Student Reader » MIMG 103L

Viral DNA Synthesis Experiment

Levi Clancy — Tue, 18 Oct 2011 16:02:18 +0000

The viral DNA synthesis experiment determines when viral DNA synthesis begins.

Infect cells, add ³H-thymidine.
Remove aliquots every 2 minutes, hybridize to filters containing cold phage DNA.

Determine when rise in viral DNA occurs.

Handed DNA encoding ORF3 of Phage 1065. How would you determine whther the .8kb mRNA for ORF3 is amplified after viral DNA synthesis? The first step is to determine when DNA synthesis occurs. See previous problem. Then perform Northern blots to determine if the .8kb mRNA appears either before or after viral DNA syntehsis Polyclonal antibody to ORF3. You also have one to ORF5. To determine if ORF3 is made before or after ORF5 in viral life cycle: infect cells; aliquot oevery 2 minutes for Western blot; probe 2 Western blots (one to ORF3 and one to ORF5) and then determine which is made first.

Common Laboratory Microbes

Levi Clancy — Fri, 19 Aug 2011 19:49:21 +0000

Pseudomonads	Optimal temperature of 30°C; strict aerobes, except in presence of nitrate or amino acids, and never fermentative; gram-negative; motile; non-sporulating; and cytochrome oxidase positive. No more than 3µm in size. Some species are capable of dissimilative nitrate reduction (denitrification) — assimilative nitrate reduction is for nitrogen as a general nutrient, instead of an energy source.
E. Coli	Rod-shaped, gram-negative, motile and diverse.
M. smeg
S. epidermidis
S. salivarius	Gram-positive chains of cocci; requires sucrose to form capsules; ferments glucose.
Streptomyces	Soil-dwelling sporulating strict aerobes, many of which secrete antibiotics.

Tissue Culture

Levi Clancy — Fri, 31 Oct 2008 00:42:14 +0000

There are two types of tissue cultures: primary cultures, derived directly from animal tissues; and transformed cell lines, which are engineered to reproduce indefinitely. Tissues cultures must be handled in biosafety hoods because they are innately considered biohazards (transformed cell lines are oncogenic), and are highly susceptible to the fungi which heavily populate breathing air.

Tissue Culture	Overview
Primary Cultures	A primary culture is harvested directly from animal tissue and then incubated in a tissue culture dish with nutrient-rich growth media enriched with blood serum. Primary cultures will frequently divide a limited number of times (due to their inherent programming) and also might contain different cell types.
Transformed Cell Line	A transformed cell line can divide indefinitely in vitro. Transformed cell lines are frequently derived from single cells, thus ensuring a culture with uniform genetic composition, growth characteristics and susceptibility to infection. Transformed cell lines are often derived from: tumors; primary cultures transformed or transfected to express oncogenes; from tissues treated with carcinogens; or from primary cultures with spontaneous oncogene mutations.

Tissue cultures are very susceptible to bacterial and eukaryotic contamination. For this reason, bacterial antibiotics are frequently added to their growth medium. In addition, media changes pH as its nutrients are consumed and cell waste accumulates. CO₂ is often kept at ∼5% in tissue culture incubators to help stabilize pH; also, a pH indicator is frequently added so that researchers know when to replace the media. Lastly, humidity is very important for tissue culture incubation so that the media does not evaporate.

Population Dynamics

Levi Clancy — Mon, 27 Oct 2008 17:42:08 +0000

In binary fission, a parent cell divides to form two daughter cells. Thus, bacterial population increase exponentially. Note the table below, demonstrating how quickly a population of bacteria can explode from just a single cell. The generation (first, second, third, etc) is denoted n and the number of cells at a given time is N (or N₀ at time zero).

Generation (n)	1	2	3	4	5	6	7	8	9	10	11	12	n
Formula	2⁰	2¹	2²	2³	2⁴	2⁵	2⁶	2⁷	2⁸	2⁹	2¹⁰	2¹¹	2^n-1
# of Cells (N)	1	2	4	8	16	32	64	128	256	512	1024	2048	N

The table below describes important formulas regarding population dynamics. They are valid for any value of x, as long as it is kept constant throughout the problem. Also, t is elapsed time and t_d is the time require for one complete cell division (for a cell to double).

