Introduction to Biotechnology

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• Genetics pertains to genes, how they carry information, how information is expressed, and how genes are replicated.

• Chromosomes are DNA-containing structures that physically carry hereditary information with genes.

• Genes are DNA segments/nucleotide sequences encoding functional products, usually proteins.

• The genetic code dictates how nucleotide sequences translate into a protein's amino acid sequence.

• Genetic code involves the degeneracy, each amino acid is coded by multiple codons.

• Genotype refers to the genetic makeup of an organism.

• Phenotype is the expression of genes.

• Genomics studies of genomes at a molecular level.

• A genome is an individual cell's complete genetic information.

• Bacteria usually have a single circular chromosome made of double stranded DNA and associated proteins.

• DNA serves as the original genetic information in a bacterial cell.

• Short tandem repeats (STRs) are repeating sequences of noncoding DNA.

• Vertical gene transfer: flow of genetic information from one generation to the next.

• Horizontal gene transfer: flow of genetic information between cells of the same generation.

• DNA forms a double helix

• It is a polymer of nucleotides: adenine, thymine, cytosine, and guanine

• It's "Backbone" consists of deoxyribose-phosphate

• Two strands of nucleotides are held together by hydrogen bonds between A-T and C-G

• Strands are antiparallel

• The nitrogen-containing bases' order forms the organism's genetic instructions

• During DNA Repliation, one strand serves as a template for a second strand's production.

• Topoisomerase and gyrase unwind and then relax the DNA strands for replication.

• Helicase separates the DNA strands at the replication fork.

• DNA polymerase adds nucleotides only to the 3' end of the growing DNA strand.

• DNA polymerase adds nucleotides in the 5' → 3' direction

• The process is initiated by an RNA primer

• The leading strand is synthesized continuously

• The lagging strand is synthesized discontinuously, creating Okazaki fragments

• DNA polymerase removes RNA primers and then the DNA polymerase and DNA ligase joins Okazaki fragment strands

• DNA replication error rate: 1 out of 109 or 1 in 1010 bases is changed (mutation).

• DNA replication has its' own proofreading mechanisms.

• Most bacterial DNA replication is bidirectional

• Each offspring cell receives one DNA molecule copy

• Replication is highly accurate due to DNA polymerase's proofreading ability

• DNA Replication energy is supplied by nucleotides

• Hydrolysis of two phosphate groups of ATP supplies energy

• RNA is ribonucleic acid

• It is a single-stranded nucleotide

• Contains 5-carbon ribose sugar

• Contains uracil (U) instead of thymine (T) nucleotides

• Ribosomal RNA (rRNA): integral part of ribosomes

• Transfer RNA (tRNA): transports amino acids during protein synthesis

• Messenger RNA (mRNA): carries coded information from DNA to ribosomes to make proteins

• Prokaryotes synthesize a complementary mRNA strand from a DNA template

• Transcription starts when RNA polymerase binds to the promoter sequence on DNA

• Transcription proceeds in the 5' → 3' direction, one DNA strand is transcribed due to DNA polymerase's 3' start point only

• Transcription stops when it reaches the terminator sequence on DNA

• Steps of Transcription

• RNA polymerase binds to DNA sequence called promoter (TATA box)

• RNA polymerase makes RNA copy of gene (transcript).

• RNA synthesis continues until RNA polymerase reaches a terminator.

• New RNA molecule and RNA polymerase are released

• In eukaryotes only, transcription occurs in the nucleus, while translation occurs in the cytoplasm

• Exons are regions of DNA that code for proteins

• Introns are regions of DNA that do not code for proteins

• Translation occurs when mRNA is translated into the "language" of proteins.

• mRNA is the template created by transcription on which protein chains are assembled.

• Codons are groups of three mRNA nucleotides coding a particular amino acid

• 61 sense codons encode the 20 amino acids

• Antisense codons are stop codons and do not code for any amino acid.

• mRNA Translation begins at the start codon: AUG (methionine)

• Translation ends at nonsense codons: UAA, UAG, UGA

• mRNA codons are "read" sequentially

• Translation occurs on the ribosome (made of two subunits, large and small).

