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Final Exam exam 16 November 2017, questions

Microbiology (University of the Free State)

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essBlue – Done – 52
Red – Study – 3
Yellow – Unsure – 31

CHAPTER 4
Mutation rates during DNA replication are remarkably low (between 10−8 and 10−11 errors
per base pair inserted), why are these errors so rare? What enzymatic activity, in addition
to polymerization, is associated with DNA polymerase III and how does it reduce errors?

Error rates are low because DNA Pol have two chances to insert the correct basepair. One is by
complementary basepairing and the other is by proofreading. Except for enzymatic activity, DNA
Pol I also have proofreading activity. DNA Pol III, a separate subunit, DnaQ, performs the
proofreading function. Incorrect base pairing can be detected because a wrongly inserted
nucleotide causes a slight distortion in the helix. Wrongly inserted nucleotides can be removed
by DNA Pol I and DNA Pol III exonuclease activity.

The start and stop sites for mRNA synthesis (on the DNA) are different from the start and
stop sites for protein synthesis (on the mRNA). Explain.

Start and stop sites for mRNA synthesis

 Start
o RNA Pol recognizes sites of initiation – promotors
o Promotors are recognized by sigma factor
o Promotors consist of 2 highly conserved regions
 Pribnow box – located -10 bases upstream of initiation site
 -35 region – located -35 bases upstream of initiation site
 Stop
o Intrinsic terminators
 Without any additional proteins
 Inverted repeat
 Stem loop
 Region of A:T
o Extrinsic terminators
 Rho protein
 Do not bind to RNA pol of promotor
 Bind to template and dissociates RNA pol and RNA from DNA
Start and stop sits for protein synthesis
 Start
o Start codon – identified by Shine-Dalgarno sequence (10 bases upstream of
AUG)
 Stop
o Stop codon – Release factors recognize a stop codon and cleave polypeptide
from tRNA

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With reference to DNA, what is meant by the terms semiconservative, complementary,

and antiparallel?

Semiconservative – After a new strand of DNA is synthesized, the new strand consists of one
new strand and one parental strand in the double helix.

Complementary – A:T , G:C

Antiparallel – One strand is from the 3’-5’ direction while the other strand is from the 5’-3’
direction.

The following questions refer to amino acids:

1. Why are amino acids called amino acids?

Amino acids are molecules that contain an —NH2 group (the amino portion) and a
—COOH group (the carboxylic acid portion); thus the term amino acid.

2. Draw a general structure for an amino acid.

3. What is the importance of the R group to final protein structure?

The R group gives an amino acid its identity. The side chain may contain a
carboxylic acid group, as in aspartic acid or glutamic acid, rendering the amino acid
acidic. Others contain additional amino groups, making them positively charged
and basic. The positions of the various R groups influence protein structure through
bonding between side chains. Nonpolar side chains tend to congregate at the
protein’s core because of hydrophobic interactions. Hydrogen bonds and ionic
bonds, though weak, also influence the structure of the protein.

4. Why does the amino acid cysteine have special significance for protein
structure?

Cysteine contains a sulfhydryl group (-SH). Using their sulfhydryl groups, two
cysteines can form a disulfide linkage (R–S–S–R) that connects two polypeptide
chains. Such covalent bonds are important in defining and maintaining protein
shape.pt

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What is the basic flow of genetic information in all cellular life? Include in your answer a
diagram that illustrates the relationships between the basic components and steps in the
flow of genetic information.

mRNA is produced from DNA through transcription while proteins are synthesized by translation
from the mRNA transcript.

Explain the difference between transcription and translation and how the processes
differ in bacteria and eukaryotes.

Transcription is the production of an mRNA transcript from a DNA template.

Prokaryotes Eukaryotes
3 promotor elements - -10, -35, upstream Many promotor elements – TATA box, initiator
elements elements, GC box, downstream core
promotor
RNA pol I RNA pol I, II, III
None Form initiation complex
Transcription and translation occur First transcription, then translation
simultaneously
None Post transcriptional modification – capping,
splicing, polyadenylation
Polycistronic Monocystronic
Termination by intrinsic or rho-dependent Termination by polyA signal and downstream
factors terminator sequence
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Translation is the synthesis of proteins using the mRNA as template.

In prokaryotes translation can take place immediately after transcription since there is no
nucleus to separate the processes.

Explain the concept of semiconservative replication and how simultaneous copying of

both strands of DNA is accomplished in prokaryotic cells.

Semiconservative replication refers to the synthesis of double stranded DNA that consists of
one parental strand and one new strand. Simultaneously copying of both strands can happen by
one strand being the leading strand and the lagging strand. The leading strand is continually
synthesised while the lagging strand is made in short Okazaki fragments. These fragments are
joined together by ligase.

Discuss how the initiation of DNA synthesis occurs in bacteria using the terms origin of
replication, replication fork, and theta structures

In bacteria a single location exists on the chromosome where DNA synthesis is initiated – Origin
of replication. Before DNA pol can synthesize new DNA, the DNA must be unwound. DNA
helicase unwinds the DNA, exposing a short single-stranded region. This is called the
replication fork. The exposed region is immediately covered with SSB to prevent the double
helix from reforming. A theta structure forms during bidirectional replication.

Explain the function of the helicases and why they are necessary.

Helicases unwinds the double helix and inserts positive supercoils. This exposes the short
single stranded region that is to be replicated.

Discuss the role of promoters in RNA synthesis

Promotors are necessary for the RNA polymerase to recognize where to bind. The promotors
are thus the site of initiation of transcription and is recognized by the sigma factor of RNA pol.

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Once the RNA Pol has bound to a promoter, transcription can proceed. Promoters consist of two
highly conserved regions, the Pribnow box and -35 region.

