Exam 2 Super Review Flashcards
i. Media: Sim tube (inoculate in a straight light)
ii. Reagent: None added
iii. Results
1. Tube is cloudy if mobile
2. Black means positive for H2S (the microbe is anaerobic)
Motility/H2S Test
a. Media: Starch agar (Tan)
b. Reagent Added: Iodine
c. Results for Positive Test: Clear halo
Starch Hydrolysis
- Starch is hydrolyzed in to maltose, glucose and dextrins by
Amylase
a. Media: Milk Agar (white)
b. Reagent Added: none
c. Results for Positive Test: Clear halo
Casein hydrolysis
- Fat, hydrolyzed into fatty acids and glycerol
lipase
a. Media: DNA agar (aqua green compared to Spirit)
b. Reagent Added: none
c. Results for Positive Test: Clear Halo
DNA hydrolysis
- Hydrolyzed nucleotides
DNAse
a. Media: Tryptophan Broth (in to test tube)
b. Reagent Added: Kovacs’ Solution (testing for presence of indole)
c. Results for Positive Test: Red/pink layer on top of the test tube
Tryptophan hydrolysis
- An amino acid, hydrolyzed into indole, pyruvic acid and ammonia by
tryptophanase
a. Media: Urea Broth
b. Reagent Added: None
c. Results for Positive Test: Turns from yellow to pink (response to high pH)
Urea hydrolysis
- Hydrolyzed into ammonia and carbon dioxide by
urease
a. Media: Bile Esculin Slant
b. Reagent Added: None
c. Results for Positive Test: Turns black
Esculin hydrollysis
- Hydrolyzed into glucose, esculatin and ferric citrate (dark brown salt) by
esculinase
- Media: Durham Fermentation Tube
- Reagent Added:
- Results: Yellow = acid; +/- for gas (see inverted tube)
Sugar Fermentation
- Media: MRVP Broth
- Reagent Added: 5 drops of Methyl Red
- Results: Red if pH 5
Mixed Acid Fermentation
- Media: Citrate Slant
- Reagent Added:
- Results: From green to intense blue
Citrate Utilization
a. Citrate used during the kreb’s cycle
b. Suggests that they engage in some form of cellular respiration if they can ingest citrate
2. Media: Citrate Slant
Citrate Utilization
- Media: Nutrient Agar Tube
- Reagent Added: Drop/o Oxidase on swab
- Results: from Red=> Blue/Black
Oxidase Test
a. Bacteria’s ability to has electron transport system
b. Suggests bacteria be engage in aerobic cellular respiration
Oxidase Test
- Media: nitrate broth
- Reagent Added: 5 drops of nitrate A+B
- Results: Red = presence of nitrite
Nitrate Reduction
a. Nitrate provides oxygen for cells that engage in anaerobic cellular respiration
b. Red means anaerobic cellular respiration, since nitrate nitrite in cellular respiration
Nitrate Reduction
- Media: MRVP
- Reagent Added: VPA 15 drops, VPB 5 drops to 1 mL broth “Barritt’s reagent” tests for acetoin, precursor to alcohol fermentation product
- Results: Red after 10 min
Alcohol Fermentation
- Media: Slant Agar
- Reagent Added: Catalase (Hydrogen Peroxide)
- Results: Bubbles or not
Catalase
a. Bubbling indicates oxygen production, confirms the presence of catalase
b. Catalase is needed to survive in the presence of oxygen
Catalase
- plasmid
- codes for beta-lactamase
- and beta-galactosidase
PGal
a. test if transformed, put on agar with ampicillin
b. will enter media, inactivate ampicillin, normal sensitive can grow
beta lactamase
a. put e coli on agar enrihched with sugars galactosides
b. exoenzyme will enter media, hydrolyze sugars, turn blue
beta galactosidase
xii. =the natural process by which some bacteria can increase their genetic variability
Transformation
- Being able to acquire naked DNA from the environment around
Competent
i. Put bacteria on the plate
ii. Incubate on ice for 10 minutes
iii. Remove and incubate in 42 degrees hot water bath for 90 seconds
iv. Return tubes to ice water bath for 1 minute
v. Add recovery broth to the tubes
vi. Incubate in 37 degree water bath for 30 minutes
vii. Inoculate controls.
viii. Make E. Coli competent try to transform competent cells from white, ampicillin sensitive bacterium to a blue ampicillin resistant bacterium.
ix. Mix E coli with plasmid pGal
Transformation
- Streptococcus pyogenes
- Staphylococcus aureus
- Clear halo forms
- All the RBC’s and hemoglobin have been destroyed
Beta hemolysis
Alcohol on Bacterial Growth
- Halo generated, not clear
- Billiverdin
a. Prouct of hemoglobin hydrolysis
Alpha Hemolysis
Do not generate a halo
Gamma hydrolysis
i. Chemically defined media
ii. We know every component and how much of that component is in the media
Synthetic
i. Infusions and extracts
ii. We use this in class a lot
iii. Secretions of ground up animal/plant stuff
iv. We don’t know the exact amount of each component beause we just kind of throw stuff in there.
