lecture exam 2

 or transverse fission: Bacteria multiply in a manner that each cell produces two new cells (binary) by splitting in half (fission)

Parent cell

Chromosome replication and cell enlargement

Chromosome and ribosome are divided and separated, 

Septum and cell membrane is created

Daughter cells created

(doubling time) time required for one cell to become two, around 30 min

2^generation to calculate cell population size

Nf=(Ni)2^n

Nf = Final number of cells

Ni = Initial number of cells

n  = number of generations

3 hours (180 min), 

# generation= 180/20= 9 

Nf=(10)(2^9)

5120

change in population over time

Lag- adjusting to the environment, growing

Exponential (log)- no limit on growth with good nutrient and environment

Stationary- new cell=dead cell (lack oxygen, nutrients, build-up of waste)

Death- new cell produced slowly, cell dying/dormant

Pro- estimate of population

Con -Live and dead cells are counted. But only a relative amount

Clear sample: high light transmitted, low absorbency

Turbid sample- low light transmitted, high absorbency

grid count

pro- easy, cheap

con- live and dead both counted

Incubated culture is sampled every interval and place in petri disk,

Pro:  live cell only.

Con: takes long time and material. 

cells with fluorescent dye, illuminated by laser,

Pro: can distinguish live and dead.

Con: cost $$$

Catabolism- big molecule(Carb protein lipid) broken into small, energy released, CO2 and H20 released

Anabolism (biosynthesis)- small molecule(amino acid & sugar) become large(carb, protein, lipid) need energy from catabolism 

They work together for cell metabolism

need to to function

(heat, pH, chemicals)

Enzyme provide physical site which substrate can fit

Enzymes lower activation energy

•Organic catalysts

•Lower the activation energy

•Speed up reactions

•Usually made of protein

•Active site: where catalysis occurs

•Not used up during a reaction (can reuse)

•Greatly affected by temperature and pH

Simple enzyme: Protein only, no cofactors or coenzymes needed

Conjugated enzyme:  Require a coenzyme /cofactor for activity

•Apoenzyme—protein only (cofactor is needed but not present)

•Holoenzyme—protein + cofactor

Exoenzyme act outside the cell

•Endoenzymes act within the cell

Constitutive enzymes always present within the cell

•DNA replication

•Anabolic functions

•

•Inducible (regulated) enzymes produced when needed

•Digestion of lactose

•Catabolic functions

Bacteria- chromosome is ⅓ of cell 

Eukaryotic genomes - located in nucleus

repeating nucleotide

phosphate, deoxyribose,

4 nitrogen bases Adenine, Guanine, Thymine, cytosine

hygrogen bond

Double helix

single strand

Uracil replace thymine

sugar is ribose

Adenine- Thymine

Guanine- cytosine

strands run in opposite directions

5 to 3

order of bond between cabon on deoxyribose and phosphate

Replication- one strand needed to create other strand DNA

transcription - DNA synthesize to RNA

Translation- RNA use to produce protein

Helicase- uncoil DNA helix

Primase- laydown RNA primer

add base to new DNA chain in 5-3 direction

Remove RNA primers and replace gaps between okazaki with correct nucleotide

Ligase- connect all dna pieces into 1 long one 

Gyrase- super coiling

creates RNA molecule from DNA

Leading strand- Created by DNA polymerase III

Lagging strand- after leading strand

 transcription

DNA->RNA 

No T, replace with Uracil

translation

RNA -> protein

RNA

•Single stranded

•Ribose

•Uracil

DNA

Double stranded

Deoxyribose

Thymine

tRNA- clover shape with amino acid and anticodon

mRNA- use uracil instead of thymine

rRNA- structure of ribosome

Carried out by RNA polymerase (RNAP)

1.Initiation: RNAP binds to promoter region of DNA

2.Elongation: RNAP synthesizes RNA in the 5'→3’ direction

3.Transcription stops when a termination sequence in the DNA is encountered.