Draw the structure of a DNA molecule with the sequence AGCT.

You isolate a piece of DNA from a microorganism you cultivated from your teeth. The
piece of DNA is 94 kbp is size and is circular. You sequence it and discover that it
contains genes for capsule formation, pili, and antibiotic resistance, as well as an origin

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of replication. What is this piece of DNA and how is it related to the other genetic
elements found in prokaryotic cells?

The piece of DNA is most likely a plasmid. It does not encode for any essential genes, but do
encode for helpful genes. It is also in the typical range of 1kbp to <1Mbp of a plasmid and
circular.

Some essential genes and DNA sequences in cells DO NOT encode for proteins but are
still essential for cellular growth and replication. Give two examples of a gene or
sequence of which this is true and explain why it is essential for growth or replication.

rRNA and tRNA. These genes are not translated into protein but are transcribed. The RNA that
is made after transcription is the final product that is critical for translation. Without rRNA and
tRNA, genes in a cell would not be able to make new proteins or grow. Other essential DNA
sequences are promoter sequences, ribosome binding sites and termination sequences.
Promoters and binding sites are never translated, but are critical to the expression of all genes
in a cell.

Explain the role of sigma factors in RNA synthesis in Bacteria

The 70S subunit consist of 5 subunits to form the holoenzyme. However, the sigma factor easily
dissociates and the rest of the factors form the core enzyme. The sigma factor recognizes the
promoter. The promoter is the initiation site of transcription.

Explain the process of RNA transcription using the terms upstream, Pribnow box, and
consensus sequence.

RNA transcription is carried out by RNA polymerase. The DNA is used as a template. No primer
is needed since RNA pol can initiate new strand on its own. Promoters are recognized by the
sigma factor. Promoters consist of two highly conserved regions namely the Pribnow box which
are located -10 bases upstream of the initiation site and the -35 region which is located -35
bases upstream of the initiation site. Strong promoters are promoters that is most like the
consensus sequence.

How is an open reading frame (ORF) identified and used to determine the sequence of
amino acids in the gene it encodes for?

A functional ORF encodes a protein.

 Locate start and stop codons, however, in frame start and stop codons may appear with
reasonable frequency
 The ORF must contain at least 300 nucleotides, 100 codons, but small genes may be
missed.
 Translation begins at start codons located downstream of ribosome binding site (Shine-
Dalgarno site)
 If codon bias differs from the usual consensus that ORF may be noncoding
 ORFs are mostly functional if the sequence is similar to an ORF in other organisms.
 If the ORF includes a sequence known to encode a protein

Speculate why the half-life of mRNA is short, while the half-lives of rRNA and tRNA are
long.

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mRNA have a short half life to permit the cell to adapt quickly to new environmental conditions.
rRNA and tRNA are very stable that can be ascribed to the highly folded secondary structures
that prevent them from being degraded by ribonucleases.

CHAPTER 6
Explain what a pathogenicity island is and how it is related to chromosomal islands.
Explain how chromosomal islands might move between different bacterial hosts.

- Pathogenicity islands are clusters of genes responsible for virulence and are present
on the bacterial cell’s chromosome/plasmid
- Chromosomal islands are a part of the chromosome and contain clusters of genes
for specialized functions that are not needed for simple survival
- In some pathogenic bacteria, chromosomal islands can encode virulence factors
- Transfer can occur by any of the mechanisms of HGT: transformations, transduction
and conjugation

What is the major difference in how duplications have contributed to the evolution of
prokaryotic versus eukaryotic genomes?

- Gene duplication is the mechanism by which most new genes evolve

- After duplication, one of the genes are free to evolve while the other copy continues
to supply the cell with the original function
- Likely fueled microbial evolution
- The ancestor of Saccharomyces duplicates its entire genome as well as Arabidopsis;
thus eukaryotes genome evolution was by means of entire genome duplication
- For bacteria, many frequent but relatively small duplications have occurred; based on
the distribution of duplicated genes and gene families

Apart from genome size, what factors make complete assembly of a eukaryotic genome
more difficult than assembly of a prokaryotic genome?

 Eukaryotic genome assembly is more complex than that of a prokaryotic genome,

because:
(1) Larger eukaryotic genome size
(2) Eukaryotic genome has a significant amount of non-coding regions (introns)
(3) Assembly of entire genome as a compact structure should be contained within
the nucleus requires the functions of several groups of proteins, collectively
known as the “mitosis promoting factors” (MPF’s).
(4) The behavior of the chromatin & chromosomes through the cell cycle phases and
in each phase of cell divisions are far more complex in eukaryotes, making the
genome assembly more complex in eukaryotes.
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Why do dideoxynucleotides function as chain terminators?

- Dideoxynucleotides are useful in nucleic acid sequencing and act as chain

terminators
- Dideoxynucleotides like deoxynucleotide counterparts but lack 3' OH so its
incorporation causes chain termination

The gene encoding the beta subunit of RNA polymerase from Escherichia coli is said to
be orthologous to the rpoB gene of Bacillus subtilis. What does that mean about the
relationship?
between the two genes? What protein do you suppose the rpoB gene of B. subtilis
encodes? The genes for the different sigma factors of E. coli are paralogous. What does
that say about the relationship among these genes?

- The gene present in E.coli which encodes for beta subunit of RNA pol enzyme is
orthologous to rpoB gene which is found in B. subtilis organism.
- The genes present in both the organism share orthologous relationship with each
other
- If the genes present in one organism are similar to genes found in other organisms,
as a result of descent from a common ancestor are referred to as orthologs.
Orthologs usually are not identical due to divergent evolution of lineage which is
followed by speciation.
- The rpoB gene which is found in B.subtiis organism will also encode for beta subunit
of RNA pol enzyme
- The genes for the different sigma factors of E.coli will share a paralogous
relationship
- These genes will not be identical due to the ability of the duplicate gene to mutate,
but these genes functions will still relate to the role of the ancestral gene.

