUNCTIONAL GENOMICS
using the entire population of mRNA as the
Transcriptomics refers to the global study of test sample
transcription and is done by monitoring the total - a part of the chip used to assay gene
RNA generated under chosen growth conditions. expression in SACCHAROMYCES
CERIVISIAE
Transcriptome the complement of all RNA
- chip holds 6000 protein-encoding genes
produced in an organism under a specific set of
of S. cerevisiae
conditions
- chip has been used to study metabolic
Two approaches: control of S. cerevisiae
1.) Microarrays APPLICATIONS OF GENE CHIPS:

IDENTIFICATION
- are small solid supports to which genes or
segments of genes are fixed and arrayed spatially - array contains set of characteristic DNA
in a known pattern (Gene chips) sequences from each of the organisms
or viruses
-- depend on DNA-RNA hybridization (Nucleic acid - can differentiate species by their
hybridization) hybridization patterns
- allows very rapid identification of
pathogenic viruses or bacteria
- can reveal how pathogenic bacteria
evolved from a non-pathogenic relatives
- Nucleic Acid probes: a labeled strand of nucleic
- Phylochips: used to asses microbial
acid that can be used to hybridize with a
diversity, contain oligonucleotides
complementary strand of nucleic acid in a mixture
complementary to 16s rRNA
(identity is known)
- FoodExpert-ID (commercial chip)
1. Gene segments can be synthesized by PCR contains 88,000 gene segments and is
or oligonucleotides are designed and used to monitor food purity in food
synthesized for each gene based on industry, can detect vertebrate by-
genomic sequence products in animal feed (that can cause
2. Attachment to the solid support then DNA prion-mediated infections)
segments can be hybridized with RNA
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2. RNA-Seq Analaysis Comparative Genomics and Proteomics
Computational techiniques:
- method in which all the RNA molecules
from a cell are sequenced 1. Sequence the genome of an organism
- the genome sequenced must be 2. Compare to genomes of other organisms
available for comparison that reveals 3. Identify similar genes
the number and what genes are 4. Different DNA sequence may not change
transcribed protein sequence
- used to measure expression of mRNA
and identify and characterized small Proteins with >50% sequence similarity same
non coding RNAs functions
- beginning to overtake microarray
Proteins with >70% sequence similarity have
analysis
almost certainly have same functions
- requires high throughput sequencing
- Clostiridium in exponential and Protein domains proteins consist of distinct
Clostridia in stationary phase were structural modules
compared
Structural proteomics proteome-wide
- used for microbial community analysis
determination of the three-dimensional structures
and can provide information on relative
transcription levels (when a genomes Interactome complete set of interactions among
sequence is not available for the macromolecules within a cell; interaction
comparison) between proteins
- can also be used in metagenomics
Metabolomics and Systems in Biology
Proteomics and interactome
Metabolome complete set of metabolic
Proteomics is the genome-wide study of intermediates and the other small molecules
structure, function and activity of an organisms produced in an organism
proteins
Mass spectrophotometry (MALDI-TOF)
Proteome all the proteins encoded by an technique to monitor metabolites
organisms genome
MALDI- Matrix assisted laser desorption
Two-dimensional (2D) polyacrimide gel ionization
electrophoresis the technique used for TOF time of flight
separating, identifying, and measuring all the
proteins present in the sample Systems Biology integration of different fields of
research to give a overview of an organism or cell
First dimension (horizontal) proteins are or even entire species or ecosystems; compares
separated by differences in isoelectric data and builds a computer model of the system
points being studied
Second dimension (vertical) proteins are
separated by size Integration of different fields of research
Liquid Chromatography used to separate protein 1. Genomics

mixtures 2. Proteomics
3. Transcriptomics
HPLC- sample is forced under pressure through a 4. Metabolomics
column packed with a stationary phase
METAGENOMICS
Mass spectrophotometry used to identify eluted
proteins - Also called environmental genomics

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- Analyzes pooled DNA or RNA from an Gene analysis three domains of life suggests that
environmental sample containing many genes present in all organisms have common
organisms that are not isolated and evolutionary roots
identified
Horizontal gene transfer and Genome Stability
- May explore pattern of gene expression
in natural microbial community In prokaryotes,
Metagenome total gene content of organisms Horizontal gene transfer (lateral gene transfer)
inhabiting an environment complicate analysis of genomes; refers to transfer
from one cell to another by means other than the
EXAMPLES:
usual (vertical) inheritance process in which
- Proteobacteria at a sampling site close genome is transferred from mother to daughter
to Hawaii in the pacific ocean cell.
Metagenomics and biome studies Extensive in nature

May cross phylogenetic domain boundaries
Microbiome: collection of prokaryotic cells
- Most prokaryotes in the gut

- Bacteroidetes and Firmicutes
- The more firmicutes (clostridium and
etc), the more fat the human or mouse
is
- Fungi such as Saccharomyces and
Candida Detectiong horizontal gene flow
- Gut fungi: Aspergillus and Trichosporon
Presence of genes found only in distantly
(inflammatory bowel disease)
related species
- Holds for probing between diseases and
Presence of DNA with GC content or codon
microbial populations In human and
bias that differs significantly from
animals
remainder of genome
Evolution of Genomes
Horizontally Transferred gene do not encode
Gene families, duplications and deletions core metabolic functions
Homologous genes/homologs genes that are Mobile DNA segments of DNA that move from
related due to shared evolutionary ancestry one location to another within host DNA
molecules; it consists of transposable elements
Gene families- group of homologous genes; it
contain both paralog and ortholog Transposons common forms of mobile DNA that
move between different host DNA molecules,
Paralogs genes whose similarity is the result of including chromosomes, plasmids, viruses by the
gene duplication at some time in the evolution of activity of an
an organism
enzyme transposase
Orthologs genes found in one organism that are
similar to genes in another organism because of Insertion sequences pieces of transposable DNA
descent from a common ancestor whose genes encode only transposition
Gene duplication mechanism by which most of Core genome vs Pan genome

new genes evolve
Core genome
Gene deletions eliminate gene no longer needed - Shared by all strains of a given species
- Typical of the species as a whole
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Pan genome Transduction, and Conjugation.
- Includes the core plus all of the optional
extras present in one or more strains but not all of - Chromosomal islands gradually lose their ability
strains of the species to move due accumulation of mutations when
-Restricted to particular strains within a inserted into the genome of a new host cell.
species
- The size of the Pan genome increases as Pathogenicity islands and the Evolution of
the genomes of more strains of a species are Virulence
sequenced. - Comparison of the genomes of pathogenic
bacteria vs. harmless relatives shows that
Horizontal gene transfer of entire genetic elements chromosomal islands that encode virulence factors,
such as plasmids, viruses, or transposable elements special proteins or other molecules or structures
is possible. that help initiate disease.
Chromosomal Islands Pathogenecity Islands

- Contains clusters of genes for specialized - Contains most of the virulence genes while
functions that are not needed for simple survival some are carried on plasmids or lysogenic
- Two strains of the same bacterial species bactriophages.
may show significant differences in genome size - Best known of the chromosomal islands
- Encode the biodegradation of pollutants - Regarded as a subclass of chromosomal
such as aromatic hydrocarbons and herbicides. islands
- Carries the essential genes for the
symbiotic relationship of rhizobia with the root - Presence or absence of of the pathogeneticity
nodules of plants. island could result to differences in genome size of
- Magnetospirillum two strains of the same bacterial species.
-A magnetosome island
- Carries genes that encode for the Staphylococcus aureus Has small pathogenecity
formation of magnetosomes, intracellular magnetic islands that encode a series of virulence factors.
particles used to orient the organism in a magnetic - Smaller than the phage genome.
field and influence the direction of its movement.
- Presumed to have foreign origin - When the islands excise from the chromosome
Why? and replicate, they induce the formation of
1) These extra regions are often flanked by defective phage particles that carry the genes for
inverted repeats, implying that the whole region the islands but are too small to carry the phage
was inserted into the chromosome by genome.
transposition. - Hence strains of S. aureus that lack the islands can
2) The base composition and codon bias in quickly obtain them and become more effective
chromosomal islands often differ significantly from pathogens.
that of the genome proper.
3) Chromosomal islands are found in some strains
Chapter 11 Genetic Engineering and
of a particular species but not in others.
Biotechnology
Genetic Engineering use of in vitro techniques to
- Chromosomal islands are typically inserted into a alter genes in the laboratory; may be reinserted
gene for tRNA; however, because the target site is into the original source organism or into some
duplicated upopn isertion, an intact tRNA gene is other host organism
regenerated during the insertion process.
Restriction enzymes or restriction endonucleases
- Transfer can presumably occur by any of the an enzyme which recognizes specific base
mechanisms of horizontal transfer: Transformation, sequences (recognition sequences) within DNA and