Important Formulas

N = N₀2ⁿ

log_x N = log_x N₀ + n (log_x 2)

log_x (N / N₀) = n (log_x 2)

log_x (N/N₀)

log_x 2

N = N₀ antilog₁₀ (.301n)

n = t / t_d

Interrupted Mating Experiment

Levi Clancy — Mon, 20 Oct 2008 06:32:58 +0000

The Interrupted Mating Experiment basically uses the following steps:

Cells are mixed, then shaken to break their pili and stop conjugation.
Streptomycin is added to stop cell division.
Cells gain certain traits depending on which genes have been transferred.
By stopping conjugation at different times, gene order of a plasmid and its inserts can be determined.

Oxidase Test

Levi Clancy — Sat, 18 Oct 2008 06:20:37 +0000

The oxidase test assays for the presence of cytochrome c in a cell’s respiratory pathway. Cells are smeared onto a sheet coated with tetrametyl-p-phenylenediamine (TMP), a colorless electron donor. A functional cytochrome c removes electrons from TMP; this oxidized TMP has an intense purple-blue color. A positive control for the oxidase test is Pseudomonas fluorescens, and a negative control for the oxidase test is S. epidermidis.

Antibiotics

Levi Clancy — Thu, 09 Oct 2008 02:41:12 +0000

An antibiotic is any chemical which inhibits microbial growth. Bleach is an antibiotic which completely sterilizes any surface it touches. However, there are other antibiotics which only affect certain cell types. These are useful for isolating bacteria, fungi and subtypes thereof. To test for antibiotic secretion, the microbe of interest is plated onto a lawn of cells. If clearings form around plated colonies, then antibiotics are secreted; if there are no clearings, then antibiotics are likely not secreted. Commonly used antibiotics are described below.

Antibiotic	Overview
Cycloheximide	Cycloheximide selectively inhibits eukaryotes by interfering with 80S ribosome-directed protein synthesis.
Chloramphenicol	Chloramphenicol selectively inhibits prokaryotes by interfering with 70S ribosome-directed protein synthesis.

Polymerase Chain Reaction

Levi Clancy — Fri, 19 Oct 2007 04:23:43 +0000

The polymerase chain reaction (PCR) replicates specific genetic sequences (DNA or RNA, in either plus- or minus-sense) so that vast quantities of a certain DNA segment can be quickly produced (amplification). PCR relies upon a few basic steps:

Obtain genetic material for PCR.
Add oligonucleotide primers complementary to the sequence you wish to amplify.
Single dNTPs are added to provide base pairs for newly synthesized DNA or RNA.
Heat-stable polymerases are added to conduct the replication.
The reaction is repeatedly heated and cooled, splitting and annealing DNA for the replication assembly to bind an conduct the replication.

Gram Stain

Levi Clancy — Sun, 18 Dec 2005 02:05:59 +0000

Gram staining distinguishes cells based on bacterial cell walls. Gram-positive organisms have a thick cell wall and outer membranes that retains crystal violet, while Gram-negative cells have no inter-glycine bridge and thin cell walls which eventually are stained pink. There are several potential problems with Gram staining: old Gram-positive cultures may stain Gram-negative; Gram-negative smears will not decolorize if the smear is too thick; samples may become over-decolorized and falsely appear Gram-negative; and Gram stains appear greenish under phase optics.