Which genomes are larger, those of chloroplasts or those of mitochondria? Describe one
unusual feature each for the chloroplast and mitochondrial genomes.

- The mitochondria genome is bigger.

- Chloroplasts are always circular and mitochondria are usually linear but are
sometimes circular.

You are interested in the minimum set of genes necessary for survival of a eukaryotic
microorganism such as Saccharomyces cerevisiae. Design an experiment to
systematically test which genes are essential for survival and which are not under high
nutrient, aerobic conditions.

Saccharomyces cerevisiae can be grown in haploid and diploid states. When growing it in
diploid state, one of the genes can be knocked out using knockout mutations to determine if the
gene is essential or not. If the organism can survive in the haploid stage without the gene, it is
not essential.

The follow question is with regards to horizontal gene transfer.

A. Discuss horizontal gene transfer in prokaryotes
Horizontal gene transfer is the transfer of DNA from one cell to another by another
means other than the usual vertical inheritance process in which the genome is
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transferred from mother cell to daughter cell. At least 3 mechanisms for horizontal
gene transfer are known: transformation, transduction and conjugation.
B. Explain how one can detected in a genome of a prokaryote
Horizontal gene transfer can be detected after genes have been annotated.
The presence of genes that encode proteins typically found only in distantly related
species. The presence of a stretch of DNA whose G/C content or codon bias differs
significantly from the rest of the genome.

Distinguish between the terms genome, proteome, and transcriptome.

Genome – The full complement of an organism’s genetic information.

Proteome – In a wider sense – all the proteins encoded by an organism’s genome. In a
narrower sense – proteins present in a cell at a given time.
Transcriptome – mRNA expressed under a set of certain conditions

Explain how codon bias and GC content can be used to detect horizontal gene transfer
within a genome.

- Horizontal gene transfer is indicated by codon bias and an abnormal GC content.

- In the case of codon bias, there are many codons for a rather rare amino acid.

Explain horizontal gene transfer and demonstrate how this phenomenon has
complicated evolutionary studies using a diagram that illustrates phylogenetic
relationships between organisms and genes.

- Horizontal gene transfer involves the transfer of a gene sequence from one cell to
another; however, this makes it difficult to track evolutionary relatedness. (p.232)

Explain the terms "core genome" and "pan genome" and describe how each contributes
to the genome of a bacterial species. Give an example of genes that are part of a core
genome versus those that are more often in the pan genome.

- Core genome: genome shared by all strains of a given species

- Pan genome: includes the core plus all of the optional extras present in one or more
strains but not all strains of that species
- A core genome is the genome content present in all strains of a bacterial species.
These are typically fundamental metabolic genes that combine to help define the
bacterial species as a whole.
- The pan genome is the genome content that is found in only some of the strains
within the species.
- The additional genes in the pan genome are what distinguish strains from one
another.
- Typical genes in the pan genome may be for accessory functions such as virulence,
degradation of a variety of substrates, or symbiosis.
- Often the pan genome contains mobile elements and plasmids

Interpret the genomic content of mitochondria in relation to their evolution. How is

mitochondrial evolution more complicated than expected?
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- Contains plasmids and chromosomal DNA

- Have both circular and linear DNA
- These fundamental differences in mitochondrial genomes further complicate even
basic comparative analyses.
- Some mitochondrial genes have also been transferred to the nuclear genome.

Design an experiment using —omic methods to test how Escherichia coli adapts to
different growth temperatures.

- You could place two separate E.coli samples in different test tubes at different
temperatures.
- You could then asses what genes are expressed (transcriptomics) and what proteins
are present (proteomics).

Recommend what type of data should be collected and analysed in a systems biology
approach to investigate how a non-pathogenic bacterial strain becomes pathogenic.
Describe what scientific fields and methods would be involved in your recommendation.

- Systems biology incorporates many fields of biology in order to make analyses.

- One could compare the genes expressed in non-pathogenic versus pathogenic
bacteria and identify which proteins and genes are related to pathogenicity.

Chromosomal islands are presumed to have a "foreign" origin based upon three
observations. What are these observations and how do they indicate that chromosomal
islands are "foreign" in origin?

 Regions are flanked by inverted repeats, whole region was inserted into the
chromosome by transposition.
 Base composition and codon bias differs greatly from genome.
 Chromosomal island is found in some strains of a particular species but not others.

Explain why organisms undergoing rapid evolutionary change often contain relatively
large numbers of mobile DNA elements, whereas once organisms settle into a stable
evolutionary niche, most of these mobile elements are lost.

- Genome diversity is high when an organism is undergoing rapid evolutionary

change.
- It will undergo horizontal gene transfer and chromosomal rearrangements.
- In contrast, organisms that are more stable in their niche will have less diversity

The intracellular parasite Nanoarchaeum equitans has one of the smallest prokaryotic
genomes. Why is the genome of N. equitans so small? How is it possible that the
genome of N. equitans contains more genes than that of Mycoplasma genitalium, which
is actually 90 kbp larger?

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- The genome of N. equitans is so small because it does not require its own genes for
energy consumption.
- It has very little noncoding DNA and no introns.
- It is adapted to live within its host.

Explain why the proteome is NOT defined as "all the proteins encoded by an organism's
genome."
- Certain proteins may not by synthesized under a particular condition.
- Not all potential ORFs will encode for proteins.
- The number and types of proteins present in a cell change in response to an
organism’s environment or other factors, such as developmental cycles.