Large and small ribosomal subunit look for AUG codon on mRNA

tRNA with anticodon corries “methionine” specific amino acid to P site. next codon to A site

Peptide bond is formed between 2 amino acids

Empty tRNA kicked out of ribosome

Stop codon UAA, UAG, and UGA reached

TAC   ACT DNA

AUG  UGA mRNA (U replaces T)

mRNA and tRNA codon creates amino acid

Methionine Typtophane

Intron splicing

•Eukaryotic genes: exons (expressed sequences) and introns (intervening sequences)

•After transcription, intron are removed (loop), and exons connected together for translation

•Vastly increases genetic variability

•Inducible operons, lactose operon, normally turned off (not transcribed)

•Transcription is blocked by the repressor protein

•When lactose is present, the repressor falls off of the DNA and RNA polymerase can transcribe the three genes

catabolic processes

•Repressible operons, arginine synthesis operon, are normally turned on (transcribed)

•As long as arginine is needed, the arginine synthesis operon is transcribed

•If arginine can be found in the environment (for free) or the cell becomes less active, it will bind to the repressor protein and the activated repressor protein will bind to the DNA, blocking transcription

anabolic processes

henotypic alternation in genotype caused by error in replication or mutagens 

addition, deletion or substitution of no more than a few bases

Substitution- One nucleotide is substituted for another. ex.

Missense mutation: different amino acid, may not change protein 

CCA-> Proline    ACA->Threonine

Nonsense- creation of stop condon, short nonfunction protein

UAG->STOP

Silent- same amino acid produced, no change to protein function

UUG-> Leucine  CUG -> Leucine

Back- undergone mutation reverses. Mutates back to its original base composition

Frameshift- addition/deletion of letter change reading frame of mRNA, non functioning protein

The big ABC ATA

DNA pol I and DNA pol III both have proofreading activity and will replace mismatched nucleotides (while it is made)

•Excision repair will remove segments of damaged or mismatched nucleotides, allowing DNA polymerase to replace them

Nonsense- creation of stop condon, short nonfunction protein

UAG->STOP

Frameshift- addition/deletion of letter change reading frame of mRNA, non functioning protein

The big ABC ATA

moving genes between organisms

genetic recombination

dna transfer along pilus, direct, drug resistance

transferred along the pilus is the F plasmid

genetic recombination

dna transfer along pilus, direct, drug resistance

transferred along the pilus is the F plasmid

genetic recombination

free dna from the environment, Indirect, 

•Rare, but common

•Used in lab, add genes to bacteria so they will produce proteins we desire

genetic recombination

dna spread by virus, indirect, exotoxin

•Occurs when a phage accidentally packages a small bit of host DNA into a virus

•Only occurs between closely related bacteria because both must serve as a host for the virus

Create drugs

Compare dna

Restriction enzymes are produced by bacteria, which use them to cut the DNA of invading viruses

palindromic

GAATTC

CTTAAG 

•DNA fragments can be separated using agarose gel electrophoresis.

•Smaller fragments move more quickly through the gel than large.

determining the order and type of base pairs in genes and genome

Amplifies the presence of a single small piece of DNA many millions of times so that it stands out from the background. determine COVID-19 in their bloodstream

1.Template DNA (your sample)

2.Two sets of DNA primers

3.A, G, T, C

4.Taq DNA polymerase

•Taq DNA polymerase from bacterium Thermus aquaticus. hot water, obligate thermophile and is used to growing at very high temperatures (inactive at cooler temps). Keep dna separated at high temp

Denature (94OC)

Anneal (60OC)

Extend (72OC)

30-35 cycles of PCR = several million DNA sequence

DNA Fingerprinting is a Combination of Restriction Enzyme Digestion of DNA and Agarose Gel Electrophoresis.

Recognition endonuclease used to cut DNA  at recognition site then loaded into agarose gel electrophoresis. Small Negative DNA is attracted to positive gel. PCR is used to increase amount of dna

Cloning vector- carries doner DNA and be accepted by cloning host (plasmid used)

Cloning host- usually ecoli, coning vector is inserted

Plasmid:  A Small, Self Replicating, Circular Piece of DNA

ORI- origin of replication, dna start to replicate

Drug resistance

Multiple cloning site- insert gene here

1000X smaller than bacterial chromosome

EX 2000 bq

Recombinant plasmid- 

Restriction enzyme cut plasmid and foreign dna. 

