Chapter 3 Flashcards
Microalgae secretes a compound that allows it to
survive and reproduce in harsh environments, which is being used for anti-aging skincare products
What is the problem with using Aspergillus?
but these cells may not make the necessary posttranslational modifications, such as glycosylation
What is Malaria caused by?
a single-celled eukaryotic protozoan from the genus Plasmodium
Anthrax is caused by the
Bacillus anthracis bacterium, whose spores are common in soil.
Plague, caused by
the bacterium Yersinia pestis
Plague is believed to be
the source of the Black Death that killed millions of people during the Middle Ages and resulted in the loss of 30-60% of Europe’s population.
Casimir Davaine and dermatologist Pierre Rayer discovered
B. anthracis and were the first to show it caused anthrax. Davaine demonstrated that anthrax could be directly transmitted from one cow to another.
When did Koch investigate anthrax
1870s
What did Koch discover?
He further investigated anthrax and established that the true cause of the disease was anthrax endospores that persisted in the soil.
Koch used the knowledge he gained from his research into anthrax (and also tuberculosis, or TB) to develop
a series of postulates to establish whether a microorganism is the causative agent in a disease.
Why did Koch recieve a nobel prize
for the identification of the bacterium Mycobacterium tuberculosis as the cause of TB
The first antibiotic was discovered in and by who
1928; Alexander Flemming
He observed a mold growing on one of his plates containing
staphylococci
Large-scale production and use of antibiotics began in the _______ to _________
1940s; to treat soliders
A pathogenic bacterium that has recently evolved is
methicillin-resistant Staphylococcus aureus, or MRSA. MRSA is a strain of S. aureus that has developed resistance to most antibiotics and is extremely difficult to treat.
Penicillin-based antibiotics prevent bacterial growth by
disrupting the formation of the bacterial cell wall by inhibiting peptide crosslinking, which makes the cell wall weak
Bacteria classified as Mycoplasma
do not take up Gram stain because they lack a cell wall and have only a thick cell membrane
Is S. aureus a facultive or obligative aerobe/anarobe?
is a facultative anaerobe that grows much faster in an anaerobic environment such as puncture wounds.
Psychrophilic bacteria grow best in cold conditions
between -15 and 10°C
Mesophilic bacteria grow best between
15 and 40°C
thermophilic bacteria grow best between
45 and 80°C.
Strains of nonpathogenic E. coli are commonly used in _____ and are grown in incubators at _____ to mimic the temperature inside the ______.
laboratory research; 37 C; human colon
Lactobacillus delbrueckii subspecies bulgaricus, or L. bulgaricus is one of the species of bacteria used to make
yogurt, cheese, sauerkraut, pickles, kimchi, and animal feed such as silage
The recombinant proteins are then
urified from the bacteria using chromatography.
When and who cloned the human insulin gene and genetically engineered E. coli bacteria to produce human insulin protein that could easily be purified and used as a treatment for diabetes?
1978; Genetech (one of the first biotechnology companies)
Bacteria, yeast, and some plant cultures are grown on ______ in _______.
solid media; petri plates, slants, and deep tubes
Slants are used to
culture bacteria for a short time but not to isolate colonies.
Bacteria grown on petri plates or slants can be stored for _____ at _____
1-2 weeks; 4 degrees celcius
Stab cultures are similar to ______, but are formed by _________
agar plates; solid agar in a test tube
Deep tubes are used
to store or study bacteria in stab cultures, where bacteria on an inoculation loop or needle are stabbed two or three times into the deep tube of solidified agar.
Stab cultures can also be used to look at
motility and gas requirements of bacteria by observing where the bacteria grow
Solid media come in many formulations, the most generic being nutrient agar that is made from
peptone and beef extract.
Eosin methylene blue (EMB) agar makes E. coli colonies turn ______, lactose-fermenting bacteria turn _________, and non-lactose fermenters, such as Salmonella enterica, remain ______
shiny green; dark blue/black; colorless
The Kirby-Bauer, or disk diffusion test, is used to determine
the sensitivity of bacteria to antibiotics. In this test, paper disks impregnated with various antibiotics are placed on an agar plate covered with the bacteria of interest. The area surrounding the disks where the bacteria are inhibited from growing is measured and used to quantify the effectiveness of the various antibiotics against the bacteria.
Broth are used to
produce large quantities of bacteria suspended in liquid, usually for protein production or for isolating plasmid DNA
Liquid media are often formulated with the same ingredients as solid media but without the
agar
Another common broth used to culture E. coli
TB (Terrific Broth)
TB for E. coli is used to
culture E. coli for plasmid purification
bacteria are grown on a large scale in specialized vats, called ________, under under controlled conditions.
specialized vats, called fermenters and bioreactors.