When analysing the sequence of genes similar to ones already known, why is the amino
acid sequence of the protein more important than the DNA sequence?

- Over time, nucleotide sequences can change, but protein sequences will always
have the same function.
- Amino acid sequence of the encoded proteins is more important because the genetic
code is degenerate.
- It can be similar on protein level but different on DNA level

The Human Microbiome Project is a large research program that aims to understand all
of the microorganisms on and in the human body. Which "-omic" methods could be
applied directly to a sample from the human body to study the microorganisms in the
sample? Propose a general experimental approach for analysing a sample containing a
complex mixture of microorganisms.

- A sample from the human body must be taken and then the DNA/RNA must be
extracted (metagenomics).
- The RNA would be converted to cDNA and the cDNA would be sequenced.

Summarize the evolution of sequencing technology beginning with the Sanger method.
Answer: The first generation of DNA sequencing used the Sanger method to determine
the DNA sequence.

1st generation – Sanger

 Used DNA pol I, deoxyribonucleoside triphospahtes, dideoxyribonucleotides
 Gel-electrophoresis

2nd generation – massively parallel methods

 454 Life Sciences Pyrosequencing
 Uses DNA pol I
 Pyrophosphate released
 Energy for luciferase
 Emits light
Illumina/Solexa
 Fluorescent tag blocks 3’ – OH group

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3rd generation – Single molecules

 HeliScope Single Molecule Sequencer
o Single 32bp fragment
o Fluorescent tags monitored on microscope
 Pacific Biosciences SMRT
o Zero-mode waveguides
o Emit flashes of light
 Pyrophosphate discarded and not incorporated into chain
4th generation – Ion torrent
 Measures release of H+
 Nanopore technology
o Narrow pores that permit single stranded DNA to move through one at a time
 Oxford Nanopore
o Pores made from protein
o Detector records change in electrical current

CHAPTER 11
Describe the basic principles of gene amplification using the polymerase chain reaction
(PCR). How have thermophilic and hyperthermophilic prokaryotes simplified the use of
PCR?
- The polymerase chain reaction (PCR) is a laboratory method for amplifying genes in
vitro (i.e., in a test tube).
- PCR involves multiple cycles of heating and cooling steps that denatures template DNA,
anneals PCR primers to DNA template, and extends primers using the template DNA
and DNA polymerase.

- Denaturation @ 94

- Annealing @ 50 – 60

- Extension @ 72
- Using DNA polymerase from mesophillic prokaryotes (such as from E. coli) in PCR
required adding DNA polymerase after each DNA denaturation step because the high
temperature also denatures the DNA polymerase enzyme.
- DNA polymerases from thermophilic and hyperthrmophilic prokaryotes, Thermus
aquaticus (Taq polymerase) and Pyrococcus furiosus (Pfu polymerase), which are stable
at high temperatures are unaffected by the high temperatures of DNA denaturation.
- In addition, the use of Taq and Pfu polymerases increases the specificity of primer
extension (i.e., reduces rates) because they can copy DNA at temperatures (72ºC for
Taq polymerase) rather than 37ºC.

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Genetic engineering depends on vectors. Describe the properties needed in a well-

designed plasmid cloning vector.

(1) small size, making DNA isolation and manipulation easier;

(2) independent origin of replication;
(3) multiple copy number, allowing for amplification of cloned DNA; and
(4) selectable markers for identification of the cells containing the cloning vector or
recombinant clone.

- Characteristics that make plasmids in particular useful for molecular

cloning (in addition to those just stated) include:
(1) a polylinker, or multiple cloning site;
(2) color selection for immediate identification of recombinant plasmids; and
(3) promoters for regulating expression of the cloned gene (e.g., M13 phage promoters
for generation of single-stranded DNA, etc.).

- The F plasmid is much too large to be useful as a cloning vector and does not
contain any selectable markers.

What is a reporter gene? Describe two widely used reporter genes.

- A reporter gene is a gene used in genetic analysis because the product it

encodes is easy to detect
- Two examples include: lacZ and GFP genes, which allow visible identification of
the expression of a cloned gene via either pigmented colonies or fluorescence,
respectively.
-
How are gene fusions used to investigate gene regulation?

- Gene fusions are single genetic constructs that contain components of two
different genes.
- The most common use of gene fusion constructs is to study gene regulation.
- For example, a promoter for a particular gene could be fused to the coding region
of a reporter gene to study transcriptional regulation of that gene.
- Alternatively, the promoter for a gene could be removed and the gene fused to a
different regulatory element to study its regulation from a different promoter.
- The reporter is thus made under conditions that would trigger expression of the
target gene.
It is used in studying gene regulation, especially if measuring the levels of the natural gene
product is difficult, expensive, or time consuming

How does the insertional inactivation of β-galactosidase allow the presence of foreign
DNA in a plasmid vector such as pUC19 to be detected?

- Vector, pUC19, contains a small part of the LacZ gene that encodes β-
galactosidase
- Insertional inactivation of a gene is the insertion of foreign DNA segment within
the coding sequence of the gene, resulting in its inactivation.
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- By cutting the foreign DNA and the vector DNA with the same restriction enzyme,
complementary sticky ends are generated that allow foreign DNA to be inserted
into the vector.
- This phenomenon is used in plasmid vectors, such as pUC19 for easy screening
of the cells containing the cloned DNA.

What is a subunit vaccine and why are subunit vaccines considered a safer way of
conferring immunity to viral pathogens than attenuated virus vaccines?