• tRNA molecules have an anticodon which recognizes codons, carrying specific amino acids to the growing protein chain.

• tRNA molecules transport required amino acids to the ribosome.

• Anticodon containing tRNA molecules base-pairs with the codon.

• Individual amino acids are then joined by peptide bonds

• Nucleic acid sequence within a gene determines the primary structure of proteins

• 2º, 3º and 4º structure then are determined by folding of the protein due to bond formation of side groups on amino acids.

• Ribosomal subunits and mRNA assemble.

• Start codon (AUG) binds to tRNA with methionine (formylmethionine for bacteria)

• Subsequent amino acids are added by translating one codon at a time during elongation.

• Ribosomes attach each amino acid to growing protein chain using peptide bond formation.

• When a stop codon is reached, translation stops, and ribosome-mRNA complex falls apart during Termination.

• In bacteria, translation can begin before transcription is complete

• Constitutive genes are expressed at a fixed rate

• Other genes are expressed only as needed; Inducible genes; Repressible genes; Catabolite repression

• Repression inhibits gene expression and decreases enzyme synthesis

• Repression is Mediated by repressors that are proteins that block transcription

• In Repression, the default position of a repressible gene is ON

• Induction turns ON gene expression using an inducer

• In Induction, the default position of an inducible gene is OFF

• Promoter: segment of DNA where RNA polymerase initiates transcription of structural genes (ex. TATA box)

• Operator: segment of DNA that controls transcription of structural genes (stop/go)

• Operon: set of operator and promoter sites and the structural genes they control

• Two operon types are Inducible and Repressible

• In an inducible operon, structural genes are not transcribed unless an inducer is present (lac operon)

• In the absence of lactose, the repressor binds to the operator, preventing transcription

• In the presence of lactose, lactose (inducer) binds to the repressor; the repressor cannot bind to the operator and transcription occurs

• In repressible operons, structural genes are transcribed until they are turned off

• Excess tryptophan is a corepressor binding and activating the repressor to bind to the operator, then stopping tryptophan synthesis

• Mutation: a permanent change in DNA's base sequence

• Mutations may be neutral (silent), beneficial, or harmful

• Mutagens: agents causing mutations

• Spontaneous mutations: occur without a mutagen (1 mutation per million replicated genes)

• Base substitution (point mutation): Most common mutation, changing one base in DNA; Silent, Missense, Nonsense, Frameshift, Base addition or deletion

• Missense mutation: Base substitution results in change in an amino acid

• Nonsense mutation: Base substitution results in a nonsense (stop) codon

• Frameshift mutation: Insertion or deletion of one or more nucleotide pairs; Shifts the translational "reading frame“

• Nitrous acid is a chemical mutagen that causes adenine to bind with cytosine instead of thymine

• Mutagens can increase mutation rate up to 1000x

• Nucleoside analog: incorporates into DNA in place of normal base; causes mistakes in base pairing (chemotherapeutic agents)

• Intercalating agents (aflatoxin, ethidium bromide) cause frameshift mutations

• Ionizing radiation (X rays and gamma rays) causes the formation of ions oxidizing nucleotides and breaking the deoxyribose-phosphate backbone

• Ionizing radiation causes deletion mutations

• UV radiation (non-ionizing radiation) damages DNA by creating thymine dimers (intra-strand bonding)

• Photolyases separate thymine dimers. Light repair enzymes use energy from visible light to fix UV light damage

• Nucleotide excision repair: Enzymes cut out incorrect bases and fill in with correct bases, repairs all mutations, cuts nonmethylated (defective) strand

• Methylase: enzyme adding methyl group to adenine of the sequence 5'-GATC-3' in newly synthesized DNA.