A biological safety cabinet (BSC) must be used
to limit the chance of an aerosol contaminating a worker
Tissue culture hoods, which are Class II BSCs, are used
to maintain sterility within the hood and reduce the chance of contamination of the culture.
A colony is
a single bacterium that has multiplied on a solid medium into millions of clones of itself and looks like a round visible dot on the solid medium
Inoculating an agar plate with a portion of the culture and counting the colonies is one way
to quantify the bacteria in the culture
The culture must be diluted at least____________, to obtain an agar plate with single colonies.
million times, or 1 ml into 1,000 L
A spectrophotometer can also be used
to determine the concentration of bacteria in a culture.
The Gram stain was invented by Hans Christian Gram in ______ to differentiate between two types of ___________________
1882; pneumonia bacteria
To assay the metabolic properties of bacteria, they are inoculated on media containing additives that result in______
a visible difference when metabolized by bacteria
the starch hydrolysis test determines
if bacteria produce amylase.
What happens to Bacillus subtilis and Bacillus megaterium during the starch hydolysis test?
When amylase-producing bacteria, such as Bacillus subtilis and Bacillus megaterium, are grown on medium containing starch, they digest the starch.
If the medium is stained with an iodine reagent containing potassium iodide (KI)
the agar turns blue if starch is still present in the medium
Identifying factors to determine the type of bacteria include
color, size, and growth pattern of the colony
A diffuse or spreading margin around the colony indicates that the bacteria are
motile
The phospholipids create a hydrophobic zone that
prevents water loss and ensures that charged particles enter and leave through transmembrane proteins
Fungi have a cell wall that is composed of
chitin, a polymer of beta glucose
In modern times, yeasts are also used in protein production since
they are inexpensive and easy to culture.
Many other fungi are used to
produce enzymes commercially
Aspergillus niger is commonly used industrially to produce
cellulases, pectinases, and amylases
Fungal enzymes are currently being investigated in
biofuel research
As the decomposers in the food chain, fungi naturally have the ability to break down
cellulose in plant cell walls
ER
has an Internal transport system and site for protein translation, the production of new membranes, proteins, and lipids, and the detoxification of poisons.
RER
is where mRNA is translated into polypeptide chains during protein synthesis.
. SER plays a role in
lipid production and protein storage
Centrosomes are made up of
centrioles near the nucleus in animal cells
Protists are being investigated as ________, because __________
potential sources of biofuels since they are so numerous and grow quickly
Common eukaryotic cells used in cell culture for protein production include
yeast and other fungi such as Aspergillus, insect cells such as the Sf9 cell line from the fall armyworm (Spodoptera frugiperda), and mammalian cell lines such as Chinese hamster ovary (CHO) cells
Antibodies are
multisubunit proteins and therefore need to be made in mammalian cells
To produce Herceptin, the gene for the humanized antibody is cloned into
CHO cells
The CHO cells to produce Herceptin are then cultured in a _______ in ____________
suspension culture; large bioreactors
The Hereceptin are then ______________ and ______________
purified from the cells; used to treat cancer patients.
What are the 3 domains?
Archaea, Bacteria, and Eukarya
Are there more bacteria or more archea?
more Bacteria than Archaea
Archaea were not recognized as a separate domain until the
1970s
Currently, there are no known human diseases caused by
Archeans
___________ are found in most habitats on earth, including the human digestive system
Archeans
Analyses of the remains of King Tutankhamun in Egypt indicate that __________ caused the kings death
Malaria
Malaria also had a role in the decline of the
Roman Empire
Almost half of the world’s population is at risk for
malaria
Anthrax rarely _________, but it could be used for ________
affects humans; terrorist attacks
Anthrax infection was frequently used as a
model system by these early scientists
What are model systems?
They are used by scientists to investigate something new in an organism or biological system they already know a lot about.
Who are recognized as founders of microbiological research?
Koch and Pasteur
TB caused
1 in 7 deaths
This “zone of inhibition” was caused by
penicillin produced by the mold
Most bacteria contain a single loop
of gDNA
The cell wall is composed of two alternating sugars ___________ in _________
NAM and NAG; a polymer that is crosslinked with small peptides.
facultative aerobes
if they just grow better in the presence of oxygen but it is not required
There are extreme thermophiles, also called hyperthermophilic bacteria, which thrive at temperatures
above 80°C
here are also pathogenic forms of E. coli responsible for
food posioning outbreaks
Salt (sodium chloride) is required for
the growth of most bacteria and to maintain the osmotic balance between their environment and the inside of the cell.