Apply Culture	If staining a broth culture, use a sterile loop to transfer a small droplet onto a glass slide. If staining a growth from a solid medium, first place a small droplet of water onto a slide and then transfer some culture into the droplet using a sterile loop. Repeat this procedure onto different parts of the slide using a gram-positive control and a gram-negative control.
Air Dry & Fix	Afterward, allow the smears to air dry and then pass the slide thrice rapidly through a Bunsen burner flame (smear side up). If you cannot read print through the smears, then they are too thick. The slide should not be so hot that it hurts to touch.
Crystal Violet	Apply a solution of crystal violet to the heat-fixed smear and let soak for about one minute. This is also called the staining step.
Water Rinse	Rinse slide under gently flowing water. Shake off excess water.
Iodine	Squirt the slide with iodine to remove excess water, then apply iodine and let soak for about one minute.
Alcohol	Gently spill a few drops of 95% ethanol onto the slide, then tilt the slide back and forth. Repeat this step twice or thrice, until purple stain ceases to drain from the smears. The alcohol washes crystal violet from thin Gram-negative cell walls, but thick Gram-negative cell walls still retain the crystal violet. This is a critical step: if not enough alcohol is applied, then all cells will appear Gram-positive; if too much alcohol is applied, then all cells will appear Gram-negative.
Water Rinse	Rinse slide under gently flowing water. Shake off excess water.
Safranin	Squirt the slide with safranin to remove excess water, then apply safranin and let soak for about one minute. This step stains the washed-out Gram-negative cells pink, and is also called the counter-stain.
Water Rinse	Rinse slide under gently flowing water. Shake off excess water.
Blot & Dry	Gently blot the slide with a paper towel. Do not rub. Rapidly pass the slide once through the Bunsen burner flame.
Examine	Examine the slide using microscopy. Gram-stained slides can be studied using oil immersion. Make sure your Gram-positive control is purple, and that your Gram-negative control is pink. Compare your sample to these controls to conclude the Gram nature of the organism.

Agarose Gel

Levi Clancy — Sun, 18 Dec 2005 01:35:21 +0000

Agarose gel (aka agar) is a very firm jelly that is laced with nutrients to grow colonies of microbes. A colony is a single cell which has reproduced to form a visible dot of cells. Note that fungal colonies are oftentimes fuzzy, while bacterial colonies are typically compact and gummy. Agar nomenclature is very simple. For example, YTA-enriched GMA is GMA enriched with a tiny amount of YTA. Also, please review types of microbes since it is crucial to create an optimal environment for the microbe you are selecting or screening. Lastly, remember that antibiotics can be added to agar to inhibit growth of a particular type of microbe.