Transformation: Join together in recombinant plasmid. Placed inside host

Selected for grow on antibiotic containing media

Bacteria cell will transcribe gene on plasmid to produce protein 

or transgenic organisms

Recombinant organisms produced through the introduction of foreign genes

•CRISPR•system that bacterial cells can use to remove integrated viral DNA 

•CAS-9 (CRISPR associated protein-9) is an endonuclease (restriction enzyme) that cuts DNA wherever CRISPR binds, allowing viral DNA to be removed

-can remove harmful gene in body of all cell 

•Healthy gene is inserted into a virus, then infect patient DNA to produce protein

correct specific genetic defects

Zolgensma cures spinal muscular atrophy

Destruction, removal, or reduction of undesirable microbe

•Highest resistance

•Bacterial Endospores, Prions (Creutzfeld-Jakob, Bacillus, Clostridium)

•Mid-level resistance

•Protozoan cysts

•Fungal Zygospores

•Naked viruses (Polio, hepatitis A) no envelop

•Some bacteria

•Mycobacterium tuberculosis

•Staphylococcus aureus

•Pseudomonas

•Lowest resistance: Everything else

•E. coli

•Coronavirus 

•Bacterial Endospores, Prions (Creutzfeld-Jakob, Bacillus, Clostridium)

Growth of microorganisms in tissues

•infection

Techniques that prevent the entry of microbes into sterile tissues

•Sterile needles, Cleaning the skin before surgery

Chemical applied to the skin to inhibit vegetative pathogens

•Antibacterial soap

Living surface- ascepsis, antiseptic, degermation

Inanimate- sterilization, disinfection, sanitization

Microbial Death:  The permanent loss of reproductive capacity, even under optimal conditions for growth

•Not all cells die at the same time

•Microbial load- # of microb

•Nature of microbes in population (type of microb)

•Temperature and pH of environment (and how much it can be changed)

•Presence of solvents, organics, inhibitors

•Is sterilization needed (disinfection enough)? Clothing no need

• reused?-

•Can item to be sterilized resist heat, acid, chemicals?

•Is control method suitable for application (stains, bad taste)? bleach on clothes

•Cost, safety?

1.Cell Wall:  Bacterial and fungal cells

  Lysozyme, detergents, alcohol

2.Cell Membrane: 

Detergents and alcohols can cause a loss of selective permeability

3.Protein and nucleic acid synthesis

x-rays, formaldehyde 

  Inhibit replication or reading of DNA

4.Protein function

  Heat, pH, heavy metals 

  Denature enzyme or block active site

Water transfers heat better than air

.Intermittent sterilization (Tyndallization)

•Used rarely for biologicals that can’t stand high temps (seeds)

•Free flowing steam   1 hour

•Incubate at 25C   24 hours (allows spores to germinate)

•Free flowing steam  1 hour

•Cycle is repeated a total of three times

•Vegetative cell to die

•Disinfection, used routinely for water

•Exposing liquids to temperatures below boiling to kill some microorganisms while not altering the taste

•Flash pasteurization: 71.6C for 15 seconds

•Ultra high temperature pasteurization 134C for 2-5 seconds

•Incineration 800-6500°C Medical waste

•Bunsen burner  1870°C Laboratory

•Dry oven 180°C Waxes, oils, are not penetrated by water

Things that can withstand heat

•Cold (and desiccation)

•Removal of energy or water

•Both typically microbistatic

•Used to store microbes

Ionizing radiation:  Gamma rays, X-rays, Cathode rays.  Deep penetration.  Works by causing breaks in DNA  

Use- vegetable, fruit meat, 

Limitation- people don’t like radiation

•Ultraviolet light (nonionizing radiation).  Works by creating abnormal bonds (thymine dimers) in DNA

•Used for: bakery, surfaces, disinfect water

Limitation-•Very little penetrating power

•Can be used for gases or liquids (mask) 

heat sensitive liquid like serum, blood product, vaccine, beer

# and kind of microb

Materials that are being treated

Time of exposure required

Strength and mode of agent

 halogens

•Exists as a gas but usually used as a liquid for safety (hypochlorite, chloramines)

•Mode of action: Forms hypochlorous acid which denatures specific amino acid in proteins. Kills endospores if given enough time.