v. Most commonly used media
Non Synthetic
a. Liquid medium containing beef extract and peptone
Nutrient Broth
a. Solid media containing beef extract, peptone, and agar
Nutrient Agar
i. Media that prevents the growth of one type of bacteria without inhibiting the growth of another type
Selective
i. Media Select for G-
ii. Inhibit G+
EMB and Hektoen
i. Selects for halophiles
SM 110
MSA Mannitol Salt agar
i. The way the organism grows on or its effect on a media helps tell the bacteria apart
Differential
i. Media Changes colors of bacteria
ChromAgar
a. EMB
b. Blood agar
c. ChromAgar
d. MSA
e. Columbia CNA
Examples of Differential Media
i. Additivevs are included to promote growth of fastidious bacteria
1. Ex:
a. TSA
b. Blood agar
Enriched media
good for propagating large numbers of organisms as well as for testing
Liquid media
shows surface growth patterns
i. Convenient for “pure culturing” organisms
Solid media
- Just the protein alone
- Has Apoenzyme core
- Cofactors
- Coenzymes
Simple Enzyme Structure
a. The protein portion
b. Contains the active site
Apoenzyme core
a. Non protein portions chemical compound that is bound tightly to an enzyme and is required for catalysis.
i. Metallic
1. Fe
2. Cu
3. Mg
Cofactors
a. Organic materials, non protein molecule that carries chemical grous between enzymes
b. vitamins
Coenzymes
i. They increase the rate of chemical reactions by lower the energy of activation.
Enzymes
ii. Only catalyze energy releasing reactions (net reaction must yield energy)
Enzymes
i. = a molecule with a shape complementary to the enzyme’s active site that, when it interacts with the enzyme, is changed by the enzyme
Substrate
ii. It binds to an active site.
Substrate
- Enzymes that do their job intracellularly (inside the cell)
Endoenzymes
- Enzymes that are secreted and do their jobs outside of the cell (extracellularly)
Exoenzymes
- Enzymes that are produced in response to the presence of a particular substrate
Induced Enzymes
- Only produced when the need is there; Turned on with changes in substrate concentration
Induced Enzymes
- Need is always there
2. Enzymes that are always produced
Constitutive Enzyme
- Enzymes that are not produced when the product of the enzyme pathway is present
Repressible
- Turned off in response to the substrate concentration
Repressible
vi. Their names are typically given based on their substrate and/or the reaction they complete
Enzymes
i. = Condensation Reactions
ii. When larger molecules are synthesized from building blocks by removing water
1. Ex: proteins from amino acids
Dehydration synthesis
i. When large molecules are split apart (digested) by adding water
Hydrolysis
i. When a molecule loses electrons/hydrogens (OIL RIG)
Oxidation
i. When a molecule gains electrons
1. Ex: during photosynthesis. CO2 is reduced to glucose C6H12O6
Reduction
i. When a phosphate is added to a molecule
1. Ex: Photosynthesis: When chlorophyll used in conversion of ADP –> ATP
Phosphorylation
i. Occurs when the final product, of an enzyme pathway, blocks further enzyme activity
ii. The product reacts with an allosteric site
iii. The shape of the enzyme’s active site is changed; and product production ceases.
Feedback Inhibition
i. An inhibitor molecule resembles the enzyme’s normal substrate and competes with the substrate for the active site.
Competitive Inhibition
i. Resemble and compete with PABA for a bacterial enzyme’s active site
ii. Is normally converted to the vitamin folic acid, by microbial enzymes.
Sulfa Drugs
adenine and guanine
Purines
Two rings
Cytosine and Thymine
Pyrimidines
One ring
i. 1 of the 4 nitrogenous bases
ii. Deoxyribose: a 5 carbon sugar
iii. A single phosphate group
Components of nucleotides
i. Sides of the DNA ladder consist of deoxyribose molecules alternating with phosphate molecules
ii. Rungs (steps) of the ladder are made by nitrogenous bonds
1. A binds to a T/U (D/R NA)
2. C binds to a G
Arrangement of nucleotides
three base sequence that codes for an amino acid or a control system
codon
a sequence of codons between a start and stop codon that codes for a protein/RNA
gene
a cluster of genes (primarily in prokarya) that operate as a unit, e.g., genes for enzymes of the same biosynthetic pathway.
operon
one DNA molecule
chromosome
the sum total of all the chromosomal DNA in the cell
genome
Composed of promoter site and operator site
Controlling site of operon
attachment for RNA Polymerase
promoter site
serves as attachment for repressor protein
operator site
a. Includes structural gene that codes for a repressor protein
Regulatory Operon
b. Includes a promoter site that serves as an attachment for RNA polymerase
Regulatory Operon for Repressor Protein
c. Has no operator site (no repressor protein for the repressor protein ha!)
Regulatory Operon for Repressor Protein
- The substrate binds to the repressor protein
- The repressor protein changes shape and has less affinity for operator site
- Now RNA polymerase can attach to promoter region; transcribe the structural genes into mRNA
Inducible enzymes
- If the substrate is absent, the repressor protein is in the wrong shape to bind/block the operator site
- Enzymes are produced
- Product is produced
- The produce forms a complex with repressor protein
- The product-repressor complex fits the operator site and blocks RNA polymerase
- – no more transcription.