- A vaccine that contains only a specific protein or two from a pathogenic

organism.
- For a pathogenic virus, this is often the coat protein because coat proteins are
highly immunogenic.
- The coat proteins are purified and used in high dosage to elicit a rapid and high
level of immunity without containing the intact pathogen.
- Sometimes poorly immuonogenic when produced in bacteria.
- More effective if a eukaryotic cloning host (yeast) is used.

What is pathway engineering? Why is it more difficult to produce an antibiotic than to

produce a single enzyme via genetic engineering?

Creating/ improving a biochemical pathway using genes from one or more organisms. Mostly
improving existing pathways rather than creating new ones. A single enzyme is often only
encoded for by a single or a few genes, whereas antibiotic production requires several
enzymes, which all are encoded by different genes. More genes are involved so it is more
complicated having to isolate each individual gene and trying to clone them. Biosynthesis
thereof has a large number of enzymatic steps –More than 72 intermediates –More than 300
genes involved! •Thus very complex biosynthetic regulation

Pathway engineering – The process of assembling a new or improved biochemical pathway

using genes from one or more organisms.

You have just discovered a protein in mice that may be an effective cure for cancer, but it
is present only in tiny amounts. Describe the steps you would use to produce this
protein in therapeutic amounts. Which host would you want to clone the gene into and
why? Which host would you use to express the protein in and why?

Describe the methodology employed when antibodies are used to detect proteins.

Antibodies can be used to detect a protein of interest. Antibodies are proteins of the immune
system that bind a highly specific way to an antigen. The protein encoded by the cloned gene is
the antigen. Because the antibody only binds to the antigen, colonies can be identified that
contain the antigen. Because a very small amount of the antigen is present in the colony, highly
sensitive methods need to be used such as radioisotopes, fluorescent chemicals or enzymes
are used.
See figure 11.8.

Describe the procedure used for colony hybridization and why it is useful.
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- Hybridization is often used to detect the presence of specific genes in genomes

that have not been sequenced, as well as the movement of genetic elements
such as transposon
- The find the specific genome location of a gene, the total genomic DNA can be
cloned
- Hybridization on the resulting colonies using a probe can detect recombinant
DNA in colonies

Describe three characteristics of an ideal cloning host for genetic engineering.

- It must be easy to manipulate and work with.

- Must be able to efficiently uptake DNA and contain necessary genes to encode
for enzymes that facilitate replication of the vector
- Grow in an inexpensive medium
- Be non-pathogenic
- Grow rapidly
- Be stable in cultures

Explain how T7 promoters regulate transcription and why cloning vectors contain them.

- In some cases the transcriptional control system may not be a normal part of the
host (e.g the use of the bacteriophage T7 promoter and T7 RNA pol to regulate
gene expression)
- In T7 expression vectors, cloned genes are placed under control of the T7
promoter
- The BL21 series of E.coli host strains are especially designed to work with the
pET series of T7 expression vectors
- In this strain of E.coli, the phage DNA has been altered by placing a copy of the
lac promoter upstream of its gene for the T7 RNA pol and was incorporated in the
E.coli genome
- Addition of IPTG to the growth medium induces expression of the T7 RNA pol
- T7 RNA pol is highly active, uses most of the RNA precursors, thereby limiting
transcription to the cloned genes
- Host genes that require host RNA pol are mainly not transcribed, cells stop
growing; translation yields the protein of interest
- This control system is very effective for generating large amounts of a particular
genome

Defend why codon usage must be considered when cloning a gene into another
organism.

Because of the redundancy of the genetic code, more than one tRNA exists for most amino
acids. If a cloned gene has a codon usage distinct from that of its expression host, it will
probably be translated ineffectively. Site-mutagenesis can be used to change the selected
codon to make it more amendable.

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When trying to make a mammalian protein in a bacterium, expression vectors are often
used. Using your knowledge of prokaryotic and eukaryotic genetics, explain why
expression vectors are so important.

- In some cases the transcriptional control system may not be a normal part of the
host (e.g the use of the bacteriophage T7 promoter and T7 RNA pol to regulate
gene expression)
- In T7 expression vectors, cloned genes are placed under control of the T7
promoter
- The BL21 series of E.coli host strains are especially designed to work with the
pET series of T7 expression vectors
- In this strain of E.coli, the phage DNA has been altered by placing a copy of the
lac promoter upstream of its gene for the T7 RNA pol and was incorporated in the
E.coli genome
- Addition of IPTG to the growth medium induces expression of the T7 RNA pol
- T7 RNA pol is highly active, uses most of the RNA precursors, thereby limiting
transcription to the cloned genes
- Host genes that require host RNA pol are mainly not transcribed, cells stop
growing; translation yields the protein of interest
- This control system is very effective for generating large amounts of a particular
genome

How can foreign DNA of greater than 300 kbp be inserted and stably maintained in BAC
vectors?
- BAC is constructed from the F plasmids (a naturally occurring plasmid of E.coli
that is 99.2 bp in size)
- BAC contains only a few genes from F necessary for replication and to keep the
copy number very low
- The plasmid contains the cat gene (confers chloramphenicol resistance on the
host) and a multiple cloning site that include several restriction sites for cloning
DNA

What is necessary for a YAC to function as a normal eukaryotic chromosome?

- fragments of DNA can be inserted

- To function like a normal chromosome, YAC must have:
1) An origin of DNA replication
2) Telomeres for replicating DNA at the ends of the chromosome
3) A centromere for segregation during mitosis

Describe a method developed by molecular biologists to easily observe the success of a

genetic engineering procedure involving the ligating of a gene of interest into a plasmid.