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cut phosphodiester backbone, resulting double- the positive electrode
stranded breaks. -gel meshwork: hinders the progress of the DNA,
-rare in eukaryotes and small or compact molecules migrate more
-protect prokaryotes from hostile foreign DNA, rapidly than large molecules
such as virus genomes -ethidium bromide: stains gel; compound that
-used for in vitro DNA manipulation binds to DNA; can make DNA fluoresce under UV
-major tool for genetic engineering light
-protect the cell from invasion by foreign DNA, -DNA ladder standard sample consisting of DNA
especially viral DNA fragments of known sizes; for comparison to
-each is partnered with a corresponding determine the size of the DNA
modification enzyme that shares the same
Nucleic acid hybridization or hybridization
recognition sequence (protection from restriction)
formed by complementary base pairing; used in
--once restriction recognition sequences are
detecting, characterizing, and identifying segments
chemically modified, it can no longer be cut by the
of DNA and RNA;
corresponding restriction enzyme
-useful for finding related sequences in different
--consists of methylating specific bases within the
genomes or other genetic elements and to
recognition sequence, which prevents the
determine if a gene is expressed into an RNA
restriction endonuclease from binding. Enzymes
transcript
are called methylases
- used to detect the presence of specific genes in
Three major classes genomes that have not yet been sequenced, as
well as the movement of genetic elements such as
Type I and Type III restriction enzymes bind to
a transposon
the DNA at their recognition sequences but cut
- also the basis of fluorescence in situ hybridization
DNA at some distance away
(FISH)
Type II restriction enzymes cleave the DNA
-on the resulting colonies using a nucleic acid probe
within their recognition sequences
can detect recombinant DNA in colonies; uses
-recognizes short inverted repeats (palindromes)
replica plating to produce a duplicate of the
of 4 to 8 base pairs
master plate on a membrane filter
EcoRIs endonuclease activity
Nucleic Acid probes or probes segments of
-E. Coli, strain RY13, Restriction Enzyme I
single-stranded nucleic acids whose identity is
-makes staggered cuts leaving short, single-
already known
stranded overhangs (sticky ends) at the ends of the
-to allow detection, probes are made radioactive or
2 fragments
labelled with chemicals that are colored
-sticky ends: beneficial for molecular cloning DNA
-Look for binding of labeled nucleic acid probe to
EcoRV
DNA from specific colonies
-cut both strands of the DNA directly opposite each
other resulting in blunt ends Southern Blot - a hybridization procedure where
DNA is in the gel and probe is RNA or DNA
Gel electrophoresis separation of fragments
-DNA is first denatured to yield single strands;
created after the DNA is cleaved; used to verify if
transferred to a synthetic membrane
the amplification of a nucleic acid was successful
-denaturant is added to the gel to prevent the
-separates charged molecules by migration in an
formation of secondary structures
electrical field (rate is based on the size and shape)
-membrane will be exposed to a labelled probe
Northern Blot RNA is in the gel; analogous

-agarose: gel, polysaccharide, separating DNA procedure; often used to identify mRNA
fragments; higher conc. greater resistance to
Fluorescence in situ hybridization (FISH) - a range
movement for larger molecules
of different fluorescent signals can be covalently
-electrical current: nucleic acid will move towards
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linked to oligonucleotide (short single-stranded 5. Looking for a specific gene
DNA or RNA molecules) probes to target specific
DNA sequences; variations in color give a visual Reverse transcription PCR (RT PCR)- Can make
indication of the specificity and power of nucleic DNA from an RNA template
acid probes. -Uses the enzyme reverse transcriptase
Quantitative PCR (q PCR) - Uses fluorescent probe
polymerase chain reaction (PCR) is essentially DNA to monitor the amplification process
replication in vitro RNaseH - a ribonuclease specific for the hybrid
-can copy segments of DNA by up to a billionfold in molecule, hydrolyzes the RNA, leaving the cDNA as
the test tube, a process called amplification template for standard PCR using an additional
-DNA polymerase - enzyme which naturally copies primer complementary to the 5 end
DNA molecules
Molecular cloning - isolation and incorporation of a
-amplifies stretches of up to a few thousand base
piece of DNA into a vector so it can be replicated
pairs (the target) from within a larger DNA
and manipulated
molecule (the template).
-result: recombinant DNA - DNA molecule that
The steps in PCR amplification of DNA contains DNA from two or more sources
-Vector - small, simple, and manipulable genetic
1. Template DNA is denatured by heating. element, such as a plasmid or virus
2. Two artificial DNA oligonucleotide primers Steps in Gene Cloning
flanking the target DNA on each strand are added
in excess. This ensures thatmost template strands 1. Isolation and fragmentation of source
anneal to a primer, and not to each other, as the DNA
mixture cools -Source DNA can be genomic DNA,
RNA, or PCR-amplified fragments
3. DNA polymerase then extends the primers using -Genomic DNA must first be
the original DNA as the template restriction digested
4. After an appropriate incubation period, the

mixture is heated again to separate the strands, 2. Insertion of DNA fragment into cloning
vector
but now the target gene is present in twice the
-Most vectors are derived from
original amount. The mixture is then cooled to
plasmids or viruses
allow the primers to hybridize with complementary
-DNA is generally inserted in vitro
regions of newly synthesized DNA, and the whole
-DNA ligase: enzyme that joins two
process is repeated
DNA molecules
Thermocycler automated PCR machine -works with sticky or blunt ends
DNA polymerase
3. Introduction of cloned DNA into host
Taq Polymerase Thermus aquaticus; organism
thermostable DNA polymerase; unaffected by the -Transformation is often used to get
denaturation phase in the PCR recombinant DNA into host
Pfu polymerase Pyrococcus furiosus; -Some cells will contain desired
hyperthermophile; has a proofreading activity cloned gene, while other cells will
have other cloned genes
Applications of PCR -Gene library: mixture of cells
containing a variety of genes
1. Phylogenetic studies -Shotgun cloning: gene libraries
2. Surveying different groups of made by cloning random genome
environmental organisms fragments
3. Amplifying small amounts of DNA
4. Identifying a specific bacteria
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-Essential to detect the correct clone Gene Disruption - occurs when cassettes are
-Initial screen: antibiotic resistance, plaque inserted into the middle of the gene
formation -causes knockout mutations: gains antibiotic
-Often sufficient for cloning of PCR-generated DNA resistance and loses genes function
sequences
Reporter Gene encodes a protein that is easy to
-If working with a heterogeneous gene library, you
detect and assay
may need to look more closely
-used to report the presence or absence of a
-If cells express the foreign gene and its expression
particular genetic element or DNA inserted within a
can be detected, then screening is relatively easy
vector
Antibodies can be used to detect a protein of -fused to the promoter of other genes so that gene
interest; proteins of the immune system that bind expression can be studied
in a highly specific way to a target molecule, -lacZ: e.coli gene, encodes the enzyme -
antigen (protein encoded by the cloned gene) galactosidase, required for lactose catabolism
-Blood serum proteins produced by animals -Xgal: cleaved by -galactosidase to yield a blue
-Made by injecting animal with specific protein color
antigen -Green fluorescent protein (GFP):widely used as a
reporter gene; a protein that glows green and is
X-ray film placed over the filter and exposed, and
widely used in genetic analysis
radioactive colonies appear as spots on the X-ray
film Gene fusions - a structure created by joining
together segments of two separate genes, in
Molecular methods of mutagenesis
particular when the regulatory region of one gene
Synthetic DNA is joined to the coding region of a reporter gene
-Systems are available for de novo synthesis of - Promoters or coding sequences of genes of
DNA interest can be swapped with those of reporter
-Oligonucleotides of 100 bases can be made genes to elucidate gene regulation under various
-Multiple oligonucleotides can be ligated together conditions
-Synthesized DNA is used for primers and probes,
- used in studying gene regulation, especially if
and in site-directed mutagenesis
measuring the levels of the natural gene product is
Site-directed mutagenesis difficult, expensive, or time consuming.
-uses synthetic DNA plus DNA cloning techniques -used to test for the effects of regulatory genes
to introduce mutation into genes at precisely 2 types:
determined sites -Operon Fusions - coding sequence that retains its
-performed in vitro and introduces mutations at a own translational signals is fused to the
precise location transcriptional signals of another gene
-Can be used to assess the activity of specific amino -Protein fusions - two coding sequences are fused
acids in a protein so that they share the same transcriptional and
-Structural biologists have gained significant insight translational start sites
using this tool
-Transcriptional control is assayed by fusing the
DNA cassettes synthtetic fragments used to transcriptional start signals of the gene of interest
mutate DNA to a reporter gene
-an artificially designed segment of DNA that -translational control is assayed by fusing
usually carries a gene for resistance to an antibiotic translational start signals of a gene of interest to a
or some other convenient marker and is flanked by reporter gene under the control of a known
convenient restriction sites promoter.
Casette Mutagenesis creating mutations by the
Cloning - allows the genetic engineer to isolate
insertion of DNA cassette
genes of interest away from their host genomes

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and insert them into carrier molecules where they versatility of the vector.
can be more easily manipulated or otherwise 8.It has a gene conferring ampicillin resistance.
studied 9.It can be inserted into cells easily by
transformation.
Plasmids are natural vectors and have useful
10.Insertion of foreign DNA into the MCS can be
properties as cloning vectors; often encode other
detected by bluewhite screening because of lacZ.
genes that confer important properties on their
hosts Blue-White Screening
-Small size; easy to isolate DNA -Blue colonies do not have vector with foreign DNA
-Independent origin of replication inserted
-Multiple copy number; get multiple copies of -White colonies have foreign DNA inserted;
cloned gene per cell successful
-Presence of selectable markers
Insertional inactivation: lacZ gene is inactivated by
- Vector transfer is carried out by chemical
insertion of foreign DNA; used to detect the
transformation or electroporation
presence of foreign DNA within the vector or
-Conjugative plasmids transferred by cell-to-cell recombinant vector; Inactivated lacZ cannot
cotact in nature; prevents unwanted movement of process Xgal; blue color does not develop
vector into the other organisms
Other plasmid vectors
pUC19 is a common cloning vector -Some vectors developed for cloning DNA products
-Modified ColE1 plasmid made by PCR
-Contains ampicillin-resistance and lacZ genes -Some vectors select for recombinant DNA using
-Contains multiple cloning site within lacZ gene viability
-multiple cloning sites (MCS) - short segment of Characteristics of Ideal hosts:
artificial DNA containing cut sites for many 1. Capable of rapid growth in inexpensive
restriction enzymes; short segment of artificial DNA medium
containing cut sites for many restriction enzymes; 2. Nonpathogenic
opens the vector at a unique location but does not 3. Capable of incorporating DNA
cut the vector into multiple pieces 4. Genetically stable in culture
5. Equipped with appropriate enzymes to
allow replication of the vector
Most useful hosts for cloning are microorganisms