Bacteria that live in extremely salty conditions are called
halophiles
Once a culture has reached the appropriate cell number or density, the bacteria are
harvested and lysed (broken open)
Humulin
which was the first drug made from a recombinant protein, is still the major recombinant human insulin used in the U.S. today
Humulin is now produced in
eukaryotic cells
Solid media are referred to as
agar
Petri plates are used to
are used to isolate individual colonies of bacteria
The most common medium used for culturing E. coli is
lysogeny broth (LB), which is also known as Luria-Bertani broth
Bunsen burners are used to heat sterilize
inoculation loops and the mouths of glass bottles
Incubators
maintain a constant temperature.
Fermentors and bioreactors have
inlets and outlets to regulate stuff
The streak plate method allows
isolation of a single colony from a bacterial culture by splitting the plate into quadrants and diluting the bacteria repeatedly.
Each individual bacterium spread on the plate will grow into a colony and is referred to as a
colony forming unit (CFU)
The value given by a spectrophotometer is referred to as
an optical density (OD)
What does the OD measure?
measures how cloudy the culture is
If a bacteria is shiny what does it mean?
it has a capsule
. Some bacteria also produce pigments and so colonies are
colored
Golgi Body
contains enzymes that modify proteins by adding phosphate or carbohydrate groups; helps the cell distinguish between self and non-self (foreign) cells.
Small vesicles often pinch off of the RER and merge with the Golgi apparatus for further
processing
Protists are a type of
eukaryotes
Many protists, including algae in the ocean are
protosynthetic primary producers
Many industrial enzymes, such as those in laundry detergents, are made using
Aspergillus
nsect cells are also used, but may not correctly perform
all the required posttranslational modifications of proteins
Currently there are multiple clinical trials in Phase 1, 2, or 3 to evaluate _____________; including ____________
the safety and efficacy of stem-cell derived cells in patients; stem-cell derived neural, adipose, and muscle cells
When did scientists induced human adult stem cells to become pluripotent or switch back to an embryonic ICM-type state?
2007
These induced pluripotent stem cells (iPSC) have huge potential since cells could be
removed from a patient, induced into pluripotency, and then differentiated into the type of cells required to treat the patient’s disease without any risk of immune rejection.
The first human embryonic stem cells (hESC) were isolated in
1998
The first transplant using tissue derived from stem cells was performed in
2017
Yeast cells are cultured in a manner similar to bacteria
on agar plates or in liquid broth
Primary cells are used by researchers to
investigate how normal cells behave
Primary cells are taken and cultured directly from
an animal
What is the disadvantage for primary cells
cultured for only a short amount of time. This makes them unsuitable for biotechnology companies that wish to manufacture therapeutic recombinant proteins
Most primary cells and cell lines need to adhere to tissue culture dishes or flasks while they are growing (_________) and stop growing when they come in contact with another cell which is a phenomenon called ___________.
adherent cultures; contact inhibition
Some cell lines have lost the requirement for adhesion and can grow in
suspension
Unlike mammalian cells, plant cells are totipotent which means
single cell can regenerate an entire plant
This forms a mass of undifferentiated cells called a
callus
The callus can then be broken up into individual cells in a ___________ or manipulated as a whole callus while the cells are _____________
suspension culture; transformed with novel DNA
Once plant cells are genetically engineered
they are given differentiation signals to produce roots and shoots, which grow into whole plants
the media used for culturing mammalian cells typically contain
a pH indicator called phenol red, which stains the media pink and changes the color of pH if it isn’t optimal
Yeast cells grow best at
30°C
mouse and human cells grow best at
37°C
Special incubators are needed for mammalian cells since they usually need to be grown in an environment with
a high level of CO2
Yeast, insect, and plant cells can be cultured in
regular incubators
Plant cells are affected by light, so
undifferentiated plant cells are often grown in the dark to discourage redifferentiation
Class II BSCs, commonly called tissue culture hoods, should be used to
prevent contamination of eukaryotic cultures.
Class I BSCs do not protect the _____________ but protect the _______________
material in the cabinet from contamination in the room; user and the environment
Traditional fume hoods protect
just the user
Cells can also be incubated with antibodies linked to fluorescent dyes that bind to specific proteins on the cell surface which
helps identify the type of cell
Selective stains can be used to
identify specific organelles using a compound microscope
methylene blue is used to
highlight the nucleus
Janus green B specifically stains the
mitochondria
Molecular biology bridges the disciplines of
genetics and biochemistry
Molecular biology also refers to an array of techniques that
relate specifically to the manipulation of DNA and is used in that context
Scientists have also used molecular biology tools to
trace strains of cultured microorganisms back to their source, solving mysteries such as the anthrax poisonings of 2001
Billions of dollars are generated each year by the ___________ through the production of _____________ for _________ using _______________
pharmaceutical industry ; recombinant proteins; medicine; molecular biology tools
Companies have used molecular biology to develop drugs like Humulin to ______________ and __________________________
treat diabetes; Protropin to alleviate deficiencies in human growth hormone.