. Vector pUC19 contains a small part of the lacZ gene which encoded for β-galactosidase. The
insertion of a foreign gene leads to the lacZ gene being inactivated by insertional inactivation.
When the colourless reagent X-gal is added, the colony that took up the foreign gene, will have
the lacZ gene inactivated and the colony remains white. Colonies who did not take up the
foreign gene, still have the activated lacZ gene and therefore codes for β-galactosidase which
will cleave the X-gal and release a blue colour.

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Defend how type II restriction endonucleases, which do not cleave random sites, can be
a part of an approach to random cloning library.

Most DNA sequences recognized by type II restriction enzymes are palindromes of 4-8bp.
EcoRI makes staggered cuts, leaving overhangs (sticky ends)

If you wanted to discover a novel biosynthetic pathway for an antimicrobial compound

and were given a handful of soil, describe the key experimental steps you would need to
take to accomplish the task and explain why alternative procedures were not performed.

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Describe the three main steps to clone a gene into an organism.

1) Isolation and fragmentation of source DNA:

- The source DNA can be total genomic DNA, DNA synthesized from an RNA
template (by reverse transcriptase), gene or genes amplified by the polymerase
chain reaction, synthetic DNA made in vitro
- If the source is genomic DNA, it must first be restriction digested into fragments

2) Inserting the DNA fragments into a cloning vector:

- Cloning vectors are small, independently replicating genetic elements used to
carry and replicate cloned DNA segments
- Most vectors are plasmids or viruses
- DNA generally inserted in vitro
- DNA ligase joins the two DNA molecules

3) Introduction of cloned DNA into a host organism:

- Transformation is often used to get recombinant DNA into cells
- Yields a mixture of recombinant constructs, some cells contain desired cloned
gene while others will have other cloned genes
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Explain why DNA fragments migrate toward the positive electrode and why some
fragments migrate more rapidly than others during gel electrophoresis. How does this
electronegativity influence observations and conclusions drawn from DNA migrated
through an agarose gel?

- Electrophoresis uses an electrical field to separate charged molecules

- Nucleic acids migrate through the agarose gel towards the positive electrode
because of their negatively charged phosphate group
- The rate of migration through the electrical field is determined by the charge on
the molecule, its size and shape
- The same DNA that has been cut by different restriction enzymes will have
different banding patterns
- Thus, small or compact molecules migrate more rapidly than larger molecules
(The higher the concentration of agarose in the gel, the greater is the resistance
to movement for larger molecules)
- Gels of different concentrations are used to separate molecules different size
ranges
- Gels can be stained with ethidium bromide (visualized under UV)
- Size of fragments can be determined by comparison to a standard called a DNA
ladder

When is it appropriate to use an artificial chromosome vector? Describe a specific

example.
- As the size of a genome increases, so does the number of clones needed to
obtain a complete sequence
- It is necessary to have vectors that can carry very large segments of DNA
- Such vectors are called artificial chromosome vectors
- BAC (Bacterial artificial chromosomes) is constructed from the F plasmids (a
naturally occurring plasmid of E.coli that is 99.2 bp in size)
- BAC contains only a few genes from F necessary for replication and to keep the
copy number very low
- The plasmid contains the cat gene (confers chloramphenicol resistance on the
host) and a multiple cloning site that include several restriction sites for cloning
DNA
- Foreign DNA of more than 300 kbp can be inserted and stably maintained in a
BAC vector

Compare how the process of cloning differs when a vector with sticky ends is used and
when a vector with blunt ends is used.

Vector with sticky ends Vector with blunt ends

- EcoRI makes staggered cuts in the - EcoRV cuts both strands of DNA
phosphodiester backbone of the DNA directly opposite each other =
= sticky ends blunt ends

Describe the usefulness of blue-white screening, also called α-complementation, in

cloning vectors such as pUC19. Include in your answer the terms polylinker, DNA ligase,
lacZ gene, insertional inactivation, Xgal, and β-galactosidase.

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The pUC19 vector contains a polylinker, also called a multiple cloning site, a short segment of
DNA which contain many restriction sites. Vector pUC19 contains a small part of the lacZ gene
which encoded for β-galactosidase. The insertion of a foreign gene leads to the lacZ gene being
inactivated by insertional inactivation. When the colourless reagent X-gal is added, the colony
that took up the foreign gene, will have the lacZ gene inactivated and the colony remains white.
Colonies who did not take up the foreign gene, still have the activated lacZ gene and therefore
codes for β-galactosidase which will cleave the X-gal and release a blue colour.

Explain the process of site-directed mutagenesis, and discuss some applications of this
technique.
- Performed in vitro and introduces mutations at a precise location
- Site-directed mutagenesis is used to manipulate protein characteristics such as
enzyme activity or protein-binding affinity
- Thus, can be used to assess the activity of a specific aa in a protein OR it can be
used to improve the activity of an enzyme for a specific substrate
- Basic procedure:
1) Synthesize a short DNA oligonucleotide primer containing the desired base
change (mutations)
2) Allow this to base-pair with single-stranded DNA containing the target gene
3) Pairing will be complete except for the region of mismatch
4) The synthetic oligonucleotide is extended using DNA pol, thus copying the
rest of the gene
5) The double-stranded molecule is inserted into a host cell by transformation
- Can be used to investigate the activity of proteins with known aa substituents
- Can be used to change a specific aa in the active site

Compare and contrast operon and protein fusions.

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CHAPTER 15
In what ways do industrial microorganisms differ from microorganisms in nature? In
what ways are they similar.