that are easily grown and for which we have much
information.
Characteristics:
Eg, Escherichia coli, Bacillus subtilis, and
1.It is relatively small
Saccharomyces cerevisiae
2.It is stably maintained in its host (E. coli) in
relatively high copy number Prokaryotic Hosts
3.It can be amplified to a very high number; - E coli is an excellent choice for initial cloning work
necessary by inhibiting protein synthesis with the BUT it is problematic as an expression host because
antibiotic chloramphenicol. it is found in the human intestine and some wild-
4.It is easy to isolate in the supercoiled form. type strains are potentially harmful.
5. Moderate amounts of foreign DNA can be - A major problem with using ANY bacterial host is
inserted, although inserts of more than 10 kilobase the lack of systems to correctly modify eukaryotic
pairs lead to plasmid instability. proteins.
6.The complete base sequence of the plasmid is - E coli, like all gram-negative bacteria, has an outer
known membrane that hinders protein secretion.
7.The MCS contains single cut sites for over a - This can be resolved by using the gram-positive B
dozen restriction enzymes, thus increasing the subtilis as cloning host instead.
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- Disadvantage: plasmid instability. It is often - Another requirement is a convenient marker to
difficult to maintain plasmid replication over many select for the plasmid in the yeast. A functional
subcultures of the organism. Also, foreign DNA is copy of the biosynthetic gene that is defective in
not as well maintained in B subttilis as in E coli the host is inserted into the shuttle vector.
thus, the cloned DNA is often unexpectedly lost.
Expression Vectors
- Host organisms must have specific genotypes to
- Tackles the problem of having poorly expressed
be effective.
genes in a foreign host cell.
ie. If the cloning vector uses the lacZ gene for - Allows the experimenter to control the expression
screening, then the host must either naturally lack of cloned genes.
lacZ gene or carry a mutation that has disabled this Objective: To obtain high levels of expression,
gene. especially in biotechnological applications.
However, when it comes to potentially toxic gene
Eukaryotic Hosts
products, a low but strictly controlled level may be
- Mainly on S cerivisiae
appropriate.
- Advantage: Already possess the complex RNA and
- Contain regulatory sequences that allow
post-translational processing systems required for
manipulation of gene expression.
the production of eukaryotic proteins.
- In practice, high levels of transcription require
- Disadvantage: Mammalian cells are very
strong promoters that bind RNA polymerase
expensive and difficult to produce under large-
efficiently. However, the native promoter of a
scale conditions.
cloned gene may work poorly in the new host.
- Insect cell lines are easier to grow, and vectors
- Expression vectors must contain a promoter that
have been developed from an insect DNA virus
functions efficiently in the host and one that is
the baculovirus
correctly position to control transcription of the
Shuttle Vectors and Expression Vectors cloned gene.
- Specialized vectors have been engineered to
= Promoters from E. coli that are used in
optimize expression of cloned genes in a given
expression vectors include lac (the lac operon
host; these are called shuttle vectors and
promoter), trp (the trp operon promoter), and tac
expression vectors
and trc (synthetic hybrids of the trp and lac
promotes)
Shuttle Vectors Regulation of Transcription from Expression

Vectors
- Vectors that can replicate and are stably
maintained in two (or more) unrelated host - Massive overproduction of foreign proteins can
organisms. damage the host cell.
- Genes carried by this can be moved between - The culture containing the expression vector is
unrelated organisms. usually grown to avoid damaging the host cells
- Importance: Allows for DNA to be easily cloned in
- Genetic switch triggers the expression of the
a genetically manipulable organism and yields a
cloned gene by regulating transcription with the
recombinant vector that can replicate in a second
help of a repressor protein.
host without any vector modifications.
- Bacterial plasmid vectors were the starting point Strong ribosome-binding site often included
and were modified to function in yeast as well. between the promoter and the cloned gene to
Since bacterial origins of replication do not allow for efficient translation.
function in eukaryotes it is necessary to provide a
yeast replication origin. - Overall result: Control of the cloned gene by the
- Shuttle vectors for eukaryotes must contain a chosen promoter together with efficient
segment of DNA from the centromere in order to transcription and translation.
be properly distributed at cell division.
12
Regulation Expression with Bacteriophage T7 Other Cloning Vectors
elements
Cloning with Bacteriophage Lambda
- In some cases the transcriptional control system
may not be a normal part of the host at all. Bacteriophage Lambda- useful cloning vector
- Bacteriophage T7 promoter and T7 RNA because:
polymerase: both for used to regulate gene
expression. 1. its biology is well understood,
2. it can hold larger amounts of DNA than

most plasmids, and
- When T7 infects E. Coli, it encodes its own RNA
polymerase that recognizes only T7 promoters. 3. DNA can be efficiently packaged into phage
- In T7 expression vectors, cloned genes are placed particles in vitro
under control of the T7 promoter.
- The gene for T7 RNA polymerase must also be
present in the cell under the control of an easily Phage Lambda- large number of genes however,
regulated system, such as lac.
Third lambda genome- not essential for infectivity
- Done by integrating the T7 RNA polymerase with
and can be replaced with foreign genome; twice
a lac promoter into the chromosome of a
the cloning capacity of typical small plasmid
specialized host strain.
vectors
- The BL21 series of E. Coli especially designed to
work with the pET series of T7 expression vectors. To facilitate the use of Lambda as molecular
- Cloned genes are expressed shortly after T7 RNA cloning vector,
polymerase transcription has been switched on by
a lac inducer, such as IPTG. 1. many of its restriction enzyme sites have
-- Since it recognizes only T7 promoters, the T7 been altered and
RNA polymerase transcribes only the cloned genes. 2. multiple cloning site (MCS) containing gene
- The T7 RNA is highly active that it uses most of for beta-galactosidase has been added to
the RNA precursors which limits the transcription select recombinant vectors
to the cloned genes.
Bacteriophage as cloning vector
Translation of the Cloned Gene
1. insertion of foreign DNA to lambda DNA
-mRNA produced must be efficiently translated. modified to contain an MCS within a lacZ
- To synthesize protein from an mRNA molecule, it gene and subsequent packaging of a
is essential for the ribosomes to bind at the correct recombinant infective lambda virion (max.
site and begin reading the correct frame. size of inserted dna -20kbp)
- If there is a high level of expression of the 2. portion of Xgal-containing agar plate
eukaryotic gene, a bacterial RBS must be showing
engineered into the vector.
- Codon usage related to the concentration of the white plaques formed by lambda phage
appropriate tRNA in the cell. containing cloned DNA and
- If a cloned gene has a codon usage pattern blue plaques formed by phage lacking
distinct from that of its expression host, it will be cloned DNA
probably be translated inefficiently in that host.
Cosmid vectors- employ specific lambda genes
amd are packaged into lambda virions; plasmid
-If the cloned gene contains introns, the correct
vectors containing the cos site for lambda genome
protein product will not be made if the expression
which yields cohesive ends when cut
host is a prokaryote.
Advantages:

13
1. can be used to clone large fragments of
DNA with inserts as large as 50kbp
Protein purification facilitated by fusion of target
2. Permit storage of DNA in phage virus protein with carrier protein
instead of as plasmids
Somatotropin and other Mammalian Proteins
Virions are more stable than plasmids
Biotechnology industry has developed genetically
Artificial Chromosome- can carry very large engineered microorganisms to produce many
segments of DNA; this allows the size of the different mammalian proteins
genome library to be manageable
Somatotropin- consists of single polypeptide
F plasmid has been used to engineer cloning encoded by a single gene, and a shortage of
vectors called Bacterial artificial chromosomes somatotropin in the body results hereditary
(BACs); dwarfism (Dwarfism- lack of the somatoropin
-contains cat gene which confers chloramphenicol receptor)
resistance on the host and multiple cloning site
Recombinant bovine somatotropin used in dairy
Yeast Artificial Chromosomes (YACs)- vectors that industry; stimulates milk production for cows
replicate in yeast like normal chromosomes but
Somatotropin has 2 binding sites:
have sites where very large fragments of DNA can
be inserted. YACs must have 1. One binds to the somatotropin receptor
and stimulates growth
1. Origin of DNA replication
2. The other binds to prolactin receptor and
2. Telomeres for replicating DNA at the ends
promotes milk production
of chromosomes
3. Centromere for segregration during mitosis

Other Mammalian Protein
Expressing MAmmalian Genes in Bacteria
Tissue plasminogen activator(TPA) - a protein that
Problems in cloning a mammalian gene in bacteria
dissolves blood clot in the bloodstream that may
1. Eukaryotic gene must be placed under form the final stages of the healing process; used in
control of a bacterial promoter heart patients
2. Any introns must be removed Transgenic Organisms in Agriculture and

Aquaculture
3. Codon bias may require edits to gene
sequences Genetically engineered plants contain transgene - a
gene from another organism
4. Many mammalian proteins require
modification after translation to yield the Ti plasmid- for the gram negative plant pathogen,
active form, and bacteria cannot perform Agrobacterium tumefaciens; can be used to
most such modifications transfer DNA directly into the cells of certain plants
Solutions: T-DNA- segment of the ti-plasmid DNA that is

actually transferred to the plant
1. Cloning the gene via mRNA
Plants can be genetically modified through:
2. Finding the gene via the protein
1. Electroporation
3. Protein Folding and Stability
2. Particle gun methods
4. Fusion proteins for improved purification