•Uses:  water, restaurant equipment, household cleaning

•Drawbacks:  Breaks down quickly in light

 halogens

Iodine:  

•Mode of action: Binds to proteins, nucleotides, and fatty acids.

•Uses: Iodophors (iodine plus soap or detergent) can be used to lessen irritation 

Weakness- irritating in high concentration and stains

•Toxic by-product of oil refining

•Mode of action: Destroys cell walls and denatures proteins

•Uses: Phenolic derivatives (phenol plus soap or detergent)

•Old-school Lysol and Amphyl.  Since discontinued due to toxicity

•Drawbacks:  Toxicity, cancer, 

•Chlorine plus phenol

•Mode of action: Destroys cell membranes and denatures proteins

•Uses: Used widely in hospitals (Hibiclens, Hibitane)

•Drawbacks:  Very few. Not sporicidal

•Only ethanol and isopropyl alcohol are safely antimicrobial

•Mode of action: Destroys cell membranes and denatures proteins.

•Drawbacks:  Not sporicidal

•Uses: Used widely in hospitals. Most effective at 70%

•Also removes oil and dirt from skin prior to injections

•Active ingredient in antibacterial soap and hand sanitizer

•H2O2

•Mode of action: Hydroxyl free radicals (-OH) destroy cell walls

•Uses: Home as antiseptic (3% H2O2 )

•High concentrations(36% H2O2) of vaporized H2O2 used to sterilize large/reusable objects in sterilization cabinets

•Drawbacks:  Very few. Also damages healthy cells so long-term use (chronic wounds) should be avoided

Glutaraldehyde, ortho-phthalaldehyde, formaldehyde

•Mode of action: Carbonyl group (C=O) binds to (alkylates) amino acids, damaging them in cellular proteins

•Glutaraldehyde and formaldehyde are sporicidal

•Uses:

•Sterilization of medical equipment

•Preservation of tissue (formalin, 37% formaldehyde)

Drawbacks:  Formaldehyde is toxic and irritating

•Ethylene oxide (ETO), propylene oxide (PPO), chlorine dioxide

•Mode of action: Alkylating agents. Bind to proteins and DNA

•Drawbacks:  ETO is explosive, carcinogenic

•Uses:   •ETO and PPO Sterilization of food, nuts, grains, plastic items

•Chlorine dioxide: Sterilization of large spaces, space probes

•Cationic detergents are most effective

•Quaternary ammonium compounds eg. benzalkonium chloride

•Mode of action: Disrupt cell membranes

•Uses:• remove organic matter before more rigorous treatment

• home cleaners

•Drawbacks: •Low level disinfection.•Easily inactivated•Requires alkaline conditions

•Mercury, Silver, Copper, Gold

•Mode of action: Bind to active sites, blocking enzyme action

•Uses: •Copper and silver added to (catheters, prostheses)

•Athletic clothing, braces

Drawbacks: •Toxic to humans•Allergenic•Neutralized by organic matter•Microbes develop resistance

•Mode of action: Denature enzymes

•Uses:

•Organic acids (citric acid, acetic acid) used to prevent mold growth and spore germination

•Drawbacks: 

•Most are too corrosive

•Bases have an unpleasant feel and flavor

•Mode of action: Cause DNA mutations

•Uses:

•select against Gram positive organisms

•e.g. EMB agar (Eosin methylene blue)

•Drawbacks: 

•Narrow spectrum

•Stain surfaces (Duh…)

administer drug to infected person that destroy infectious agent without harming host cell

Fungi

•Penicillium, Cepahalosporium

Bacteria

•Bacillus, Streptomyces, Micromonospora, Chromobacterium