- Industrial microbes have been selected to produce metabolic products, whereas cellular
growth is the main attribute of microbes isolated from nature.
- For industrial use, a microbe must be genetically stable, be easy to grow in pure culture on
inexpensive media
- Possess a reasonable rate of growth and product synthesis (in commercial processes time is
money),
- not produce harmful products along with the desirable product (and non-pathogenic)
- be easily removed from the production medium
- be susceptible to genetic manipulation.
- The industrial microbe is a specialist, frequently possessing metabolic imbalance and other
altered traits that would probably be deleterious to survival in nature.

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- In their more general activities, industrial and conventional microbes are similar regarding
general physiological phenomena, genome, and mechanisms for transcription and translation

List three major types of industrial products that can be obtained with microorganisms,
and give two examples of each.

1) Antibiotics - secondary metabolites (penicillin, tetracycline)

2) Food additives (vitamins, amino acids)
3) Chemicals (biofuels, citric acid)
4) Alcoholic beverages (beer, wine)

Compare and contrast primary and secondary metabolites, and give an example of each.
List at least two molecular explanations for why some metabolites are secondary rather
than primary

Primary metabolite = forms in the exponential phase, formation is proportional to growth

Secondary metabolite = near the end of growth, near or in stationary phase, non-proportional to
growth
Why some are secondary metabolites
1) Non-essential for growth and reproduction and their production is dependent on growth
conditions
2) Often over-produced and produced as a group of closely related compounds
3) Products of spore forming microorganisms (antibiotics are all produced by spore forming
fungi or prokaryotes)

How does an industrial fermenter differ from a laboratory culture vessel? How does a
fermenter differ from a fermenter?

Fermentor = vessel where an industrial microbiology process takes place

Fermenter = Microbe that carries out fermentation, large-scale microbial process itself, doesn't
have to be biochemically a fermentation
Industrial fermenter is:
1) Production is on a much larger scale so the fermenter is much larger
2) Fermenters large, closed cylinder, culture may be grown under aerobic/anaerobic conditions
3) Cooling jacket (steam for sterilisation, water for cooling so that the temperature is optimum
for growth)
4) Aeration system, high density of microbes lots of demand for 02 - aerator (sparger) and a
impeller which stirs the mixture
5) Computers monitor pH, temperature and oxygen levels because of its large size

List three examples of antibiotics that are important products of industrial microbiology.
For each of these antibiotics, list the producing organisms and the general chemical
structure

1) Streptomycin, Streptomyces griseus, aminoglycoside, binds to 30S subunit, prevents fmet

tRNA from binding so inhibits protein synthesis.
2) Tetracycline, Streptomyces rimosus, naphthacene, binds to 30S ribosome subunit, inhibits
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protein synthesis
3) Erythromycin, Streptomyces erythreus, macrolide, binds to 23S rRNA blocking exit of growing
peptide and inhibiting translation
4) Penicillin, Penicillium chrysogenum , beta-lactam, inhibits cell wall synthesis

Compare and contrast the production of natural, biosynthetic, and semisynthetic

penicillins

Natural penicillins – If the peniciilin fermentation is carried out by without addition of side-chain
precursors, a group of related products will form.

Biosynthetic penicillins - The final product of natural penicillins can be specified by adding a side
chain-precursor to the culture medium so that only one type of penicillin is produced in the
greatest amount.

Semisynthetic – To produce penicillins with the most clinical use, protection against Gram
negative bacteria, semisynthetic penicillins are produced. A natural penicillin is treated to yield
6-APA that is then chemically modified by the addition of a side chain.

What unusual characteristics must an organism have if it is to overproduce and excrete

an amino acid such as lysine

Has to be a mutant so that no feedback inhibition can occur, so it is resistant to AEC (enzyme
inhibitor). AEC binds to allosteric site and inhibits enzyme activity, (non-competitive) if no
feedback inhibitor of the enzyme occurs then the lysine will be overproduced and excreted.

What are extremozymes? What industrial uses do they have?

An extremozyme is an enzyme that function at some environmental extreme such as high or low
temperature or pH. Many industrial catalysts operate best at high temperatures so
extremozymes from hyperthermophiles are widely used in the industry. Besides Taq and Pfu
polymerases used in PCR, thermostable proteases, amylases, cellulases, pullalanes and
xylanases are used. Enzymes isolated from psychrophiles, halophiles, alkaliphiles and
acidophiles are used.

What is high-fructose syrup, how is it produced, and what is it used for in the food
industry

Starch converted to glucose by amylase, then glucose is converted to fructose by glucose

isomerase. End product is high fructose syrup, much sweeter than glucose and used in the food
industry to sweeten juices.

In what way is the manufacture of beer similar to the manufacture of wine? In what ways
do these two processes differ? How does the production of distilled alcoholic beverages
differ from that of beer and wine?

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Similarities
1) Saccharomyces yeast used as fermenters
2) Involve anaerobic conversion of sugar to ethanol
Differences
1) Different saccharomyces species yeast used.
2) Starting products (grapes for wine, malt from barley seeds and hops for beer)
3) Addition of sulphite to wine to kill the wild yeasts present on the grapes
4) 2 types of brewing strain yeasts (top and bottom)
5) In brewing, need to convert the starch of the grain to sugar (mashing), sugar is
already present in the crushed grape
6) Wine is aged for years but beer is only aged for weeks
The fermented liquids are heated to volatise the alcohol. The distillate is then distilled,
much higher alcohol content than fermentation alone.

What are the size differences among a laboratory fermenter, a pilot plant fermenter, and a
commercial fermenter? How is proper aeration ensured in a large-scale fermentation?

Laboratory fermenter – 1 -10l

Pilot plant fermenter – 300 – 3000l
Commercial fermenter – 10 000 – 500 000l

Oxygen dynamics are carefully measured to determine how increases in volume affect oxygen
demand in the fermentation. Two devices are used. One is a sparger, an aerator and the other
is an impeller, which mixes the gas bubbles produced by the sparger and organisms throughout
the medium.