14
3. Use of plasmids from bacterium - cells infected by recominbinant vaccinia virus
Agrobacterium tumefaciens (with disrupted tdk gene) grow long enough to
yield a new generation of virions = look for viruses
Areas targeted for genetic improvements in plants- with tdk genescontains a cloned insert of foreign
resistance to herbicides, insects and microbial DNA
disease as well as improved product quality - dont really need thymidine kinase to replicate
- can still infect human cells and express any
One widely used approach for genetically foreign genes they carry
engineered insect resistance in plants involves - can be engineered to carry genes from multiple
viruses
introducing: Bacillus thuringiensis- genes encoding
the toxic protein
SUBUNIT VACCINES
- contain only a specific protein or two from a
Transgenic animals- used for improving livestock
pathogenic organism
and other animals for human consumption
- For a pathogenic virus, this is the coat protein
GENETICALLY ENGINEERED VACCINES *coat protein are highly immunogenic and are
purified and used to elicit a rapid and high level of
Recombinant Vaccines immunity
- Genetic Engineering can be used in vaccine - can be used to produce large amounts of
development by modifying the pathogen itself immunogenic proteins without intact of pathogen
- genes can be deleted from a virus or bacteria that * If vaccines are poorly immunogenic it is
is pathogenic nonglycosylated
- genes can also be added from a pathogenic virus *Glycosylation is necessary for the proteins to be
to another immunologically active
- Eukaryotic host is necessary for the glycosylation
VECTOR VACCINES
- vaccine made by inserting genes from a ENVIRONMENTAL GENE MINING
phatogenic virus to a harmless carrier virus Gene Mining: process of isolating potentially useful
novel genes from the environment without first
POLYVALENT VACCINES culturing the organisms that carry them
- vaccine that immunized against two different - DNA/RNA is isolated directly from environmental
disease at the same time. samples and cloned into suitable vectors to
construct a genomic library
*if RNA, need to be converted to DNA by reverse
transcriptase
- lipases such as chitinases, esterases have been
isolated by this approach
Example: - Enzymes with improved resistance to industrial
> Fowlpox virus - a pox virus production conditions (pH, temperature and
> Newcastle disease - viral disease that is often oxidation) are desirable and valuable
fatal - BACs (Bacterial artificial Chromosome) can carry
- was first modified by deleting virulence genes but large inserts of DNA and are useful for screening
not those that elicit immunity. The immunity samples from rich environments (such as soil)
inducing genes from the Newcastle virus were
inserted inti the modified fowlpox virus
Viccinia Virus - widely used to prepare ENGENEERING METABOLIC PATHWAYS

recombinant vaccine for human use.
- not pathogenic Pathway Engeneering process of assembling a
- used as a vaccine against smallpox virus new or improved biochemical pathway using genes
- thymidine kinase - gives a selective marker from one or more organisms
required for cloning -most are done with bacteria
- 5 - bromodeoxyuridine was produced by the *genetic engineering of bacteria is simpler than of
converation of thymidine to thymidine higher organisms
triphosphate which kills cells that express
thymidine kinase

15
- engineered microbes are used to make products CHAPTER 15
(alcohol, solvent, food additives, dyes and Industrial Microbiology
antibiotics) - products are produced on a large scale by
- may be also used to degrade agricultural waste, microorganisms
pollutants, herbicides and other toxic undesirable - contrasts with biotechnology
materials
EXAMPLE: production of Indigo from E.coli Industrial Products and the Microorganisms That
Make Them
SYNTHETIC BIOLOGY
-Refers to the use of genetic engineering to create
novel biological systems out of available biological
parts biobricks (promoters, enhancers,
operators, riboswitches, regulatory proteins,
enzyme domains, signal receivers)
EXAMPLES:
Mycoplasma capricolum cell first self-
replicating synthetic bacterium by synthetic Desirable Properties of an Industrial Microorganism
biologists in J. Craig Venter Insitute in 1. Capable of growth and product formation
California on a large scale culture
photographs produced by E.coli 2. It should produce spores or other
- required biology of 3 genetic modules reproductive cell.
1. a light detector and signaling module 3. Able to grow in liquid culture medium
2. a pathway to convert heme obtainable at large quantities at a low price
3. an enzyme encoded by a gene whose 4. Not pathogenic
transcription can be switched on and off 5. Amenable to genetic analysis
to make the dark pigment Many industrial microbiological processes use
*light detector is a fusion protein waste carbon as major/supplemental ingredients
*outer half is the light detecting part of for large scale culture media. These include:
the phytochrom protein = Synechocystis
*inner half is the signal transmission 1. Corn steep liquor
domain of EnvZ sensor protein from - Rich in N2 and growth factors
E.coli - Product of corn wet-milling industry
*EnvZ activates the DNA-binding 2. Whey
protein OmpR then activated OmpR - Contains lactose and minerals
binds to the promoter - Waste liquid of dairy industry
- pigment made by E.coli results from -
the activity of B-galactosidase (lactose Production and Scale
degrading enzyme)-> lacZ (target gene) Microbial metabolites of interest to industrial
encodes this enzyme Microbiology:
*only in the dark where lacZ is
expressed and B-galactosidase is made. 1. Primary metabolite
- enzyme cleaves to lactose analog Xgal o grows during exponential growth
present in growth medium to release phase of microorganism
galactose and a black dye o (ex) alcohol forms in parallel w/
* this is not applicable in the light growth
Ethanol product of
fermentative metabolism of
yeast and certain bacteria

16
Small vessel (1-10 liters) test temp, pH, and other
parameters at low cost
Pilot plant stage (300-3000L fermenters) close to

actual commercial
2. Secondary metabolite
o forms near end of growth; commercial fermentor
at/in/near stationary phase
o non essential for growth and Commercial fermentor (10,000-500,000L)
reproduction; formation is highly
dependent on growth condition Is penicillin a primary or a secondary metabolite?
o often produced as a group of closely How can you tell by looking at Figure 15.1?
related compounds; often Secondary metabolite; forms near stationary phase
overproduced What are the size differences among a laboratory
o products of spore-forming fermentor, a pilot plant fermentor, and a
microorganisms commercial fermentor? How is proper aeration
ensured in a large-scale fermentation?
o Penicillum chrysogenum Use of sparger and impeller for proper aeration;
mold; produces penicillin small vessel (1-10 litres), pilot plant ( 300-
not made until after 3000litres)
exponential phase What parameters in an industrial fermentation are
typically monitored and why would adjustments
Fermentors and Characteristics of Large-Scale need to be made in real time by automated
Fermentations systems?
During an actual production run, fermentors are
Fermentor vessel where the industrial monitored in real time for temperature, oxygen,
microbiology process is carried out pH, and the levels of key nutrients, such as
ammonia and phosphate. This is done because it is
Fermentation refers to any large-scale microbial often necessary to alter the conditions in the
process fermentor as the fermentation progresses.
Computers are used to process environmental data
Bc sterilization and removal of heat are vital, as the fermentation proceeds and are programmed
fermenters are fitted with: to respond by signaling for nutrient additions,
increases in the rate of cooling water, impeller
- external cooling jacket steam (sterilization) speed or sparger pressure, or changes in pH or
and water (cooling) can be run other parameters, at just the right time to maintain
- internal coil for very large fermentors; high product yield.
steam and water can be piped
Antibiotics: Isolation, Yield, and Purification
*O2 is poorly soluble in H2O; fermentors with high Antibiotics
density of microbial cells require large amount of - produced by microorganisms; kill or inhibit
oxygen; aeration system is critical growth of bacteria
- typically secondary metabolites
to ensure adequate equation - produced by filamentous fungi or bacteria
1. sparger of the Actinobacteria group
aerator; series of holes; produce
bubbles Cross-streak method
2. impeller - to assay antibiotic production
stirring device; mixes gas bubbles
made by sparger; mixes organisms to
ensure uniform access to nutrients
Scale-up transfer of process from a small-scale
laboratory equipment to a commercial large-scale
lab equipment
Laboratory flask
17
- Produced in 20,000-400,000 litre
fermenters
Production:
o Highly aerobic
o Begins once carbon source is nearly
exhausted
o Corn steep liquor as major ingrdt
o High glucose = repress production
o High lactose = does not repress
Lactose (from whey)

- Added to corn steep liq in large amounts as
a carbon source
Pyrogens Production of tetracylines

- cause severe or fatal rxns in patients Tetracycline
treated with co-purified (w/ pyrogen) - Contains four-membered naphtacene ring
drugs/antibiotics
Chlortetracyline
Penicillins - Streptococcus aureofaciens (producing
- class of beta lactam antibiotics organism in chlortetracycline fermentation)
- produced by Penicillum and Aspergillus - Its synthesis is repressed by both glucose
- uses strains of mold Penicillum and phosphate
chrysogenum - Corn steep liquor and sucrose rather than
lactose as carbon source
other beta lactam antibiotics Catabolite repression

o cephalosporins (Cephalosporium - Transcriptional control mechanism where
acremonium) glucose is strongly avoided
o 6-aminopenicillanic acid (6-APA)
- Parent structure of penicillins What are the major groups of microorganisms that
- Has thiazolidine ring w/ condensed beta produce antibiotics?
lactam ring Actinobacteria
- Variable side chain in position 6 What is meant by the word screening in the
context of nding new antibiotics?
Natural penicillin carried out without sidechain By using indicator organisms, screening allows for
precursor the detection and isolation of microorganisms
producing antibiotics (when there are zones of
Biosynthetic penicillin inhibition).
- produced by adding side chain precursor to What chemical structure is common to both the
culture medium penicillins and the cephalosporins?
Beta lactam ring
Semisynthetic penicillin Why would corn syrup not be useful in the
- produced by treating natural penicillin with production of tetracyclines?
6-APA, then chemically modified by adding Lol corn syrup contains glucose which represses the
side chains production of antibiotics
- they are broad-spectrum antibiotics
useful against a Vitamins and Amino Acids
variety of pathogenic Vitamins
bacteria - Supplements for food and animal feeds
can be taken orally - Vitamin B12 and Riboflavin are most
and do not require important
injection
- (ex) ampicillin widely prescribed drug Vitamin B12
Penicillin G
18
- Synthesized in nature exclusively by o ^ done by isolating mutants
microorganisms; required as growth factors resistant to S-aminoethylcysteine
by all animal (AEC), a lysine analog which binds to
- (as a coenzyme) imp. role in methyl allosteric site of aspartokinase and
transfers inhibits its activity
o AEC-resistant mutants are obtained
Pernicious anemia and modified to form modified
- Vitamin B12 deficiency aspartokinase whose allosteric site
- Low production of RBCs and nervous no longer recognizes AEC or lysine.
system disorders
Which amino acid is commercially produced in the
Propionibacterium freudenreichii and greatest amounts?
Pseudomonas Glutamic acid
- Main commercial producers Why is a mutant derivative of the bacterium
Corynebacterium glutamicum required for
Metal Cobalt commercial lysine production?
- Increases production of vitamin B12 mutant deriv no feedback inh
Riboflavin Enzymes as Industrial Products