• Spontaneous mutation rate: 1 in 106 (1 in 1,000,000) replicated genes, 10-6

• Mutagens increase the mutation rate by a factor of 10–1000. This changes the rate to 10-3 -10-5

• Positive (direct) selection detects mutant cells as they grow or appear differently than unmutated cells

• Negative (indirect) selection detects mutant cells that cannot grow or perform a certain function

• Auxtotroph: mutant w/a nutrition requirement that is absent in the parent via replica plating identification

• An Ames test exposes mutant bacteria to mutagenic substances to measure mutation reversal rate

• Genetic recombination: exchange of genes between two DNA molecules that creates genetic diversity

• Crossing over: Two chromosomes breaking and rejoining, causing insertion of foreign DNA into the chromosome

• Vertical gene transfer: genes transfer from an organism to its offspring

• Horizontal (lateral) gene transfer: genes transfer between same-generation cells resulting in genetic recombination

• Mechanisms of horizontal gene transfer: Transformation; Conjugation; Transduction

• Transformation: genes transferred from one bacterium to another as "naked" DNA

• Conjugation: plasmids transfer from one bacterium to another that requires cell-to-cell contact via sex pili

• Donor cells contain the plasmid (F factor), termed F+ cells; Recipient cells (F-), then become F+

• In some cells the F factor integrates into the chromosome (Hfr cell) that then transfers R plasmids (R factors) through conjugation

• Transduction transfers DNA from a donor cell to a recipient via a bacteriophage (transducing phage)

• Generalized transduction: random bacterial DNA is packaged inside a phage and is transferred to a recipient cell

• Specialized transduction: specific bacterial genes are packaged inside a phage and transferred to a recipient cell

• Plasmids are self-replicating circular DNA pieces, 1-5% the size of a bacterial chromosome

• Plasmids often code for proteins enhancing a bacterium's pathogenicity, encoding antibiotic resistance (Resistance factors, R factors).

• Mutations and recombination create cell diversity raw materials for evolution

• Natural selection acts on populations of organisms, ensuring survival of organisms fit for a particular environment

• Some mutations can be beneficial, over time creates genetic diversity keeping populations healthy, though many mutations have no effect at all. </existing_notes>

Please update these study notes with any new information from the following text:

Terminology of Microbial Control

• Sepsis refers to bacterial contamination.
• Asepsis is the absence of significant contamination.
• Aseptic surgery techniques prevent the microbial contamination of wounds.
• Sterilization removes/destroys all microbial life.
• Disinfection destroys harmful microorganisms on objects.
• Antisepsis destroys harmful microorganisms from living tissue.
• Nosocomial refers to hospital-acquired infections.
• Degerming is the mechanical removal of microbes from a limited area like an injection site.
• Sanitization lowers microbial counts on eating utensils to safe levels.
• Biocides (germicides) are treatments that kill microbes.
• Bacteriostasis inhibits microbial growth without killing.
• Commercial sterilization kills Clostridium botulinum endospores from canned goods using heat.
• A sterilant is a sterilizing agent.

Rate of Microbial Death

• Effectiveness of treatment depends on the number of microbes present, environment (organic matter, temperature, biofilms), exposure time, and microbial characteristics, including endospores.
• Microbial death occurs at an exponential rate.
• Decimal reduction time (DRT): Minutes to kill 90% of a population at a given temperature.

Actions of Microbial Control Agents

• Agents can alter membrane permeability, leading to leakage.
• Agents damage proteins and enzymes by breaking bonds, causing denaturation.
• Damage to nucleic acids can be caused by heat, radiation, and chemicals.

Physical Methods of Microbial Control: Heat

• Heat denatures enzymes.
• Thermal death point (TDP) is the lowest temperature at which all cells in a liquid culture are killed in 10 minutes.
• Thermal death time (TDT) is the minimal time for all bacteria in a liquid culture to be killed at a particular temperature.

Moist Heat Sterilization

• Moist heat denatures proteins.
• Methods include boiling and free-flowing steam.
• Autoclaving uses heat and steam under pressure and is a common sterilization method.
• Autoclaving typically occurs at 121°C at 15 psi for 15 minutes.
• Autoclaving kills all organisms and endospores.
• Steam must contact the item's surface.
• Large containers require longer sterilization times when autoclaving.
• Autoclaving cannot sterilize heat-labile substances.
• Test strips are used to indicate sterility after autoclaving.