Minimal Agar	MA	A simple medium containing a few salts.
Glucose Minimal Agar	GMA	Minimal agar with glucose.
Yeast Tryptone Aga	YTA	A complex agar containing yeast extract and tryptone.
Blood Agar		Usually just a mixture of sterile sheep blood and agar, blood agar is useful for screening based on α- or β-hemolysis. Many bacteria secrete enzymes that destroy blood cells, causing a clear lytic zone to form around the colony on an otherwise red plate. α-hemolysis is caused by overall harmless bacteria that degrade hemoglobin to greenish-yellow bile pigments. However, β-hemolysis is caused by pathogens such as Streptococcus and causes completely clear lytic zones by a massively destructive battery of lytic enzymes.
Hektoen-Enteric Agar	HE	A selective and differential agar containing bile salts, bromothymol blue and acid fuschin. Bile select for Salmonella and Shigella by killing gram-positive and coliform species. Bromothymol blue and acid fuschin are indicators: coliforms appear orange/salmon; and non-lactose fermentors form blue-green (Salmonella) or green (Shigella) raised and moist colonies.
Levine’s EMB Agar		A selective & differential media for isolating gram-negative enteric bacteria. EMB agar contains eosin Y, methylene blue and lactose. This selects for gram-negative cells because methylene blue inhibits gram-positive growth. Also, this agar is differential because eosin Y is an indicator: lactose fermentors like Enterobacter appear pink/brown; E. coli (also a lactose fermentor) appears metallic green; and non-lactose fermentors are translucent and amber or colorless.
Sabourauds Agar	SA	Saboraud’s agar (SA) is a complex media that selects for molds and yeasts via acidity (pH 5.6) and high sugar content (2% glucose).
Glucose, Yeast & CaCO₃	GYC	Contains glucose, yeast extract and calcium carbonate (GYC). GYC is an indicator for glucose fermentation; lactic acid, a byproduct of glucose fermentation, reacts with calcium carbonate for form clearings.
Starch Agar		Secreted amylase breaks down starch into maltose and other sugars, forming a clearing around amylase-secreting colonies. Pouring iodine onto a starch agar plate will reveal undigested starch (blue) and digested starch (reddish brown). A common positive control is Bacillus subtilis.
Gelatin Agar		Secreted proteolytic enzymes hydrolyzes gelatin (a protein) into constituent amino acids. Trichloroacetic acid precipitates undigested gelatin — if poured on the plate, hydrolyzed gelatin appears as a clearing amidst an opaque precipitate of undigested gelatin. A common positive control is Bacillus subtilis.
Egg Yolk Plate		Secreted phospholipases hydrolyze lecithin, the major phospholipid in egg yolk. Lecithin hydrolysis releases long chain fatty acids, visible as an insoluble waxy buildup around colonies. A common positive control is Pseudomonas fluorescens.
O/F Media		Usually used with agarose, oxidation/fermentation media (O/F media) consists of tryptone (an amino acid source), 1% glucose and bromothymol blue (an indicator dye). Despite its name, bromothymol blue is green at neutral pH. Anaerobic conditions are created by pouring oil over the agar; aerobic conditions are created by not pouring oil. Oxidation and fermentation of glucose both result in acid byproducts which turn bromothymol blue a yellow color. Deamination of amino acids leads to alkaline byproducts which turn bromothymol blue a blue color.
King’s B Medium		King’s B Medium (KBM) with 0.2% KNO₃ is one of many agars used for distinguishing pseudomonads. Positive controls such as P. fluorescens fluoresce in KBM, while negative controls such as P. aeruginosa do not. Also, a nitrite reagent can be added after incubation to assay for reduction of nitrate to nitrite.

Auxotrophy

Levi Clancy — Sun, 18 Dec 2005 04:16:41 +0000

Auxotrophs are unable to synthesize all their own biomolecules (nutrients, such as amino acids). For example, a bio^- mutant is unable to synthesize biotin. Auxotrophs can be studied to understand pathways for the synthesis of amino acids. Also, they can be used for other experiments (such as the Ames test and R-factor experiment).

Isolating a lysine auxotroph, from a mixture of auxotrophs and prototrophs, is a simple procedure. You can easily modify this procedure to isolate almost any specific kind of auxotroph.

Grow cells in media containing penicillin (which will kill dividing cells) and all amino acids except lysine.
Remove the penicillin-laced media and replace with media containing lysine
Screen for lysine auxotrophs
Further assays may be performed to determine the location of the mutation in the lysine synthesis pathway

Another, more efficient procedure is to:

Plate auxotroph/prototroph mixture on enriched GMA
Screen for tiny colonies
Pick tiniest colonies
Grow on GMA and YTA
Colonies able to grow only on YTA are auxotrophs

Auxotrophs form tiny colonies because there is only a small amount of complex media in enriched GMA. This is an example of screening, because you distinguish between the two different colony types (auxotrophs and prototrophs) based on colony morphology.

However, if you want to isolate any cell exhibiting auxotrophy (from a batch of overwise prototrophic cells) then the procedure is different. This can be helpful if you want to determine the pathway for synthesizing a certain compound. The procedure for outlining the biosynthetic pathway for lysine is given below. You will use E. coli as your start culture because WT E. coli are prototrophic.

Inoculate minimal broth with E. coli
Mutagenize culture, resulting in mixture of auxotrophs and prototrophs
Add penicillin
Replace media with minimal broth + glucose + lysine
Plate onto enriched GMA
Pick tiny colonies, inoculate GMA and GMA+lysine
Colonies that grow only on GMA+lysine are lysine auxotrophs
Perform a —- test.