- Parent compound of flavins FAD and FMN Exoenzymes
Coenzymes; - excreted into envt
imp role for - digest insoluble polymers
redox rxns Proteases
- used as additives in laundry detergents
o Ashbya gossypii naturaly produces (also amylase and lipases)
riboflavin - help remove stains by degrading polymers
into water soluble components
Amino Acids
- Used as additives (food and animal Alkaliphilic bacteria
husbandry) - where enzymes (optima 9-10pH; remain
- Used as nutritional supplements ( active bc of alkaline ph of laundry det) are
natraceutical) mostly isolated
- Bacillus (main producing); like Bacillus
Glutamic acid licheniformis
- Flavor enhancer (Monosodium Glutamate,
MSG) Amylase and glucoamylase
- Produced by Corynebacterium glutamicum - Used in production of glucose from starch
- Amylase + glucose isomerase to convert
To overproduce corynebacterium glucose to fructose; final product is high-
- Starved for biotin; membrane weakens and fructose syrup
becomes leaky and susceptible to
glutamate excretion Extremozymes
o Biotin imp for fatty acid synthesis - Produced by extremophiles
- Capable of functioning at envtal extremes
Asp and Phe
- Synthesizes aspartame, a non nutritive and Immobilized enzyme
non carbohydrate artificial sweetener - When enzyme is attached to a solid surface
Lys
- Also produced by Corynbacterium 3 ways of immobilizing
glutamicum 1. Enzymes can be bonded to a carrier
- Can feedback inhibit aspartokinase activity 2. Enzymes can be linked by a chemical rxn
- Production is controlled at the level of with a cross-linking reagent (dilute
aspartokinase glutaraldehyde)
- To overproduce.. 3. Enzymes can be enclosed in
o Isolate C. glutamicum mutants mircocapsules
where aspartokinase is no longer
subject to feedback inhibition How are enzymes of use in the laundry industry?
19
they degrade polymers into h20 soluble
components
Alcoholic Beverages and Biofuels

Wine
Dry wines Malic acid
- sugars of juices are almost completely o Sharp, bitter
fermented o Fermented into lactic acid
o Soft and
Sweet wines smoother
- some of the sugar is left or additional sugar acid; more
is added after fermentation complex and
palatable
Fortified wine o Catalyzed by species of lactic acid
- brandy or other alcoholic spirit is added bacteria (Lactobacillus, Pediococcus,
after fermentation e.g. sherry and port Oenococcus) which are extremely
acid-tolerant
Sparkling Wine
- CO2 is present e.g. champagne Brewing and distilling
Brewing
Wine Production - Production of alcoholic beverages from
Must malted grains
- Juice from crushed grapes
White wine (Chabilis, Rhine Wine, sauterre, Malt
chardonnay) - Prepared from germinated barley seeds;
- From white grapes or skinless grapes has natural enzymes that digests starch of
grains and convert it to glucose
Red wine (Burgundy, Chianti, claret, zinfandel, Mashing
cabernet, merlot) - Process in preparing fermentable liquid for
- Pomace(skins, stems, seeds) are left in brewing
- Stronger flavor bc more tannins (chemically - Necessary to digest starch
extracted into juice from the grape skins
during fermentation When mash is heated, enzymes from the mash
will digest starch and produce glucose, which will
2 Types of Yeast be fermented by the yeast
1. Wild yeast
o Present on grapes Wort
o Less alcohol tolerant; produce - Aqueous mixture
undesirable compounds
o Sodium metabisulfite (Na2S2O5) kill
wild yeast
2. Strains of cultivated wine yeast, Hops
Saccharomyces ellipsoideus - Herb from female flowers; added to wort
after mashing
Racking - Adds flavor to wort; has antimicrobial
- wine is separated from sediment containing ppties
yeast cells and precipitate
Brewery Yeast Strains
bouquet odor 1. Top fermenting
o Remain uniformly distributed in
Malolactic Fermentation wort and carried to the top by CO2
- 2ndary fermentation gas
- Full-bodied dry red wines o Used in brewing of ales
o Saccharomyces cerevisiae
o Ferment at higher temp (14-23
degrees)
o Shorter fermentation time
20
Biofuel
2. Bottom fermenting - Fuel is made from fermentation of plant
o Settle at the bottom material
o Used to make lager beers - Biodiesel (made from vegetable oils)
o Saccharomyces carlsbergensis - Algal fuels (from green algae)
Gasohol
- Produced by addition of 10% ethanol to
Home brewing gasoline
- Same as commercial brewing except hop- - Produce lower amount of carbon monoxide
flavored malt extract is used directly as the and nitrogen oxide
wort
E-85
- EtOH rich fuel
- 85% ethanol, 15% gasoline
Most beers contain 3-6% alcohol - On modified engines only
American style lager 3.5% - Reduces nitrogen oxide emission by 90%
Munich-style dark 4.5% Petroleum Biofuels
Bock beers 5%
Ales more than 12%
Botrycoccus braunii
Distilled alcoholic beverages - Excretes long-chain hydrocarbons
- Distillates, product of heating fermented - Crude oil consistency
liquids to volatilize alcohol - 30% of cell dry weight is petroleum
Distilling How can yeast help to solve global energy

- Process where distillate is condensed and problems?
collected Yeast produces ethanol via fermentation. Ethanol
- Higher alcohol content is obtained can be used as biofuel
Brandy Products from Genetically Engineered

- From distilled wine Microorganisms
Insulin
Whiskey - First commercialized product
- From distilled malt brews
Major problems revolved around the challenge of
Rum expressing eukaryotic genes in bacteria.
- From distilled fermented molasses 1. the genes must be placed under control of
a bacterial promoter
Vodka 2. the introns must be removed;
- From distilled fermented grain or potatoes 3. codon bias affects the efciency of
translation; and
Gin 4. many mammalian proteins are modied
- From distilled fermented grain and juniper after translation, and bacteria lack the
berries ability to perform most such modications
To eliminate undesirable flavor, product is aged, Expressing Mammalian Genes in Bacteria

usually in oak barrels. Aging times commonly 5-10 Expression vectors
years, if expensive, 20 or more. - to express eukaryotic genes in bacteria
Gene Cloning via mRNA

To achieve very high levels of expression of
Biofuels eukaryotic genes in prokaryotes, the expressed
Ethanol gene must be free of introns. This can be
- Most important global biofuel accomplished by synthesizing cDNA from the
- Brazil as major producer mature mRNA encoding the protein of interest or
by making an entirely synthetic gene.
21
from which it was derived naturally
Mature mRNA
- uninterrupted coding sequence - contructed genes are free of introns; mRNA
does not need processing
Eukaryotic mRNA has poly(A) tail at 3 end which
makes it easy to isolate. If a cell extract is passed Protein Folding and Stability
over a chromatographic column containing strands Protein synthesis in foreign host is subject to other
of poly(T) linked to a cellulose support, most of the problems:
mRNA separates from other RNAs by the specic - degradation by intracellular proteases
pairing of A and T bases. The RNA is released from - toxicity of eukaryotic proteins to
the column by a low-salt buffer, which gives a prokaryotic host
preparation greatly enriched in mRNA. - formation of inclusion bodies
Reverse transcription
- conversion of an RNA sequence into the Fusion Proteins for Improved Purification
corresponding DNA sequence Fusion protein
- enzyme: reverse transcriptase - genetically engineered protein made by
fusing 2 DNA sequences encoding different
Newly synthesized cDNA has hairpin loop; bc after proteins together into a simpler gene
enzyme copies mRNA, it starts to copy the new - 2 genes fused to yield single coding
DNA sequence
- loop provides convenient primer for - Short segment is recognized and cleaved by
synthesis of new cDNA (by DNA pol I); later protease
removed by single strand specific nuclease - Then, release target protein from carrier
protein
Result is double-stranded DNA, one strand is - Carrier protein can be chosen to have ideal
complementary to mRNA; contains coding properties for purification
sequences; lacks introns; can be inserted into
plasmid Production of Genetically Engineered Somatotropin
Insulin
cDNA library - First human protein made via genetic
- contains sequences found in a mRNA engineering
population from a particular tissue and
represents only those genes expressed in
that tissue.
Somatotropin
Finding the Gene via the Protein - Growth hormone; single polypeptide
encoded by single gene
- knowledge of amino acid sequence of a - Cloned as cDNA from mRNA
protein can be used to make a probe or
synthesize a new gene Dwarfism
- Lack of somatotropin (may be treated with
- amino acid sequence of protein can be used recombinant human somatotropin)
to design and synthesize oligonucleotide - Also absence of somatotropin receptor (no
probe that encodes it effect in administration of somatotropin)
- complicated by degeneracy of genetic code Recombinant bovine somatotropin (rBST)