Heat

• Pasteurization reduces spoilage organisms and pathogens, but does not sterilize.
• High-temperature short-time (HTST) pasteurization uses 72°C for 15 seconds.
• Thermoduric organisms can survive pasteurization.

Dry Heat Sterilization

• Dry heat kills by oxidation.
• Methods include flaming, incineration (effective for prions), and hot-air sterilization.

Filtration

• Filtration involves the passage of a substance through a screen-like material.
• It is used for heat-sensitive materials.
• High-efficiency particulate air (HEPA) filters remove microbes larger than 0.3 µm, often used in rooms or hoods.
• Membrane filters remove microbes larger than 0.22 µm.
• Pore size for bacteria: 0.22 – 0.4 µm.
• Pore size for viruses: 0.01 µm.

Physical Methods of Microbial Control

• Low temperature has a bacteriostatic effect.
• Methods include refrigeration, deep-freezing, and lyophilization (freeze-drying).
• High pressure denatures proteins and alters CHO structure, killing vegetative bacteria.
• Desiccation, or the absence of water, prevents metabolism but organisms can remain viable.
• Osmotic pressure uses salts and sugars to create a hypertonic environment, causing plasmolysis.

Radiation

• Ionizing radiation (X rays, gamma rays, electron beams) ionizes water to create reactive hydroxyl radicals.
• Ionizing radiation damages DNA by causing lethal mutations and is commonly used using Cobalt-60 radioisotope.
• Salmonella and Pseudomonas are particularly sensitive to ionizing radiation.
• Ionizing radiation is used for sterilization of heat-sensitive materials like drugs, vitamins, herbs, suture material, and Petri plates.
• Some use ionizing radiation as "cold pasteurization" of food.
• Nonionizing radiation (UV, 260 nm) damages DNA by creating thymine dimers.
• Actively dividing organisms are more sensitive to nonionizing radiation.
• Nonionizing radiation use to limit air and surface contamination at close range on directly exposed microorganisms.
• Germicidal lamps employed in operating rooms or cafeterias are examples of using nonionizing radiation.
• Microwaves kill by heat, not primarily antimicrobial, through indirect killing via H2O energy absorption.

Principles of Effective Disinfection

• Key factors for effective disinfection include the concentration of disinfectant, organic matter present, pH, and contact time.
• Disinfectants are regulated by the EPA.
• Antiseptics are regulated by the FDA.

Evaluation of Disinfectant

• In dilution tests, metal cylinders/rings dipped in test bacteria are dried, placed in disinfectant for 10 minutes at 20°C, then transferred to culture media to check for survival.
• The disk-diffusion method evaluates chemical agents' efficacy using filter paper disks soaked in a chemical that is then placed on a culture.
• The disk-diffusion method identifies efficacy by looking for a zone of inhibition around disks.

Phenol and Phenolics Disinfectants

• Phenol and Phenolics injure lipids in plasma membranes, causing leakage.
• Bisphenols contain two phenol groups connected by a bridge.
• Examples of Bisphenols include hexachlorophene (pHisoHex) and triclosan.
• Phenol and Phenolics are active in presence of organic matter.

Biguanides Disinfectants

• Chlorhexidine is in surgical hand scrubs.
• Biguanides disrupt plasma membranes.
• They are effective against Gram-positive and Gram-negative bacteria (except pseudomonads), and some enveloped viruses.
• Biguanides are used on skin and mucous membranes.
• Alexidine is used to replace Betadine as a halogen disinfectant.

Alcohols Disinfectants

• Alcohols are effective against bacteria and fungi.
• They denature proteins and dissolve lipids.
• Alcohols have no effect on endospores and nonenveloped viruses.
• Ethanol and isopropanol require water to be effective, at a concentration of 60-80% solutions.
• They are used in hand sanitizers and cosmetics.

Heavy Metals and Their Compounds Disinfectants

• Oligodynamic action: Very small amounts exert antimicrobial activity.
• Heavy metals denature proteins by targeting -SH bonds.
• Examples include silver, mercury, copper, and zinc.
• Silver nitrate is used to prevent ophthalmia neonatorum.
• Mercuric chloride prevents mildew in paint.
• Copper sulfate is an algicide.
• Zinc chloride is found in mouthwash.