_____________ coined the phrase the “central dogma of molecular biology” in ______
Francis Crick; 1956
There are special circumstances in which genetic information flows differently like
RNA viruses, such as the influenza virus, can replicate their own RNA using a virus-encoded RNA-dependent RNA polymerase. Other viruses, such as the human immunodeficiency virus (HIV), can copy RNA into DNA. Retroviruses like HIV use reverse transcriptase, an enzyme that has become an important tool in cloning
___________ deciphered the structure of DNA, scientists have been studying how ___________. This research has been the foundation of the ____________, leading to the ________________________.
James Watson and Francis Crick; its structure leads to its function; recombinant DNA revolution; development of genetic engineering and the polymerase chain reaction (PCR).
it is the complementarity between bases that is the basis of
PCR and DNA sequencing
The phosphate group of the nucleotide is attached to ___ and the base is attached to __________.
carbon 5; carbon 1
Nucleic acids are synthesized in ________ only in a 5’ to 3’ direction
vivo
As the strand grows, each new nucleotide is added to the _____________ of the sugar. the relative positions are usually noted as being either upstream (toward the __ end) or downstream (toward the __ end) from one another.
3’-hydroxyl group (attached to carbon 3); 5’; 3’
Modern biotechnology is based on the premise
that DNA is virtually the same in all cells and can be read and acted on (or expressed) in all cells.
In the pharmaceutical industry, recombinant proteins are
produced and sold as therapeutic drugs.
In medicine
people with genetic diseases can be helped by gene therapy where a recombinant gene is expressed in specific tissues to counter a genetic defect.
The first gene therapy to treat leukemia was approved in the ________ in _______ and others are _________
US; 2017; in the clinical trial stage
In _____ bacterial enzymes that cut DNA in very predictable locations were discovered
late 1960s,
The discovery of restriction enzymes by_________ started the _______.
Werner Arber, Hamilton Smith, and Dan Nathans; DNA revolution.
How do phages often kill bacteria?
Phages inject their DNA into bacteria and then use the bacterial cellular machinery to reproduce more copies of themselves, frequently killing the bacteria.
How does bacteria destroy the page?
Bacteria produce restriction enzymes to cut the phage DNA, thereby destroying the phage.
Why are restriction enzymes named the way they are?
Restriction enzymes were so named because they limit or restrict the growth of phages; at the time, scientists did not understand how these enzymes functioned.
Since the bacteria’s own DNA may contain the same restriction sites, bacteria protect their DNA from being cut by ______________The presence of it changes ____________ and prevents ________
adding methyl groups to the DNA bases; the bacterial DNA conformation; restriction enzymes from recognizing and cutting the restriction sites on the DNA
Specific restriction enzymes are named for the bacteria from which they were originally isolated.
The name uses the first initial of the genus and the first two letters of the species name of the bacterium, followed by the order in which the enzymes were discovered in that particular strain
EcoRI
The first restriction enzyme isolated from Escherichia coli RY13 strain
HindIII
The third restriction enzyme isolated from Haemophilus influenzae Rd strain.
PstI
The first restriction enzyme isolated from Providencia stuartii bacteria.
Restriction enzymes are also called
endonucleases
In general, restriction sites are
palindromic
By chance, a 4-base sequence occurs every ________ in a DNA sequence, while a 6-base sequence occurs every __________.
few hundred base pairs; several thousand base pairs
Ligase catalyzes
the formation of a phosphodiester bond between the free 5’-phosphate end on the backbone of a DNA fragment and the 3’-hydroxyl group of another DNA fragment.
To prevent plasmids from self-ligating
the phosphate on the 5’ end of the plasmid DNA is removed using an enzyme called phosphatase in a process called dephosphorylation.
The DNA fragments can also ligate to one another. To reduce this occurrence,
the molar ratio of plasmid to DNA fragment is controlled during a ligation reaction and is usually, but not always, 1:1.
After the ligation is complete
the ligated DNA is transformed into bacteria.
The use of restriction enzymes relies on __________ and it also requires several separate reactions, so it can be ______________
the presence of restriction sites in the DNA sequences to be cloned; time-consuming, error-prone, and inefficient.
Type IIs restriction enzymes (for example, BsaI)
cut DNA outside of their recognition sites
The topoisomerase I cuts DNA at a specific sequence much like a restriction enzyme. It then stays with the DNA (for example, the plasmid) and .
ligates any DNA fragment with compatible overhangs to create a recombinant molecule
Enzyme concentration is often given in
units of activity (U) rather than the concentration of protein.
Most enzymes are also supplied with reaction buffers that
provide the optimal reaction conditions and provides the optimal salts and pH for that specific enzyme
Many enzymes should be stored at the recommended temperature, usually _____and are usually supplied in _______
20°C; glycerol so that they do not freeze.
Freeze-dried enzymes are designed
for a single use