(kaya many probes are possible for a given - Used in dairy industry; stimulate milk
polypeptide sequence) so, the best region production but does not make them grow
of gene to synthesize as a probe is the one larger
that encodes the protein rich in amino acid - somatotropin has 2 binding sites
specified by only one codon. o To the somatotropin receptor
stimulates growth
- to produce short peptide hormone like o To the Prolactin receptor
insulin, it is better to construct the artificial stimulates lactation
gene that encodes for the final hormone - Use of site-directed mutagenesis to
rather than the larger precursor protein generate somatotropin that does not bind
22
to prolactin receptor (used to remedy - Made by insertion of genes to pathogenic
human growth defect) to avoid side effect virus to another harmless virus called a
of prolactin activity carrier virus
- Several amino acids needed for binding to - safer than killed vaccines bc it is impossible
prolactin receptor were altered to transmit disease in vaccine
- more reproducible bc genetic makeup can
Other Mammalian Proteins and Products be monitored
Tissue plasminogen activator (TPA) - prepared faster
- Blood protein; scavenges and dissolves - less expensive
blood clots
- Used by heart patients or others suffering Polyvalent Vaccine
from poor circulation to prevent - Immunizes against 2 different diseases
development of clots
-Vaccinia virus
Blood clotting factors VII, VIII, and IX o Not pathogenic for humans; widely
- Important in formation of blood clots used to prepare recombinant
- For hemophiliacs who suffer from the vaccine
deficiency of one or more blood clotting o Against related smallpox virus
factors o Cloning genes to vaccinia requires a
selective marker, provided my
DNAse I thymidine kinase
- Treat DNA-containing mucus in patients o Thymidine kinase
with cystic fibrosis Converts thymidine to
- Formation of mucus is often accompanied thymidine triphosphate
by lung-infections by P. aeruginosa; form Also converts 5-
biofilms within the lungs which make drug bromodeoxyuridine to a
treatment difficult nucleotide
- DNA is released when bacteria lyses Lethal rxn
- DNAse digests DNA and decrease viscosity Cells expressing this enzyme
of mucus are killed by 5-
bromodeosyuridine
Alginate Lyase
- Other treatment for cystic fibrosis Subunit Vaccine
- Degrades polysaccharide produced by P. - contain only a specic protein or two from a
aeruginosa pathogenic organism
- Also contributes to lung mucus; its - protein is coat protein which is highly
hydrolysis relieves respiratory symptoms immunogenic; coat protein is purified and
used in high dosage to elicit rapid and high
Rennet level of immunity
- Enzyme used to make cheese - used to produce large amount of
- Product of slaughtered animals immunogenic proteins without possibility of
Recombinant Rennet purified product containing the pathogenic
- Produce vegetarian cheese organism
Genetically Engineered Vaccines
Vaccines
- Elicit immunity to particular disease when
injected to animal
- Suspensions of killed or modified
pathogenic microorganisms or viruses
- Surface protein elicits the immune response
Recombinant Vaccines
- Delete pathogen genes that encode Preparation:
virulence factors; leave those who elicit - fragmentation of viral DNA by restriction
immune response enzymes
- cloning viral coat protein genes into a
Vector vaccine suitable vector
23
- providing proper promoters, reading frame, o Need to find hot envt polluted by
and ribosome-binding sites; and target compound, the pollutant
- reinsertion and expression of the viral o Assuming microorganism capable of
genes in a microorganism degradation of target compound is
there, DNA from envt would be
DNA Vaccines isolated and cloned
- genetic vaccine o Host bacteria (like E.coli) containing
- use genome of pathogen itself to immunize the clones would be screened for
the individual growth on the target compound
- key genes which encode for immunogenic o Once identified, enzyme extracts are
protein is cloned into plasmid or viral vector tested in vitro at high temp
and delivered by injection
o when DNA is taken by animal cells; it Engineering Metabolic Pathways
may be degraded or transcribed and Production of small metabolites by genetic eng
translated typically involves multiple genes that must be
o if translated, protein produced is coordinately expressed.
immunogenic, thus animal is
effectively immunized against the Pathway engineering
pathogen - Process of assembling new or improved
- immune response is made against the biochemical pathway using genes from one
protein encoded by the vaccine DNA. The or more organisms
DNA itself is not immunogenic. - (ex) production of indigo by E.coli
- Escape surveillance by host immune system - Indigo
bc nucleic acids are poorly immunogenic; o Blue Dye used for treating wool and
prevents autoimmune disease cotton
- DNA is more stable than vaccines; avoid o Blue jeans (cotton dyed with indigo)
need for refrigeration o Previously extracted from plants but
is now produced chemically
o Structure is very similar to
naphthalene; enzymes that
Mining Genomes oxygenate naphthalene also oxidizes
Metagenome indole to form indigo
- Collective genomes of an environment o Such enzymes are present on
plasmids found in Pseudomonas and
Gene Mining other soil bacteria; genes from
- Process of isolating potentially useful novel plasmids were cloned into E. coli;
genes from the environment blue cells had picked up the genes
- Instead of being cultured in the lab, DNA or from the enzyme, naphthalene
RNA is taken directly from environment oxygenase
sample and cloned into suitable vectors to - 4 steps in indigo pathway
construct metagenomics library o 2 enzymatic, 2 spontaneous
o If RNA is isolated, convert to DNA
first via reverse transcriptase; time
consuming
Bacterial Artificial Chromosomes (BACs)

- Vector; able to carry large inserts of DNA
Targeted Gene Mining

- Metagenomics can screen directly for
enzymes with certain properties
- (ex) you need enzyme capable of degrading
a certain pollutant at high temperature

24
Improving Livestock and Other Food Animals
- pigs genetically engineered to express the
reporter gene that encodes the green
uorescent protein are, as expected, green
- genes that encode the enzymes of the

metabolic pathway for making methionine,
a required amino acid, could remove the
need for this amino acid in the animals diet
- fast-growing salmon - the promoter for

the growth hormone gene was replaced
with the promoter from another sh that
grows at a more or less constant rate all
year round
- insertion into pigs of a gene from

Transgene Escherichia coli that helps degrade organic
- Gene from one organism that has been phosphate. The resulting Enviropig no
inserted into a different organism longer needs phosphate supplements in its
feed
Transgenic Organism
- Contains a transgene, foreign DNA Gene Therapy in Humans
- Related term is genetically modified
organism (GMO), but GMOs may or may Human hereditary diseases
not contain foreign DNA Gene Tharapy
- non functional or dysfunctional gene in a
Genetic Engineering of Animals person is replaced by a functional gene
- use of microinjection to deliver cloned genes to - strictly speaking, it is NOT the GENE that is
fertilized eggs replaced but its FUNCTION
o actual gene replacements in germ
Genetic recombination incorporate foreign DNA line cells cannot be applied to
into genome of the egg humans; raise ethical questions
- Therapeutic wild-type gene is inserted in
Mice the genome; its gene product corrects the
- First transgenic animals genetic disorder
Transgenic Animals in Pharming Severe combined immune deficiency (SCID)

Pharming - Caused by the absence of adenosine
- Process where genes coding for proteins of demeanase (ADA)
pharmaceutical value are inserted into host - ADA
animals o Enzyme of purine metabolism in
bone marrow cells
Transgenic Animals in Medical Research - Crippled immune system
knockout mice - First genetic disease approved for gene
- have had both copies of a particular gene therapy
inactivated by genetic engineering o Uses retrovirus as a vector to carry
Myostatin wild-type copy of ADA gene
- protein that slows muscle growth o T cells are removed
- if the mice lacks both copies, it would o Retrovirus carry marker gene,
develop massive muscles encoding resistance to antibiotic,
- if the mice would overproduce this protein, neomycin; T cells carrying inserted
it would show reduced muscle mass retrovirus may be identified
25
o Engineered Tcells are then placed
back into body
o T cells have limited life span so
therapy must be repeated every few
months
o (for infants) ADA gene is inserted to

stem cells from umbilical cord blood;
bc stem cells continue to provide
fresh supply of T cellslong term
cure
Transgenic Plants in Agriculture

Plants can be genetically modified via:
- Electroporation
- Particle gun methods
- Use of plasmids or bacterium
Agrobacterium tumefaciens
- A. tumefaciens
o Naturally transfers DNA directly into
the cells of certain plants
o Gram negative plant pathogen
o Contains large, Ti Plasmid Herbicide and Insect Resitance
o Responsible Tobacco
for virulence - First GM crop produced commercially
o Contains - Engineered for resistance to viruses
genes that
mobilize DNA Main GM crops today:
for transfer - Soybeans, corn, cotton, and canola
o to the plant
o Contracts Herbicide resistance
crown gall - Engineered to crop plant for protection
disease from herbicides applied to kill weeds
Herbicide glyophosate (RoundupTM)

T-DNA - Kills plants; inhibit enzyme producing
- Segment of Ti Plasmid that is transferred to aromatic amino acids
the plant gene
Insect Resistance: Bt toxin
Binary vector B. thuringiensis
- Common Ti vector system; used for - Produces Bt toxin, a crystalline protein
transfer of genes to plants; does not work Bt toxin
with monocots - Toxic to moth and butterfly larvae
- Two plasmid system - Pest control in plants
- Has cloning vector and a helper plasmid
o Cloning vector contains 2 ends of Bt transgene
DNA flanking a multiple cloning site - Inserted directly into plant genome
o 2 origins of replication so it can o Natural Bt toxin is cloned into
replicated both E.coli and A. plasmid vector under chloroplast
tumefaciens rRNA promoter
o 2 antibiotic resistance markers; one o Transferred in plant chloroplast by
for selection in plants and the other microprojectile bombardment
for bacteria - Prob.. may kill helpful, nontarget insects,
resistance to herbicides and insecticides
- Harmless to mammals, including humans
o Cooking food destroys the toxin
26
o Ingested toxin is digested; - Shewanella and Geobacter species (metal
inactivated in GI tract of mammals reducing bacteria), Desulfovibrio species (sulfate
o Bt toxin binds to specific receptors reducing) COUPLE THE OXIDATION OF ORGANIC
in insect intestine that are absent in MATTER AND H2 TO THE REDUCTION OF
other organisms URANIUM
*If U4+ (uraninite) is exposed to oxic conditions, it
Other Uses Of Plant Biotechnology oxidizes.
Genetic engineering can also be used to develop
GM plants that are more nutritious, and have PETROLEUM AND HYDROCARBON
characteristics desirable to consumers. BIOREMIDATIOBN
- Petroleum is a rich source of organic matter =
GM Tomato microorganisms easily attack hydrocarbons when
- Spoilage is delayed; increase shelf life petroleum comes into contact with air and
- First GM grown in US moisture
- in bulk petroleum tanks, microbial growth is
Transgenic plants can also be employed to produce undesirable and can be promoted by addition of
human proteins for pharmaceutical use inorganic nutrients
- E.g. human protein, interferon - In Oxic conditions, oxygenase (enzymes)
- Plantibodies (antibodies) have potential as introduces oxygen into the Hydrocarbon thus
anticancer or antiviral drugs making the Hydrocarbon remediation effective in a
- CaroRx (antibody) blocks bacteria causing short time
dental caries from attaching to teeth - Hydrocarbon oxidation us most extensive if the
Also for production of vaccines.. temperature is warm enough and supplies of
- P. vivax proteins elicit immune response to inorganic nutients (N and P) are sufficient.
humans; used to produce vaccine for
malaria Oil oxidizing grows in oil films and slicks
- Plasmodium vivax - oil is insoluble in water and floats to the surface
o parasite causing malaria and forms slicks
- the hydrocarbon - degrading bacteria attaches to
- edible vaccines the droplets and decomposes the oil and disperses
o amflora potato the slick
transgenic plant - Alcanivorax borkumensis: oil degrading bacteria
not intended for eating that grows on Hydrocarbons, fatty acids and
makes only amylopectin, no pyruvate, it produces surfactant chemicals that
amylose break up the oil and solubilize it
approval is still under - if oil is solubilize, it can be catabolized as an
consideration electron donor and carbon source
BIOREMEDIATION *In large oil spills, microbes consume oil by