Surface-Active Agents Disinfectants

• Soap is a surface-acting agent works by degerming and emulsification.
• Acid-anionic sanitizers/detergents have anions react with the plasma membrane.
• Quaternary ammonium compounds (quats) are bactericidal, denature proteins, and disrupt the plasma membrane via cations.

Chemical Food Preservatives Disinfectants

• Sulfur dioxide prevents wine spoilage.
• Organic acids inhibit metabolism.
• Sorbic acid, benzoic acid, and calcium propionate prevent molds/bacteria in acidic foods and cosmetics.
• Nitrites and nitrates prevent endospore germination.
• They are used in cold cuts/hot dogs but can convert to nitrosamine (carcinogenic).

Halogens Disinfectants

• Halogens are effective against bacteria, endospores, fungi, and viruses.
• Tincture is a solution in aqueous alcohol (wound antiseptic).
• Iodophor is combined with organic molecules (Betadine).
• Iodine impairs protein synthesis and alters membranes.
• Oxidizing agents shut down cellular enzyme systems.
• Bleach contains hypochlorous acid (HOCl).
• Chlorine is widely used in pool and drinking water.
• Chloramine contains chlorine and ammonia.

Antibiotics Disinfectants

• Nisin and natamycin prevent spoilage of cheese.
• Bacteriocins are proteins produced by one bacterium that inhibits other bacteria.
• They are tasteless, non-toxic, and easily digested.

Aldehydes Disinfectants

• Aldehydes inactivate proteins by cross-linking with functional groups (-NH2, -OH, -COOH, -SH).
• Used for preserving specimens and in medical equipment.
• Formaldehyde (virus inactivation for vaccines) and ortho-phthalaldehyde
• Glutaraldehyde is one of the few liquid chemical sterilizing agents, killing S. aureus in 5 min; M. tuberculosis in 10 min.
• Cidex is an example, for hospital equipment is bactericidal, tuberculocidal, and virucidal.

Chemical Sterilization Disinfectants

• Gaseous sterilants cause alkylation, replacing hydrogen atoms of a chemical group with a free chemical radical.
• Sterilants cross-link nucleic acids and proteins.
• They inhibit cellular function.
• Used for heat-sensitive material.
• Ethylene oxide is an example of Chemical Sterilization.

Plasma Disinfectant

• Fourth state of matter: Consisting of electrically excited gas.
• Uses free radicals to destroy microbes.
• For tubular instruments.

Supercritical Fluids

• CO2 with gaseous and liquid properties (supercritical state achieved by compression).
• Used for medical implants (bone, tendons, or ligaments taken from donor patients).

Peroxygens and Other Forms of Oxygen Disinfectants

• Oxidizing agents.
• Used for contaminated surfaces and food packaging, specifically inanimate objects.
• Examples include O3, H2O2, and peracetic acid.
• H2O2 is used as a 3% solution or higher.
• Especially effective against anaerobic bacteria.
• Effervescent action may be useful for wound cleansing through removal of tissue debris but is not as effective as an antiseptic for open wounds.

Microbial Characteristics and Microbial Control

• Glutaraldehyde has fair effectiveness on Endospores and good effectiveness against Mycobacteria.
• Chlorines have fair effectiveness on both Endospores and Mycobacteria.
• Alcohols have poor effectiveness on Endospores and good effectiveness against Mycobacteria.
• Iodine has poor effectiveness on Endospores and good effectiveness against Mycobacteria.
• Phenolics have poor effectiveness on Endospores and good effectiveness against Mycobacteria.
• Chlorhexidine has no effectiveness on Endospores and only fair effectiveness against Mycobacteria.
• Bisphenols effectiveness on Endospores and Mycobacteria is none.
• Quats effectiveness on Endospores and Mycobacteria is none.
• Silver effectiveness on Endospores and Mycobacteria is none. </existing_notes>