Repressible enzymes
a. DNA supercoiling is relaxed by
a. DNA helix unwinds and unzips
b. Synthesis of complementary strands
DNA replication steps
i. Topoisomerase in eukarya
ii. DNA gyrase in prokarya
DNA supercoiling relaxed by
i. Starts at a single “origin of replication” in prokaryotes
1. Unzips in both directions
ii. (Multiple origins in Eukarya)
iii. Both catalyzed by helicase
dna helix unwinds and unzips
i. Each strand of DNA acts as a template for the arrangement of complement nucleotides
1. Remember A to T and C to G
complementary strands
builds RNA primer that’s necessary to start the complementary strand
RNA Polymerase
- Takes over after the primer is built
- Catalyzes the addition of DNA nucleotides to the growing DNA molecule
a. Archaea have a special DNA polymerase
i. Used in PCR
DNA Polymerase
- The fragments are ultimately attached to one another via an enzyme called a ligase
- DNA Pol can only add to a strand on its 3’ end, meaning it “writes” from 5’ to 3’
a. Has to then read a strand from 3’ to 5’
DNA replication is continuous on the 3’ strand and discontinuous on the 5’ strand
- Unzips the DNA helix
Helicase
- Synthesizes an RNA Primer
Primase/RNA Polymerase
- Adding bases to the new DNA chain
2. Proofreads the chain for mistakes
DNA Polymerase III
- Removes RNA primer
- Closes gaps
- Repairs mismatches
DNA Poly I
- Final binding of nicks in DNA during synthesis and repair
Ligase
- Supercoiling
Gyrase
i. Source: synthetic agents
ii. Effect: block DNA gyrase (the enzyme responsible for relaxing the DNA supercoiling prior to the unwinding of a DNA strand)
1. Prevents relaxation of the DNA
a. Naladixic acid and ciprofloxacin
Interfere with DNA Replication
i. Source: synthetic nucleotide mimic that has a base similar to guanine
ii. Target: DNA Polymerase
iii. Effect: Competitive inhibition of DNA synthesis
iv. Microbe affected:
1. Genital Herpes
2. Chickenpox
3. Shingles
v. Toxicity
1. Brain seizures
2. Confusion and skin rash
Interfere with DNA Replication
Acyclovir
i. = Azidothymidine (and related synthetic drugs)
ii. Source:
1. Synthetic thymine analogue
a. Mimics thymine nucleosides
iii. Target:
1. Reverse transcriptase
a. (has a lesser affinity for DNA Polymerase)
iv. Effect:
1. Competitively inhibits the transcription of HIV RNA into HIV DNA
v. Microbe Affected
1. Retroviruses (like HIV – the AIDS virus)
vi. Toxicity
1. Causes anemia and immunosuppression
a. Inhibits mitochondrial DNA replication
Interfere with DNA Replication
AZT
are antiviral drugs that are analogs of purines and pyrmidines (nucleoside mimics)
AZT and Acyclovir
how to make mRNA
transcription
i. DNA untwists and unzips to expose the codons
ii. RNA polymerase attaches to promoter site on one of the DNA strands
1. Specifically the template strand, which is the strand from which mRNA can be generated continuously
iii. Complementary RNA nucleotides are attached sequentially until a “stop” codon is reached
iv. There are no introns to remove in prokaryotes and finished mRNA may represent more than one protein
v. Completed m-RNA
Transcription
fold introns, remove them and splice remaining exons together.
Spliceosomes
Eukaryotes
a. mRNA Sent out of the nucleus into the cytoplasm where it will encounter 80s ribosomes
Eukaryotes
Completed mRNA
can be translating the same m-RNA at the same time
Polyribosomes
i. Happens as the the m-RNA is being
70s ribosomes find and attach to mRNA
Prokaryotes
v. There are no introns to remove in prokaryotes and finished mRNA may represent more than one protein
fact.
- A transfer RNA (tRNA) with an appropriate complementary anticodon and carrying an activated amino acid attaches at the start codon
Translation
- Begins when 30s ribosome subunit binds to the start codon (AUG) on the m-RNA
Initiation
Translation
- the transcript RNA
- Consists of a single chain (half a ladder) of nucleotides
a. Has the same bases as DNA except Thymine replaced by uracil
b. Uses the sugar ribose instead of deoxyribose
mRNA
- derived from a large precursor RNA molecule that is split into not only ribosomal RNA, but also transfer RNA
- Combines with protein to form the 50s and 30s subunits of ribosomes
rRNA
- made from a number of different precursor RNA molecules
- consists of about 80 nucleotides with one exposed codon
a. this is called the anti-codon - is the “decoder” molecule
a. transports ATP activated amino acids to the appropriate, complementary codon on the mRNA.
b. Responsible for the translation of the mRNA code into an actualy amino acid sequence
tRNA
- Can be doing transcription/translation at the same time
- Do not have introns and exons
- Finished mRNA may represent more than one protein
Prokaryotes
- Have removal of introns via spliceosomes
2. Transcription and translation are different times and location (nucleus vs. cytoplasm)
Eukaryotes
a. E
i. Linear chromosomes
b. B
i. 1 circular
c. A
i. 1 circular
DNA
a. E
i. histones
b. B
i. none
c. A
i. Histone like
Histones
i. Introns in m-RNA
ii. Operons rare
iii. Topoisomerase (relaxes the supercoiling)
iv. Replicates through mitosis
E
i. no introns in m-RNA
ii. Operons
iii. DNA gyrase relaxes the supercoiling
iv. Binary fission for replication
B
i. No introns in mRNA
ii. DNA gyrase for relaxing the supercoiling
iii. Has Unique DNA Polymerase that tolerates extreme temperatures
A
i. M-RNA processing in the nucleus
ii. Transcription is separate from translation
E
i. One class has only one type of RNA polymerase; the other has >1
ii. No m-RNA processing
iii. Transcription and Translation happen at the same time.