- Refers to the clean up of oil, toxic chemicals, or oxidizing it to Carbon dioxide
other environmental pollutants
*Pollutants: petroleum products, xenobiotic DEGRADATION OF STORED HYDROCARBONS
chemicals, synthetic chemicals produced by - Sulfate - reducing bacteria can grow in tanks and
organisms. can consume hydrocarbons under anoxic
conditions
Common goal of bioremediation: to change the - H2S (sulfide) produced is corrosive and causes
mobility of inorganic materials, making them less pitting and subsequent leakage tanks with souring
likely to move with groundwater and contaminate of the fuel
the surroundings - Aerobic degradation are sealed and has little
dissolved O2 which makes it less of a problem
BIOREMEDIATION OF URANIUM
- reduction of uranium 6+ to uranium 4+ BIOREMEDIATION OF ORGANIC POLLUTANTS
- Uranium 6+ is soluble
- Uranium 4+ is an immobile uranium (uraninite) PESTICIDE CATABOLISM
- Uranium 4+ is better because it cannot move in - Xenobiotics (pesticides, polychlorinated
the groundwater biohenyls, munitions, dyes and chlorinated

27
solvents) differ chemically and biodegrade very Chapter 18
slowly. Methods in Microbial Ecology
- Pesticides (herbicides, insecticides,
fungicides) include chlorinated, aromatic, nitrogen
and phosphorus containing compounds, and can be Microbial ecology focused on how
used as Carbon and Energy sources of microbial communities interact with each
Microorganisms other and their environment
*Highly chlorinated = most resistant to microbial - Major components: biodiversity and
attack (sample: 2,4-D) microbial activity
- Environmental factors (temperature, pH,
aeration, organic content of the soil) influence the I. Culture-Dependent Analyses of Microbial
rate of pesticide decomposition Communities
- some pesticides can disappear by Volatilization, - More than 99% have never been grown in
leaching, spontaneous, chemical breakdown laboratory cultures
- Some pesticides degrade with the presence of an Culture-dependent analyses tools and
organic material that could be used as a primary methods used in culturing (enrichment) in
energy dource (cometabolism) a selective growth medium
- Cometabolism isnt always good because it could
vreate new microbes that are more hard to Culture-independent techniques that can
degrade because the pesticides is only partially tell about the structure and function of
degraded microbial communities in the absence of
actual laboratory cultures
DECHLORINATION
- Burkholderia dechlorinates the pesticide 2,4,5-T
aerobically releasing Cl in the process Enrichment
- Dioxygenase enzyme breaks the aromatic ring to
Isolation the separation of individual
yield compounds that can enter the Citric acid
organisms from the mixed community
cycle and yield energy
- Reductive Dechlorination in anoxic conditions Enrichment cultures - selective for desired
- anoxic conditions develop polluted microbial organisms through manipulation of medium
habitats and incubation conditions (Figure 18.1)
- Other compounds that can be reductively - duplicate as closely as possible the resources
dechlorinated: and conditions of a particular ecological niche
dichloroethylene, trichloroethylene, tetrachloroeth
ylene, chloroform, DCM and polychlorinated
Inoculum - the sample from which
microorganisms will be isolated
biphenyls
- Several brominated and fluorinated compounds Martinus Beijerinck conceptualized the
(highly toxic and cancerous) can be dehalogenated enrichment culture technique
including PCBs (insulators) - used enrichment cultures to isolate for the
first time the nitrogen-fixing bacterium
PLASTICS Azotobacter
- Polymers of various chemistries resources (nutrients) and conditions
- unaltered for long periods in landfills, refuse (temperature, pH, osmotic considerations,
dumps, and litter in the environment and the like) must mimic those of the
- Microbial plastics = replacement for Synthetic habitat to give the best chance of obtaining
plastics the organism of interest
- Polyhydroxyalkanoates (PHAs) are bacterial
storage polymer and can be biosynthesized in Enrichment cultures
various chemical forms
- selective for desired organisms through
- PHA copolymer containing poly-B-hydroxybutate
manipulation of medium and
and poly-B-hydroxyvalerate = plastic substitute
incubation conditions
- synthetic plastics are cheaper than microbial
- resources (nutrients) and conditions
plastics that's why a lot of people still buy those
(temperature, pH, osmotic
- Ralstonia eutropha (bacterium) produces PHAs in
considerations, and the like) must
high yield
mimic those of the habitat to give the

28
best chance of obtaining the organism - to target one or a small group of organisms or
of interest a particular pathogen (use highly selective
- Can prove the presence of an organism media + incubation conditions)
in a habitat but cannot prove that an - to get a general estimate of viable cell
organism does not inhabit an numbers (using simple media)
environment
Some enrichment cultures yield
nothing because the organism capable Roll tube method uses tubes containing a
of growing under the enrichment thin layer of agar on their inner surface that
conditions specified is absent from the can be streaked for isolated colonies;
habitat; Alternatively, even though the - used for the isolation of anaerobic
organism of interest exists in the prokaryotes, Because the tubes can be flushed
habitat sampled, the resources and with an oxygen-free gas during streaking
conditions of the laboratory culture Pure/Axenic culture can be verified by:
may be insufficient for its growth - Microscopy
- The ability to isolate an organism from - Observation of colony characteristics
an environment says nothing about its - Tests of the culture for growth in other
ecological significance media
Laser tweezers
The Winogradsky column - consist of an inverted light microscope
- with a strongly focused infrared laser and a
- An artificial microbial ecosystem
micromanipulation device
- Serves as a long-term source of - traps single cells by force created by laser
bacteria for enrichment cultures beams pushing down on the cell and holding it
- Named for Sergei Winogradsky in place. When the laser beam is moved, the
trapped cell moves along with it; can be
Enrichment bias moved away from contaminants and then
isolated
- Microorganisms cultured in the lab are
- Isolating slow-growing bacteria from
frequently only minor components of
the microbial ecosystem mixed cultures
Reason: the nutrients available

in the lab culture are typically
Flow cytometry
much higher than in nature
- Uses lasers
Dilution of inoculum is
- Suspended cultures passed through
performed to eliminate rapidly
specialized detector; can also sort out
growing, but quantitatively
single cells based on measured criteria
insignificant, weed species
- Cells separated based on fluorescence
Isolation
High-throughput methods - use of robotic systems
Pure cultures - contain a single kind of
microorganism
- Obtained by: streak plate, agar shake, Culture-Independent Microscopic Analyses of
or liquid dilution (Figure 18.3) Microbial Communities
Environmental genomics
Agar dilution tubes
method for assessing the entire
- mixed cultures diluted in molten agar; gene complement of a habitat,
colonies embedded in the agar revealing both the biodiversity
- Useful for purifying anaerobic and metabolic capabilities of the
organisms microbial community at the
Most-probable-number technique same time
- Serial 10x dilutions of inoculum in a
liquid medium
General Staining Methods
- Used to estimate number of
Fluorescent Staining with Dyes that Bind
microorganisms in food, wastewater,
Nucleic Acid:
and other samples
29
- DAPI - bright blue (Figure 18.6a) complementary in base
- Acridine Orange - orange or greenish orange sequences to rRNA sequence
(Figure 18.6b) - Target: rRNA
- penetrate cells without lysing them and
- SYBR Green I - green (Figure 18.6c)
hybridize with rRNA directly in the ribosomes;
ribosomes are scattered throughout the cell
fluoresce under UV light (prokaryotes), making the entire cell fluoresce
- By targeting sites in the rRNA that are variable
for the enumeration of microorganisms in
between different organisms, phylogenetic
samples
stains can be designed to be very specific and
nonspecific and stain nucleic acids react with only one species or a handful of
Cannot differentiate between live and dead related microbial species
cells - by targeting conserved stretches in the rRNA
they can be made more general and react
with, for example, all cells of a given
Viability stains: differentiate between live phylogenetic domain
and dead cells
- FISH technology can also employ
- Two dyes are used multiple phylogenetic probes Used in
- Based on integrity of cell membrane microbial ecology, food industry, and
(dead = cell membrane no longer clinical diagnostics
intact) CARD-FISH
- Green cells are live - FISH can be used to measure gene
- Red cells are dead expression in organisms in a natural
- Can have issues with nonspecific sample (Figure 18.12)
staining in environmental samples - Target: mRNA (much less abundant than
Green fluorescent protein can be rRNA; therefore standard FISH cannot be
genetically engineered into cells to applied and needs amplification)
make them autofluorescent (Figure - A FISH method that enhances the signal is
18.8) called catalyzed reporter deposition FISH
- Can be used to track bacteria (CARD-FISH)
- also useful in phylogenetic studies of
- Can act as a reporter gene
prokaryotes that may be growing very slowly
- requires O2 to become fluorescent; not
since they contain few ribosomes compared to
suitable for tracking cells introduced into
actively growing cells; standard FISH yields
strictly anoxic habitats
only a weak signal
Limitations of Microscopy:
- Small cells can go unnoticed
- Difficult to differentiate live from dead cells Culture-Independent Genetic Analyses of
- Does not reveal phylogenetic diversity Microbial Communities
- specific genes can be used as measures of
biodiversity and metabolic capacity. Some
Fluorescence In Situ Hybridization (FISH) genes are unique to particular organisms.
Detection of such a gene in an environmental
Nucleic acid probe - DNA or RNA sample implies that the organism is present.
complementary to a sequence in a target
gene or RNA
- when the probe and the target come together,
they hybridize
- can be made fluorescent by attaching PCR Methods of Microbial Community Analysis
fluorescent dyes
- used to identify organisms that contain a Major steps in PCR:
nucleic acid sequence complementary to the 1. Two nucleic acid primers are hybridized to a
probe complementary sequence in a target gene
FISH: fluorescence in situ hybridization 2. DNA polymerase copies target gene
3. Multiple copies of the target gene are made
- Phylogenetic Staining Using FISH
by repetition
Fluorescing oligonucleotides
30
- Restriction enzymes are used to cut the
- Suitable target genes: genes encoding small PCR products
subunit ribosomal (SSU) rRNAs, and genes ARISA: automated ribosomal intergenic
that encode enzymes for metabolic functions spacer analysis
unique to a specific organism - Related to T-RFLP
- Uses DNA sequencing
Orthologs genes that have changed in sequence - ARISA does not require a restriction
enzyme digestion following PCR
over time as species have diverged
amplification
Phelotypes organisms that share the same or - automatically identifies and assigns
sizes to each dye-labeled fragment
very closely related orthologous genes
-Next-generation DNA sequencers do not
require a cloning step
-PCR products can be used directly for
In a typical community analysis experiment, sequencing
total DNA, which is a mixture of genomic
-Allows for the detection of minor phylotypes
DNA from all microbes from the habitat, is
isolated. PCR is used to amplify target gene.
How are phylotypes sorted: Results of PCR phylogenetic analyses
1. Physical separation by gel - Several phylogenetically distinct
electrophoresis prokaryotes are present
2. Clone library construction rRNA sequences differ from
3. Next-generation sequencing those of all known laboratory
cultures
Specific genes can be used as a measure of - Molecular methods conclude that
diversity fewer than 0.1% of bacteria have been
- Techniques used in molecular cultured
biodiversity studies
Microarrays for Analysis of Microbial Phylogenetic