Lac Operon

Levi Clancy — Thu, 15 Dec 2005 23:40:19 +0000

Native to E. coli, the lac operon allows cells to use lactose (a sugar found in milk) for energy. Found only in prokaryotes, an operon is a contiguous row of genes transcribed from a single promoter. This means that genes within an operon are activated together and inactivated together. Most operons — including the lac operon — encode polycistronic mRNA, which is a continuous mRNA molecule encoding proteins involved in the same pathway. The anatomy of the lac operon is detailed below.

Gene	Product	Overview
I		Not part of the operon. Encodes a tetrameric repressor called lacI that binds to the operator (O) and blocks σ factor from binding to the promoter (P).
P_lacZYA		A weak promoter for genes Z, Y and A that binds RNA Polymerase.
O		O (the operator) is where the repressor lacI binds. A mutation in the operator leads to constitutive expression, meaning constant high-level expression even without an inducer.
CBS		Where CRP binds, misleadingly titled CAP Binding Site (CBS).
Z	β-Galactosidase	β-galactosidase breaks lactose into glucose and galactose.
Y	Lac Permease	Lac permease transports lactose into the cell via an electrochemical gradient.
A	Thiogalactoside Transacetylase	Thiogalactoside transacetylase detoxifies the cell, since this process is toxic to the cell.

The various factors involved in lactose metabolism were identified via direct analysis. Since these reactions are performed even in extracts lacking any cells, product formation was analyzed at every step of the way under various conditions. There are two regulatory forces: the repressor (lacI) and the activator (CRP). The repressor, lacI, binds to the operator and prevents transcription by blocking σ factor from binding to the promoter. The activator, CRP, is composed of cyclic AMP (cAMP) and CAP and binds to the CAP Binding Site. Once CRP binds to the CAP Binding Site, RNAP binds with high affinity to initiate transcription.

Glucose is easier for cells to catabolize (digest) than lactose, and the lac operon has evolved so the cell catabolizes only the best energy source available. Catabolization of glucose generates catabolites (byproducts) which lead to reduced cAMP levels. Without any cAMP to bind to CAP, no CRP can form and the lac operon is not activated. Thus, glucose concentrations are inversely proportional to cAMP concentrations — high levels of glucose lead to low levels of cAMP, and vice versa.

Β-Galactosidase	When inserted randomly into the mouse genome without associated control sequences, -galactosidase is not expressed in any tissue. However, when ligated to a 1kb sequence conserved in fish, Ligate and inject into fertilized mouse egg to generate a transgenic mouse with this DNA inserted randomly into some region of the genome. (Not gene targeting by homologous recombination.) Noncoding regions near a gene that is highly conserved across species (determined via comparative analysis) often a site for transcriptional control.
Allolactose	Allolactose is a byproduct of lactose metabolism that binds lacI (the repressor) to keep it form binding to the operator.
cAMP Receptor Protein	cAMP Receptor Protein (CRP) is a dimeric activator protein composed of cyclic AMP (cAMP) and CAP. CRP binds binds next to the promoter and helps RNA Polymerase bind the promoter, thus increasing transcription of lac and other operons.
Adenylate Cyclase	Adenylate Cyclase (cya) produces cAMP form ATP, and is inhibited when glucose is present.
Glucose	Glucose activates the phosphotransferase system (PTS). One component of the PTS — IIa — inhibits production of cAMP from ATP, thus lowering cAMP concentrations and preventing CRP (the activator) from forming.
Lactose	Lactose is transported into the cell by lac permease, then induces the lac operon to produce β-galactosidase. β-galactosidase breaks lactose down into glucose and galactose.
ONPG	Ortho-Nitrophenyl-β-galactoside is a colorless chemical cleaved by β-galactosidase to form galactose and orthonitrophenol (a yellow compound). This color formation allows for assay of β-galactosidase activity; however, ONPG does not induce the lac operon.
IPTG	Isopropyl β-D-1-thiogalactopyranoside mimics allolactose, a byproduct of lactose metabolism which induces the lac operon. IPTG does not require lac permease to enter the cell, and CRP must also be present to activate transcription.
cAMP	cyclic AMP (cAMP) is necessary for CRP to form and bind CBS; in absence of cAMP (even without repression) transcription is not activated. cAMP alone is not enough to activate transcription; the repressor has to be deactivated, or the operator (where the repressor binds) must be mutated.
Maltose	Not used preferably.
Mannitol	Mannitol is preferable over lactose; glucose is more preferable than mannitol.