B+A
a method for rapidly generating identical strands of DNA
Polymerase Chain Reaction
b. Heating a DNA strand of 94 degrees C for 30 seconds,
i. causes the DNA to unwind and unzip
PCR
c. Cooling the DNA to 50 degrees C
i. will allow primers to attach to the two separate DNA strands
PCR
d. Raising the temperature to 72 degrees C for 2 minutes and providing DNA polymerase
i. promotes the synthesis of complementary strands
PCR
i. Provide the unique heat resistant DNA Polymerase used for this
Archaea
PCR
a. Involve environmental mutagens
i. Viruses
ii. UV radiation
1. Dimers are the most common mutations
iii. Compounds foreign to the cell
Induced mutation
a. Involve DNA Replication errors
Spontaneous Mutation
a. When, within a codon, one nucleotide is replaced by another with a different base
b. If the new codon codes for the same amino acid as the original codon, no change in he protein coded by the gene will occur
c. If the substitution results in a code for a different amino acid, the resultant protein may be defective
i. Hemoglobin in sickle cell anemia differs from normal hemoglobin by one aino acid
Point mutation
Base Substitution
a. Due to insertions or deletions of nucleotides
b. Can result in changing all the codons that follow the insertion or deletiono
c. Change the encoded protein COMPLETELY
Point mutation
Frameshift
- Found naturally in sunlight
- Wavelengths of 40nm-390nm
- Causes the redistribution of electrons and protons in thymines and cytosine, making them highly reactive
a. Bonding of adjacent thymines and/or cytosines creates dimers, which distort the DNA
b. Interferes with DNA replication and transcription
c. Most common mutation***
Non Selective Agents that Damage DNA
UV light
a. Create hyperactive ions which react with nucleotides causing the release of bases and the breakage of the DNA
b. Create free radicals
i. Highly reactive molecular fragments that have an unpaired electron; attack DNA
c. Have high penetration, but sterilization is often not possible because of negative effects on the media
d. Kill salmonella and E. coli in food products
e. can be used to sterilize plastic equipment and pharmaceutical products, as well as preventing food spoilage
Non Selective Agents that Damage DNA
Ionizing radiation
Gamma rays
a. Can create free radicals that attack DNA
b. Have considerable energy and penetration
Non Selective Agents that Damage DNA
Ionizing Radiation
x rays
- Increase the penetration power of water
2. Are either anionic (negatively charged) or cationic (positively charged)
Non Selective Agents that Damage DNA
Soaps and detergents
i. Usually bad
mutations
ii. Discovered by Barbara McClintock in 1948.
Other sources of genetic variation
transposons
i. Special DNA segment that have the capability of moving from one location in the genome to another – “jumping genes.”
ii. Discovered by Barbara McClintock in 1948.
iii. Causes the rearrangement of the genetic material
iv. Can move from one chromosome site to another, from a chromosome to a plasmid, or from a plasmid to a chromosome.
v. May be beneficial or harmful
vi. Thought atht 45% of our genome could be from these.
vii. A “jumping gene” that can “jump” between chromosomes or plasmids
viii. Enters the host genome via the enzyme transposase generating recombinant DNA (DNA generated from two or more sources)
Other sources of genetic variation
transposons
a. Where plasmids are transferred via a mating bridge involving sex pili in G- cells
i. attaches to a receptor on a target cell retracts, pulling the cells together
Other sources of genetic variation
conjugation
- Plasmids are…
a. G+ cells gain intimate contact and exchange DNA through a conjugation bridge
b. donor transfers a copy of plasmid through pilus
Other sources of genetic variation
conjugation
a. A copy of the chromosome can begin the passage to a second cell via mating/conjugation bridges
b. The passage is usually interrupted and the chromosomes breaks such that only a partial transfer occurs
c. Transferred chromosomal DNA can integrate itself into the new cell’s genome, creating recombinant DNA
Other sources of genetic variation
conjugation
involving chromosomes
- Conjugation can be accelerated when the cells are exposed to environmental stress
Other sources of genetic variation
conjugation
truth
- = the transfer of genes from one bacterium to another via a virus=bacteriophage
Other sources of genetic variation
transduction
a. Some viruses can infect several species and even genera
Other sources of genetic variation
a. Random fragments of disintegrating host DNA are picked up by the phage during assembly
i. Any gene can be transmitted this way
b. The bacterial genome breaks into fragments and some of the newly forming viruses pick up a bacterial DNA fragment instead of viral DNA
c. Bacterial DNA is then injected into another bacteria
Other sources of genetic variation
Generalized Transduction
a. A highly specific part of the host genome is regularly incorporated into this virus
b. Viral DNA is integrated into a bacterial genome at one specific site
i. =lysogenized
c. The virus excises itself along with the neighboring bacterial DNA
i. A restricted group of bacterial genes are transferred to new host cell, instead of a random fragment.
Other sources of genetic variation
Specialized transduction
b. Viral DNA is integrated into a bacterial genome at one specific site
Other sources of genetic variation
transduction
=lysogenized
- Chromosome fragments from a lysed cell are accepted by a recipient cell; the genetic code of the DNA fragment is acquired by the recipient
a. Donor and recipient cells can be unrelated
b. Useful tool in recombinant DNA technology
Other sources of genetic variation
Transformation
- Where Naked DNA is taken up by bacteria and recombines with the main bacterial genome
Other sources of genetic variation
Transformation
the term used when eukarya take up naked DNA
Other sources of genetic variation
Transfection
a. For bacteria, competency can occur for a short time during the log phase of growth
b. Is facilitated by a competence factor that can be released into the medium by the bacterial cells or displayed as a surface protein that binds to the DNA
Cells must be competent in order to take up naked DNA
i. Live strain w/ capsule killed rat
ii. Heat killed strain/w capsule rat survives
iii. Live strain, no capsule rat lives
iv. Live no capsule + dead capsule = dead rat
1. Both strains are found in dead rat LIVING with isolated
Griffith’s work
Transformation
Streptococcous Pneumoniae
- When genes from a cell are transferred to another existing cell
a. i.e. when genes from a bacterium are transferred to a neighboring bacterium
Other sources of genetic variation
Lateral Gene Transfer
is when genes are passed to two daughter cells that result from mitosis or binary fission
Other sources of genetic variation
Vertical Gene Transfer
- Conjugation, transduction, and transformation (transfection)
Other sources of genetic variation
all result in lateral gene transfer
- About 80% of microbe genes have been laterally transferred at some point in their history.