DNA isolation and sequencing
and Functional Diversity
PCR
Phylochip: microarray that focuses on
Restriction enzyme digest phylogenetic members of microbial
Electrophoresis community
Molecular cloning Functional gene microarray: microarray that
focuses on genes of biochemical significance
- Encompasses many different metabolic
PCR Methods of Microbial Community Analysis
pathways
DGGE: denaturing gradient gel - GeoChip contains 50000 gene
electrophoresis separates genes of the same sequences few more than 290 gene
size based on differences in base sequence categories
- Denaturant is a mixture of urea and
formamide
Phylochips and functional gene microarrays
- Strands melt at different denaturant
like the GeoChip circumvent many of the
concentrations
time-consuming stepsPCR, DGGE, cloning,
- Once DGGE has been performed,
and sequencing
the individual bands are excised and
sequenced
Environmental Genomics and Related Methods
Environmental genomics (metagenomics)
- DNA is cloned from microbial
T-RFLP: terminal restriction fragment length
community and sequenced
polymorphism
focused on capturing random fragments of
- Target gene is amplified by PCR environmental DNA in small- or large-insert
31
plasmids - Can measure a wide range of activity
- pH, oxygen, CO2, and others can be
measured
- Small glass electrodes, quite fragile
(Figure 18.22)
- Detects as many genes as possible - Electrodes are carefully inserted into
- Yields picture of gene pool in the habitat (e.g., microbial mats)
environment
Measurements taken every 50
- Can detect genes that are not amplified 100 mm
by current PCR primers, no need for - Using a bank of microsensors, each sensitive to a
cloning DNA; could now be sequenced
different chemical, simultaneous measurements of
directly from total DNA
several transformations in a habitat can be made
- Powerful tool for assessing the
phylogenetic and metabolic diversity of Stable isotopes: nonradioactive isotopes of
an environment an element
- metabolized differently by
> It is not the immediate goal of environmental microorganisms and can be used to
genomics to generate complete and finished study microbial transformations in
genome sequence, the idea is to detect as many nature
genes as possible encoding recognizable proteins - Isotope fractionation
Carbon and sulfur are commonly
Metatranscriptomics used
- Analyzes community RNA
Lighter isotope is incorporated
- The isolated RNA is converted into
preferentially over heavy isotope
cDNA by reverse transcription
before sequencing Indicative of biotic processes
- Reveals genes in a community that are Isotopic composition reveals its
actually expressed past biology (e.g., carbon in
- Reveals level of gene expression plants and petroleum;
Metaproteomics The activity of sulfate-reducing
- Measures the diversity and abundance bacteria is easy to recognize
of different proteins in a community from their fractionation of sulfur
in sulfides
- an even more direct measure of cell
function than is metatranscriptomics Linking Genes and Functions to Specific Organisms:
because different mRNAs have SIMS, Flow Cytometry, and MAR-FISH
different half-lives and efficiencies
of translation, and thus will not all
Analysis of cells by secondary ion mass
yield the same number of protein
spectrophotometry (SIMS)
copies
- High-energy ion beam impacts a
sample; the elemental and isotopic
Measuring Microbial Activities in Nature composition of released materials can
be obtained
Chemical Assays, Radioisotopes, and Microsensors - Secondary ions are released
(sputtering)
In many studies, direct chemical
- Mass spectrometry of secondary ions
measurements are sufficient
- Higher sensitivity can be achieved with
radioisotopes NanoSIMS
Proper killed cell controls must - SIMS devices that yield information on
be used because some isotopic single cells
transformations might be due - Uses multiple detectors to provide
to abiotic processes simultaneous analysis of ions
Microsensors Flow cytometry and multiparametric analysis
32
- Natural communities contain large
populations
- Flow cytometer examines specific cell
parameters very fast
Cell size
Cell shape
Fluorescence
- Advantage: Parameters can be
combined and analyzed
(multiparametric analysis)
Radioisotopes are used as measures of

microbial activity in a microscopic technique
called microautoradiography (MAR)
Radioisotopes can also be used with FISH

- Microautoradiography FISH (MAR-
FISH)
Combines phylogeny with
activity of cells
Linking Genes and Functions to Specific Organisms:
Stable Isotope Probing and Single-Cell Genomics
Stable isotope probing (SIP): links specific

metabolic activity to diversity using a stable
isotope
- a method that employs stable isotopes
such as 13C or 15N or even 18O to
label DNA of organisms in a community
- Microorganisms metabolizing stable
isotope (e.g., 13C) incorporate it into
their DNA
DNA with 13C can then be used
to identify the organisms that
metabolized the 13C
- SIP of RNA also possible
Single-cell genomics
- Multiple displacement amplification
(MDA)
Links metabolic function to
individual cells (Figure 18.32)
- key to single-cell genomics and is
used to amplify chromosomal DNA
from a single cell isolated
- Amplifies DNA from a single organism
Uses cell sorting
Uses bacteriophage DNA
polymerase

33

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Chapter 6- Microbial Genomics -Separated by Gel Electrophoresis (based on

charges and size/molecular weight)

-reveals organisms function and Primers- short segments of DNA/RNA that

Sanger Dideoxy Method Illumina/Solexa sequencing

SOLID/ Applied Biosystems methods

Genome Annotation-conversion of raw sequence

Large Prokaryotic Genomes RubisCO- enzyme that catalyzes the key

Yeast Genome *mRNA must be measured when studying

1.) Microarrays APPLICATIONS OF GENE CHIPS:

Liquid Chromatography used to separate protein 1. Genomics

(2016:GIM Prelims) Arcilla, Cada, Misola, Montemayor, Ortega, Quilang, Sampang

Metagenomics and biome studies Extensive in nature

- Most prokaryotes in the gut

Gene duplication mechanism by which most of Core genome vs Pan genome

Chromosomal Islands Pathogenecity Islands

(2016:GIM Prelims) Arcilla, Cada, Misola, Montemayor, Ortega, Quilang, Sampang

Northern Blot RNA is in the gel; analogous

4. After an appropriate incubation period, the

(2016:GIM Prelims) Arcilla, Cada, Misola, Montemayor, Ortega, Quilang, Sampang

Most useful hosts for cloning are microorganisms

Shuttle Vectors Regulation of Transcription from Expression

2. it can hold larger amounts of DNA than

(2016:GIM Prelims) Arcilla, Cada, Misola, Montemayor, Ortega, Quilang, Sampang

3. Centromere for segregration during mitosis

2. Any introns must be removed Transgenic Organisms in Agriculture and

Solutions: T-DNA- segment of the ti-plasmid DNA that is

(2016:GIM Prelims) Arcilla, Cada, Misola, Montemayor, Ortega, Quilang, Sampang

Viccinia Virus - widely used to prepare ENGENEERING METABOLIC PATHWAYS

(2016:GIM Prelims) Arcilla, Cada, Misola, Montemayor, Ortega, Quilang, Sampang

(2016:GIM Prelims) Arcilla, Cada, Misola, Montemayor, Ortega, Quilang, Sampang

Pilot plant stage (300-3000L fermenters) close to

Lactose (from whey)

Pyrogens Production of tetracylines

other beta lactam antibiotics Catabolite repression

Riboflavin Enzymes as Industrial Products

Alcoholic Beverages and Biofuels

Distilling How can yeast help to solve global energy

Brandy Products from Genetically Engineered

To eliminate undesirable flavor, product is aged, Expressing Mammalian Genes in Bacteria

Gene Cloning via mRNA

- complicated by degeneracy of genetic code Recombinant bovine somatotropin (rBST)

Bacterial Artificial Chromosomes (BACs)

Targeted Gene Mining

(2016:GIM Prelims) Arcilla, Cada, Misola, Montemayor, Ortega, Quilang, Sampang

- genes that encode the enzymes of the

- fast-growing salmon - the promoter for

- insertion into pigs of a gene from

Transgenic Animals in Pharming Severe combined immune deficiency (SCID)

o (for infants) ADA gene is inserted to

Transgenic Plants in Agriculture

Herbicide glyophosate (RoundupTM)

BIOREMEDIATION *In large oil spills, microbes consume oil by

(2016:GIM Prelims) Arcilla, Cada, Misola, Montemayor, Ortega, Quilang, Sampang

(2016:GIM Prelims) Arcilla, Cada, Misola, Montemayor, Ortega, Quilang, Sampang

Reason: the nutrients available

Microarrays for Analysis of Microbial Phylogenetic

Radioisotopes are used as measures of

Radioisotopes can also be used with FISH

Stable isotope probing (SIP): links specific

(2016:GIM Prelims) Arcilla, Cada, Misola, Montemayor, Ortega, Quilang, Sampang

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