What is the major liquid biofuel made worldwide? How is it currently being made in the
United States? Why is it necessary for new feedstock to be developed?

Ethanol. Saccharomyces yeast ferments glucose from cornstarch, and the ethanol is separated
by distillation. Economically, new feedstocks are needed in order for the price of human food
and livestock feed to not go up, as using plant material wastes land that could be used to grow
food.

CHAPTER 21
Describe the general processes that occur in a sludge digester. What is an advantage of
sludge digestion over activated sludge?

Anaerobes use polysaccharides, proteases and lipases to digest suspended solids and large
macromolecules into soluble compounds.
These soluble compounds are fermented to yield a mixture of fatty acids, H2 and CO2.
The fatty acids are further fermented by syntrophic bacteria to produce acetate, CO2 and H2.
These are then used as substrates by methanogenic Archaea, fermenting acetate to produce
methane and CO2 – the major products of anoxic sewage treatment.
The CH4 is burned off or used as fuel to heat and power the wastewater treatment plant.

Compare the operation and function of a trickling filter system and activated sludge.
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Trickling filter system – A bed of crushed rocks about 2m thick. The wastewater is sprayed on
top of the rocks and slowly passes through the bed.
Organic material adsorbs to the rocks and microorganisms grow on the large exposed rock
surfaces. The complete mineralization of organic matter to CO2, ammonia, nitrate, sulphate and
phosphate takes place in the extensive microbial biofilm that develops on the rocks.

Activated sludge – The wastewater is continuously aerated in large tanks.

Slime forming anaerobic bacteria including zoogloeal ramigera and others grow and form
aggregated masses – flocs
Protists, small animals, filamentous bacteria and fungi attach to the flocs Oxidation of organic
matter occurs on the floc as it is aggregated and exposed to air.
The aerated effluent containing the flocs is pumped into a holding tank or clarifier where the
flocs settle. Some of the floc material – activated sludge – is then returned to the aerator as
inoculum for new wastewater. The rest is pumped to anaerobic sludge digester or is removed,
dried and burned, or used for fertilizer.

Identify (stepwise) and discuss the process of purifying drinking water. What important
contaminants are targeted by each step in the process?

Physical and chemical purification:

Raw water is pumped from the source to a sedimentation basin.
Anionic polymers, alum and chlorine are added.
Soil, sand, mineral particles and other large particles settle out – sedimentation
The sediment free water is pumped to a clarifier or coagulation basin – polymers form large
particles from the smaller suspended solids – flocculation
The large, aggregated particles – floc- settle out by gravity, trapping microorganisms and
adsorbing suspended organic matter and sediment.
The clarified water undergoes filtration to remove organic and inorganic solutes, suspended
particles and microorganisms. The filters consist of thick layers of sand, activated charcoal and
ion exchangers.

Describe floc formation in the activated sludge process.

Slime forming anaerobic bacteria, including Zoogloea ramigera and others grow to form
aggregated masses called flocs.

Trace the flow of raw water through a typical drinking water purification scheme.

Same as question above?

Which microorganisms commonly thrive in drinking water distribution systems and how
might they impact human health?

Despite sufficient chlorination of drinking water, biofilms persist on the walls of pipes in water
distribution systems. The biofilms consist mostly of non-pathogenic bacteria, but can harbor
opportunistic pathogens such as Naegleria, Acanthamoeba, Legionella, Pseudomonas, and
Mycobacterium species. There are also large numbers of protists present that graze on (eat) the
bacteria in the biofilm. Legionella, Pseudomonas, and Mycobacterium species can live inside
these protists, increasing their ability to persist in the environment. Although these organisms
are present in drinking water systems, they are usually opportunistic pathogens and thus may
not affect the health of people with normal immune systems. The impact of these organisms and
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how they might be distributed through showerheads and other mechanisms is an area of current
research.

Compare and contrast secondary anaerobic and secondary aerobic wastewater

treatment

Secondary anaerobic wastewater treatment: Sludge digester

Anaerobes use polysaccharides, proteases and lipases to digest suspended solids and large
macromolecules into soluble compounds.
These soluble compounds are fermented to yield a mixture of fatty acids, H2 and CO2.
The fatty acids are further fermented by syntrophic bacteria to produce acetate, CO2 and H2.
These are then used as substrates by methanogenic Archaea, fermenting acetate to produce
methane and CO2 – the major products of anoxic sewage treatment.
The CH4 is burned off or used as fuel to heat and power the wastewater treatment plant.

Secondary aerobic wastewater treatment: Activated sludge or trickling filter

Activated sludge:
The wastewater is continuously aerated in large tanks.
Slime forming anaerobic bacteria including Zoogloea ramigera and others grow and form
aggregated masses – flocs
Protists, small animals, filamentous bacteria and fungi attach to the flocs. Oxidation of organic
matter occurs on the floc as it is aggregated and exposed to air.
The aerated effluent containing the flocs is pumped into a holding tank or clarifier where the
flocs settle. Some of the floc material – activated sludge – is then returned to the aerator as
inoculum for new wastewater. The rest is pumped to anaerobic sludge digester or is removed,
dried and burned, or used for fertilizer.

Trickling filter:
A bed of crushed rocks about 2m thick. The wastewater is sprayed on top of the rocks and
slowly passes through the bed.
Organic material adsorbs to the rocks and microorganisms grow on the large exposed rock
surfaces. The complete mineralization of organic matter to CO2, ammonia, nitrate, sulphate and
phosphate takes place in the extensive microbial biofilm that develops on the rocks.

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