- Bacterial speciation is more driven by LGT than from mutations. Pretty cool!
- About 80% of microbe genes have been laterally transferred at some point in their history.
- Bacterial speciation is more driven by LGT than from mutations. Pretty cool!
i. =the intentional removal of genetic material from one organism and combining it with that of a different organism
ii. Bacteria picks up plasmid by trransformation
Recombinant DNA Technology
- Objective is CLONING, which requires that the desired donor gene be selected, excised by restriction endonucleases, and isolated
- The gene is inserted into a vector (plasmid, virus) that will insert the DNA into a cloning host
- Cloning host is usually bacterium or yeast that can replicate the gene and translate it into a protein product.
Recombinant DNA Technology
A gene source
i. A vector = what you use to transfer the gene to the bacteria
ii. A cell in which the recombinant can proliferate =clone
Genetic Engineering
requirements
- The gene must include a start codon, a stop codon, AND a controlling site
- Gene product must not be toxic to a host cell
Genetic Engineering
gene source
- DNA into which the desired gene is spliced so the gene can be inserted into a host cell
a. Since host cells won’t usually accept a foreign gene all by itself
Genetic Engineering
vector
- bacterial and yeast plasmids as well as viral DNA are often used
a. must be able to replicate
b. must have a gene that allows selection of the DNA that is recombinant
i. usually two genes for antibiotic resistance
Genetic Engineering
vector
= viruses, have the natural ability to inject their DNA into bacterial hosts through transduction
Genetic Engineering
= bacteriophages
iii. A cell in which the recombinant can proliferate
Genetic Engineering
=clone
i. Construction of the recombinant DNA
ii. Introduction of the recombinant into the host cell via a vector
iii. Selection of a host cell with a recombinant DNA
iv. Cloning and expression of the gene involves
Steps in Genetic Engineering
a. Bind DNA (DNA in the vector as well as well as DNA in the gene source) and uct it in one or more places at specific sites
i. Each species or strain of bacteria has a specific restriction endonuclease that normally breaks down foreign DNA
Steps in Genetic Engineering
i. Construction of the recombinant DNA
Restriction endonucleases
a. Cuts DNA so it has staggered ends (sticky ends)
b. is used to prepare DNA so a gene can be spliced in to generate recombinant DNA
Steps in Genetic Engineering
i. Construction of the recombinant DNA
Restriction endonuclease
is used as a vector to carry and incorporate genes into mammalian cells
Steps in Genetic Engineering
ii. Introduction of the recombinant into the host cell via a vector
viral genome
a. are the most common techniques for delivering “naked DNA” to new hosts
b. The host cells need to be made competent
c. Eukaryotic plant cells can be transfected (infected by the new recombinant DNA) by the gram negative bacteria Agrobacterium tumefaciens
Steps in Genetic Engineering
ii. Introduction of the recombinant into the host cell via a vector
Transformation of bacterial cells
Transfection of eukaryotic cells
- Are usually made competent via exposure to chloride salts solutions and temperature extremes or via electroporation
a. Cells are exposed to pulses of electrical fields that open small pores in cell membranes - Plasmids are used as vectors to transform bacteria
Steps in Genetic Engineering
ii. Introduction of the recombinant into the host cell via a vector
Truth.
- The vector, with the gene of interest, needs to include two genes for antibiotic resistance
Steps in Genetic Engineering
iii. Selection of a host cell with a recombinant DNA
- The host cells are plated on a media that contains the 2 antibiotics
Steps in Genetic Engineering
iii. Selection of a host cell with a recombinant DNA
- Cells with appropriate antibiotic resistance, that is, resistance to 1 of the antibiotics (and therefore has the gene of interest) can be identified
Steps in Genetic Engineering
iii. Selection of a host cell with a recombinant DNA
- Harvesting the resistant cells that contain the recombinant (gene of interest)
- Allowing the cells to multiply (gene amplification)
Steps in Genetic Engineering
iv. Cloning and expression of the gene involves
Significance of Genetic Engineering
- Factor VIII
a. For hemophilia - Growth hormone and insulin
- The gene for normal hemoglobin
- The gene for normal chloride ion channels
a. Cystic fibrosis - Interleukin II (Bubble Boy) for the treatment of SCID (Severe Combined Immunodeficiency)
i. Genetically engineer human proteins and cells to correct hereditary diseases
Significance of Genetic Engineering
- Alpha interferon for treatment of Hairy Cell Leukemia
- Vaccines
a. Hepatitis B (HBV)
i. The gene for a viral surface protein is inserted into a yeast plasmid
Genetically engineered drugs
Significance of Genetic Engineering
= use of an organism’s biochemical and metabolic pathways for industrial production
Biotechnology
Significance of Genetic Engineering
i. Gene for herbicide resistance
ii. Bacteria transformed
iii. Transfection of plant cells
iv. 99% of corn, soy, and cotton are genetically engineered.