Growth Media

Levi Clancy — Fri, 16 Dec 2005 07:33:10 +0000

Minimal media consists of merely a carbon source and trace elements (P, S, K, Mg, Ca, Na). Iron is used for cellular respiration. N₂ is used for N-fixing bacteria. Growth factors needed n small amounts: vitamins, amino acids, purines and pyrimidines.

Simple Media	Contains precise amounts of highly purified organic and/or inorganic chemicals. For example, simple media might contain a single (or none) carbon source as well as mineral sources of nitrogen and sulfur. An organism growing in such a simple media could be cultivated from sand or other meager environment.
Complex	Precise chemical composition is unknown. Complex media typically include all amino acids, vitamins, purines and pyrimidines (these are all usually extracted from yeast, meat or milk). Organisms growing on complex media without sugar use amino acids for energy and biosynthesis, typically creating foul odors in the process. Organisms necessitating complex media with sugar perform glycolysis.
Differential	Differential media is an indicator media used for screening — meaning dyes are added to the media to identify certain microbes.
Enriched	Complex media with additional nutrients like blood. Enriched media is useful for isolating organisms from a body.

Microbes

Levi Clancy — Fri, 16 Dec 2005 07:11:38 +0000

Colony Form	Description
Pinpoint	Smaller than 1 mm.
Circular	A circle larger than 1 mm.
Irregular	A non-circular shape.
Filamentous	Resembling a bunch of strings.
Colony Elevation
Cushion
Raised
Flat
Raised Center
Colony Surface
Smooth	Smooth colony surface.
Rough	Rough colony surface.
Ringed	A colony surface with rings.
Colony Texture
Amorphous	A typical textureless colony.
Granular	Chunky and sand-like.
Fluffy	A fluffy texture, like bread or a smoothie.
Mucoid	Runny, like mucous.
Colony Optics
Opaque	Light does not penetrate the colony.
Translucent	Light somewhat penetrates the colony.
Transparent	Light can fully penetrate the colony.
Iridescent	Colony is iridescent, like foil.

Cell Morphology	Description
Coccus	Sphere
Baccilus (Rod)	Rod.
Spirilium	Curved baccilus. Spirilium is also spelled spirrilium.
Spirochete	Spiral.
Appendaged	Bacteria possessing extensions as tubes or stalks.
Filamentous	Bacteria which form long, thin cells or chains of cells.
Endospore	Endospore nucleuses can be centered (central), at one end (terminal) or off-center (subterminal).
Flagella	Peritrichous flagella are located all across the cell surface; polar flagella are located at one location.
Capsule	Capsules have a thick polysaccharide layer surrounding the cell.


Environment	Microbe
Very Cold	Psychrophile
Cold Viable	Psychrotolerant
25º-37º	Mesophile
Hot	Thermophile
Extremely Hot	Hyperthermophile
pH < 5	Acidophile
5 < pH < 9	Neutrophile
pH > 9	Alkaliphile
Dies w/ O₂	Obligate anaerobe
Dies w/o O₂	Obligate aerobe
Viable w/ & w/o O₂	Facultative aerobe
Viable w/o O₂	Tolerant anerobe
High [NaCl]	Halophile


Metabolism	Microbe
Uses Unfixed Carbon	Autotroph
Needs Fixed Carbon	Prototroph
Ferments Lactose	Coliform

There are three cardinal temperatures: minimum, optimum, and maximum. The minimum and maximum are the most extreme temperatures at which the microbe can grow. At the optimum temperautre, the microbe can grow the most. The optimum temperature is generally near the maximum.