eukaryotic plants can be transfected by the G- bacteria, Agrobacterium Tumefaciens, plant pathogens
Significance of Genetic Engineering
i. Correct or repair a faulty gene in humans
gene therapy
Significance of Genetic Engineering
a. Normal gene cloned in vectors, tissue removed from the patient
ex vivo
Significance of Genetic Engineering
a. Naked DNA or vector is directly introduced to the patients tissue
in vivo gene therapy
- Glycolysis
ii. Incomplete oxidation of glucose/carbs in the absence of oxygen
iii. Yields small amount of ATP - But less competition in areas w/o oxygen
iv. Formation of acid, gas, and other products by action f various bacteria on pyruvic acid
v. Glycolysis - Organic compounds are the final electron acceptors
vi. Products include - Swiss cheese with CO2
a. Makes the holes
Fermentation
i. Glycolysis
ii. Kreb’s cycle
iii. Respiratory chain
iv. Yields about 28ATP
Aerobic Respiration
i. Glycolysis
ii. Kreb’s cycle
iii. Respiratory chain
1. Molecular Oxygen is NOT the final electron acceptor
2. Has oxygen containing ions but not free oxygen
3. Nitrate (NO3) and nitrite (NO2-)
iv. Yields fewer ATP (~20)
Anaerobic Respiration
- O2-
- Superoxide
- Free radical
i. All organisms exposed to oxygen produce the toxic molecule
ii. Obligate aerobes plus facultative and microaerobes have enzyme which converts O2- into
hydrogen peroxide
a. Breaks O2- into hydrogen peroxide
Superoxide dismutase
a. Breaks hydrogen peroxide into H2O and O2
Catalase
i. Anaerobic and can be independent of the sun
ii. Can occur in extreme temperatures and pressures
iii. H2 oxidised to form sugar
iv. Sulfur compounds are oxidized to form sugar
v. Rocks are oxidized to form sugar
chemosynthesis
- Methane as byproduct
- Found in variety of ecological niches
a. Seqage slude
b. Marine and lake sediments
c. Geothermal springs
d. Deep sea hydrothermal vents
e. Animal intestines (cow farts)
f. Archaea photosynthesis
Methanogens
i. Archaea
1. Bacteriorhodopsin
ii. Only produces ATP
iii. No Calvin Benson Cycle – no sugar
iv. No ETC
Archaea photosynthesis
Light dependent reactions
Oxygenic photosynthesis
- Light
a. Cyclic Phosphorylation
i. ATP - +CO2
- Calvin Benson Cycle (light indp)
- Sugar
b. +H2O + NADP
i. Non Cyclic Phosphorylation - ATP/NADPH/O2
a. Calvin Benson Cycle Sugar/H2O/RuBP
Oxygenic photosynthesis
ight dependent reactions
i. Done by green and purple sulfur bacteria
ii. Uses the pigment bacteriochlorphyll
iii. Uses hydrogen sulfide or hydrogen gas for replacement electrons
iv. No O2 released
Anoxygenic photosynthesis
i. Breakdown of Glucose to pyruvic acid
ii. For one glucose,
1. 2 ATP IN, 4 ATP out = net 2ATP gain
2. 2 NADH (for electron transport chain)
3. 2 pyruvic acid
Glycolysis
i. Pyruvic acid Acetyl CoA before begins this
ii. Processes pyruvic acid and generates
iii. 3 CO2 molecules
iv. NADH and FADH2 generated
v. ATP
vi. Makes 6 NADH, 2FADH2 (per glucose; goes to ETC)
vii. Makes 2 ATP and 6CO2 (per glucose)
Kreb’s cycle
i. Final processing of electrons and hydrogen and the major generator of ATP
ii. Chain of redox carriers that receives electrons from reduced carriers (NADH and FADH2)
iii. Shuttles electrons down the chain
1. Energy released
2. Subsequently captured
iv. Used by ATP synthase complexes to produce ATP
1. Oxidative phosphorylation
v. Accepts electrons from NADH and FADH
vi. Generates energy through sequential redox reactions called “oxidative phosphorylation”
vii. It’s a total of 10 NADH and 2FADH2/glucose give electrons and hydrogens to the ETS
1. Electrons pass along a group of coenzymes
2. Hydrogens pumped to to exterior of the cell membrane, creating a gradient
viii. NADH 3 ATP
ix. 1 FADH2 2 ATP
Electron Transport System
+ Phosphorylation
- Algae and Plants
a. Chlorophyll a and b
Oxygenic photosynthesis pigment
- Cyanobacteria
a. Phycobiliprotein
b. Chlorophyll a and b Sometimes
photosynthesis pigment
bacteria
oxygenic photosynthesis
a. Bacteriochlorophyll
photosynthesis pigment
bacteria
anoxygenic photosynthesis
i. Archaea
1. Bacteriorhodopsin
ii. Only produces ATP
iii. No Calvin Benson Cycle – no sugar
iv. No ETC
photosynthesis pigment
Archaea photsynthesis
i. The ultimate source of all the chemical energy in cells comes from the sun
1. 6CO2 + 6 H2O –light glucose (C6H12O6)+6O2
Photosynthesis in general
- Electron released from pigment returns to its original photosystem (photosystem 1) via the ETS
- Only generates ATP
Cyclic photophosphorylation
- If electrons lost from PS1 are picked up by a molecule of NADP
- The electrons are released from chlorophyll in PSII
a. Used to replace those lost by chlorophyll in PS 1
b. Are delivered to PS1 via ETS that generates ATP - H2O provides final replacement electrons (and O2)
a. Photolysis of H2O with loss of flectrons to PSII
b. PSII powerful oxidizing agent, removes electrons from hydrogens in water
i. Electrons are used to replace electrons lost form chlorophyll in PS II
ii. ATPs generated in ETC
iii. H+ are used for the reduction of NADP- to NADPH
4. Generates ATP and NADPH to go to the Calvin Benson cycle
noncyclic photophosphorylation
i. Light independent
1. Uses ATP and NAPH to fix CO2 and convert it to glucose
Calvin Benson cycle
- Must obtain carbon in an organic form made by other living organisms
a. Proteins
b. Carbohydrates
c. Lipids
d. Nucleic acids
Carbon Source
Heterotroph
- =self feeder
- An organism that uses Co2, an inorganic gas, as it’s carbon source
- Not nutritionally dependent on other things
- Makes its own sugars
Carbon source
Autotroph
- Gains its energy from chemical compounds
a. Glycolysis and breaking down glucose
Energy source
chemotroph
- Gain energy through photosynthesis
Energy source
phototroph
- Don’t use light so have to each other things as a source of nutrients
- Have organic compounds as its carbon source
- Differ based on their final electron acceptor
Chemoheterotrophs
Chemoheterotrophs
i. All animals
ii. Most fungi
iii. Protozoa
iv. Bacteria
Chemoheterotrophs
O2 electron acceptor
Chemoheterotrophs
i. Separated by
ii. Inorganic compounds
1. Like Clostridium
iii. Organic compounds
1. Fermentation of Streptococcus for instance
Chemoheterotrophs
No O2 electron source
- The ones that do chemosynthesis
- Energy source chemicals
- Carbon source CO2
- Archaea and Bacteria
Chemoautotrophs
- Light as energy source
- Organic compounds for C source
a. Green and purple, nonsulfur bacteria
b. Archaea
Photoheterotrophs
- Light energy source
- Co2 carbon sourc
- Use H2O to reduce CO2?
Photoautotrophs
Photoautotrophs
i. Oxygenic photosynthesis
1. Plants/algae
2. cyanobacteria
Photoautotrophs
Use H2O to reduce CO2
Photoautotrophs
i. Anoxygenic photosynthesis
1. Green purple sulfur bacteria
Photoautotrophs
Do not use H2O to reduce CO2
- Lowest temperature that permits a microbe’s growth and metabolism
Minimum temperature
- Highest termperature that permits a microbe’s growth and metabolism
Maximum temperature
- Promotes the fastest rate of growth and metabolism
Optimum temperature
i. Optimum temperature
1. 15 degrees C
2. Capable of growth of 0 degrees C Refrigerator spoilage
psychrophile
i. Optimum temperature 20-40degrees
ii. Most human pathogens
mesophile
i. Optimum temperature greater than 45 degrees
ii. compost
Thermophile
i. A mesophile that can grow at 0 degrees
ii. Cause for disease and spoilage
Psychrotolerant
i. Greater than 80 degrees
ii. Includes Extremophiles (which are happy <0 degrees C)
Hyperthermophiles
80 degrees C
Extremophiles
i. Cannot grow without oxygen
ii. Can utilize oxygen and detoxify it
Obligate aerobe
i. Utilizes oxygen but can also grow in its absence
1. Staphylococcus
2. E. Coli
Facultative Anaerobe
i. Requires ony a small amount of oxygen
ii. Helicobacter species
Microaerobes
i. Lacks the enzymes to detoxify oxygen so cannot survive in an oxygen environment
Obligate Anaerobe
i. All organisms exposed to oxygen produce the toxic molecule
1. O2-
2. Superoxide
3. Free radical
truth
i. Oxygen level at which organism switches from aerobic to anaerobic metabolism
Pasteur Point
ell environment results in cellular dehydration and plasmolysis with protein precipitation
High solute concentration
a cell’s environment result in water entering the cell such that cells may lyse
Low solute concentration
- Require an environment with high concentration of organic molecule
- Sugar
osmophiles
- Do not require high concentration of solute but can tolerate it when it occurs
osmotolerant
- Require environment with high salt concentrations to stabilize their membranes
halophiles
- Parent cell enlarges, duplicates its chromosomes, and forms a central transverse septum dividing the cell into two daughter cells.
binary fission
a. = Time required to complete fission cycle (one parent two daughter cells
generation time for bacteria
i. “flat” period of adjustment
ii. Enlargement
iii. Little growth
1. Cells are increasing in cell mass
Lag phase
i. Period of maximum growth
ii. Continues as long as cells have adequate nutrients and a favorable environment
iii. Proportional to the rate of energy metabolism
iv. Very susceptible to antibiotics and other chemicals at this point.
Log phase
Exponential Growth phase
i. Rate of cell growth = rate of cell death
ii. Caused by depleted nutrients and O2,
iii. Caused by excretion of organic acids and pollutants
iv. Cells in survival mode, making defensive proteins
Stationary Phase
i. As limiting factors intensify, cells die exponentially
ii. Remain are those most resistant and endospores
Death Phase
i. Most simple
ii. Turbidity = degree of cloudiness
1. Reflects the relative population size
Turbidometry
i. Dilutions and plating allow plate to have accurate counting of plates
ii. Remember the dilutions and you can multiply that and see how many colonies were in the original sample
Plate count
- Reduce the number of pathogens off inanimate objects
reduces number of microbes
Disinfectant
- To reduce or inhibit microbes on living tissue
reduces number of microbes
Antiseptic
- Removes or kills all microbes
a. They are incapable of reproducing
Eliminates microbes
You sterilize PLUS remove all microbial toxins
Decontaminations
i. Inhibits bacteria
Bacteriostatic
Kills bacteria
Bacteriocide
Kills fungi
Fungicide
Kills microbes
Germicide
Destroys bacterial or fungal spores
Sporicide
i. Sterilizes
ii. Like what we do to inoculating loop and inoculating needle
Dry heat
oven and incineration
- Sterilizes if spores are not present
Boiling
- Sterilizes if spores are present
Tyndallization
- Pressure cooker
- Results in temperatures about boiling
- Good penetration
- The most practical and dependable
- Sterilizes, DOES NOT CONTAMINATE
Autoclave
- Does not sterilize
- Used to eliminate pathogens from food products
a. Usually beverages like milk - Controlled heat BELOW boiling (72 degrees for milk) for 15 seconds
- Media that are concentrated or contain fats and sweeteners require higher temperature
a. Skim milk vs. cream
Pasteurization
i. Shortest time required to kill all microbes in a suspension at a given temperature.
Thermal Death Time
i. The lowest temperature at which microbes are killed in 10 minutes
Thermal Death Point
i. Bacteria are added to tubes with different dilutions of a chemical agent and then incubated
ii. Identify agents that prevent growth at the great dilution
iii. Minimal inhibitory concentrations
1. Serial dilution of the agent plus
a. A suspension of the organism
2. = the tube with the lowest amount of agent that is without visible growth
Dilution tests
i. Filter paper method
ii. Look for a clear area around the paper disks soaked in a given agent.
1. This is where bacteria growth has been inhibited
iii. The media used in determining sensitivity should be comparable to tissue fluids of the body
iv. Staphylococcus Aureus is the usual test organism
v. Can’t tell if the organisms are dead or inhibited
vi. Various agents diffuse through agar at differet rates
Sensitivity Discs
i. Occasionally acquired during spontaneous mutation in critical chromosomal genes
ii. OR
iii. Acquisition of new genes or sets of genes via transfer from another species.
1. Originates from resistance factors (plasmids) encoded with drug resistance, transposons
iv. Survival when there was a high concentration of antibiotics
How resistance of genes are spread
a. ajor contributors to resistance
i. Hospitals (immunosuppressed, sick, old)
ii. Daycare centers (immature immune systems, high exposure)
iii. Antibiotic use in livestock
iv. Third world countries (no controls on use, war, and poverty)
v. First world countries
1. Given unnecessarily
2. Instructions not followed
3. Antibiotics in products
Where do humans most often encounter resistant microbes?
Immunocompromised
Kids
Health care workers
who’s at risK?
a. Produce enzymes that activate antibiotics
b. Prevent antibiotics from reaching the target
c. Alter antibiotic targets
d. Change the enzyme’s pathway
e. Note that resistance tends to increase with time
f. Humans pool antibiotics from trillions of bacteria
Major resistance strategies by microbes
ii. Actual results are the same when treating livestock with antibiotics such that we can acquire resistant bacteria from animal products. Body flora of livestock represent the 3rd major reservoir of genes for antibiotic resistance.
iii. The more effective a treatment is at eradicating a susceptible population fof bacteria, the more it will promote the development and spread of resistant bacterial populations.
Successful Antibiotic treatment
i. The sensitive bacterial pathogen and benign normal body flora are both killed by the antibiotic
1. The “shield” that normal body flora provide against new pathogens entering the body is weakened
2. Resistant normal body flora
a. Will flourish without competition and create super infections
i. E.g. Clostridium difficile
b. Surviving resistant normal body flora act as a reservoir for resistance genes that can be transferred to antibiotic sensitive pathogens
Successful antibiotic trreatment
i. Surviving resistant pathogens may replicate leading to a renewal of the disease
ii. The antibiotic fails
1. The disease is now harder to treat
2. Re-emergence of disease and an increase in untreatable infections
iii. Wrong antibiotic given or wrong dosage
iv. Instructions not followed
Unsuccessful antibiotic treatment
i. Hospitals (immunosuppressed, sick, old)
ii. Daycare centers (immature immune systems, high exposure)
iii. Antibiotic use in livestock
iv. Third world countries (no controls on use, war, and poverty)
v. First world countries
1. Given unnecessarily
2. Instructions not followed
3. Antibiotics in products
Major contributions to resistance
i. Increased health costs
ii. Re-emergence of disease
iii. Loss of work
iv. Higher death rates
v. Increased side effects
Consequences of antibiotic resistance
a. Use less antibiotics!
b. Practice prevention
c. Reduce exposure in hospitals and daycares
d. Survey diseases and resistance
e. Eliminate vectors that spread disease
f. Educate people (and doctors) on the proper use of antibiotics
Best strategy to reduce number of resistance bacterial populations
a. Because resistance is on a plasmid, it slows replication rate.
b. Would be a disadvantage in an environment without anti biotics
c. Allows the sensitive bacteria to outgrow and outcompete the resistant ones
d. Loss of plasmids becomes favored (plasmids not copied, unevenly distributed to daughter cells).
e. Resistance still maintained but on a lower level.
Explain why resistant bacteria are disadvantaged when antibiotic use is discontinued.
