Lecture 30, 11/28; Immunology

Here is the audio for today's lecture.

• Be sure to have your lab number on your write up of AIDS week
• Lab practical next teus. About 80minutes.
o 8 lab 2
o 10 lab 3
o 12 lab 4
o 2pm+4pm either lab 1+5

• • IMMUNOLOGY
• o Back of page 2 from handout from 11/28 “Immunology”
• o B cells are associated with plasms cells. Plasma cells produce all those antibodies. T cells tend to be regulatory in nature. We know cd4+. That is the cell that HIV likes. T-helper helps the B cells. HIV destroys the communication between these two cells.
• o Side notes: candida albicans (budding)– thrush
• • Toxoplasmosis – protozoan
• • Valley fever - fungal
• • PCP – fungal
• • Herpes – virus
• o Vaccines
• • Cowpox vaccine is given instead of smallpox. They share antigens
• • Engerix-b – recombinant vaccine
• • Handout from 11/26 “Infection and Disease” - definitions
• o Toxoids - diphtheria, tetanus
• o vaccine – simple suspensions of antigens. When given to an individual the production of antibodies is stimulated
• o virulence – capacity or ability to cause disease. Highly virulent organisms – rabies, ebola. Low virulence organism – candida albicans (must be given an opportunity to cause disease)
• o zoonosis – diseases of animals that can be transmitted to man. Example – Lyme, Anthrax. Fungal zoonosis – ringworm, microsporum canis. Protozoan zoonosis – taxoplasmosis gondii. Prion zoonosis – mad cow disease. Viral zoonosis – rabies. Most of our emerging diseases are zoonosis.
• o Nosocomial infections (from the hospital) – if you see puss it is either a staph or a strep. Bacteria that account for 70% of nosocomial infections (these are the main 4): E. coli → UTI, Staph aureus, enterococcus faecalis. pseudomonas aeruginosa – blue green puss
• • STAGES OF INFECTIOUS DISEASE
• o Incubation period – time of exposure to time of symptoms
• o Prodromal stage – highly communicable, disease can spread easily at this stage.
• o Clinical stage – peak symptoms
• o Decline stage – first signs of recovery, could also become latent or hidden. Disease that does this → herpes. You are also are developing antibodies.
• o Convalescent stage – return to recovery
• • SUMMARY OF EVENTS IN THE TRANSMISSION OF DISEASE
• o Portals of exit
• • Feces
• • Urine
• • Discharges from mouth and nose
• • Saliva – rabies, mononucleosis
• • Blood
• o Portals of entry: skin, respiratory

Micro Lab 11/27 - Review Session with Eric Wells

I recorded the review session with Eric during lab 3. Here is the audio.

DONT TRUST MY SPELLING!!!

• • Selective – only grows certain types of organisms. Ex. EMB selects for gram – organisms and inhibits the growth of gram +.
• • Differential – what organisms do when they grow. Ex. EMB is differential based on lactose fermentation
• • Enriched media – don’t know exactly what is in it, really good for fastidious organisms (organisms that require a lot of ingredients)
• • Chemically defined – all components are known. Organisms that grow on these are the complete opposite of fastidious. Ex. M-9, E. coli grows on this
• • General purpose – use this if you want to grow an organism and don’t need anything special to do it
• • NA Plates – general purpose. We used to isolate organisms from our body and from dust.
• • TSA (triptic soy agar – has soybean digest and triptone which is a digestive kasin. Kasin is the protein that makes milk white)– general purpose. We used it to isolate organisms from our body and from dust.
• • Nutrient Agar – has beef extract and peptone for nutrients.
• • Motility test agar stab tube – differential, used to determine motility. Percent of agar used is 0.5%. The plates have 1.5% agar.
• • Sab-Dex Plates – selective because they only grow fungi. High sugar and low pH around 6 which is great for fungi growth.
• • Agar block plates – made with sab-dex. Used to identify different molds.
• • Blood agar – enriched media. Differential because you can look at hemolysis. Alpha hemolysis – partial clearing of red blood cells. Two organisms that did that are streptimoni and strep salivarius. Beta hemolysis – complete clearing. Two organisms that do this are streptococcus pyogenes and staph aerues.
• o How do you make blood agar/chocolate agar → start with a base (TSA,NA, Brain Heart infusion). Heat it and melt it. Cool it down to 35-40C before you add 5% sheep blood.
• o Chocalate Agar is the same process except you cool it to 80C when you add the blood. This lyses the blood cells and turns it a chocalatey color.

• • Chocalate Agar – enriched media. You can grow even more fastidious organisms on it. It is the same principle as cooking food → it helps the organism digest nutrients easier. You would grow strep pnemonea, any legionella, heamophilus influenza, neisseria gonohrrea.

• • EMB plates – selective and differential. Selective because of the dyes (eosin and methaline blue → inhibit gram (+) this means they select for gram (-)). They are differential because of lactose.
• o EMB – e. coli has metallic green sheen
• o Klebsiella – gooey fish eye
• o EA – looked normal
• o Proteus Mirabilis – negative
• o Staph Aureus – had trouble growing
• o Ent. Feacalis – had trouble growing
• o B. sub – had trouble growing but had endospores and survived.
• • MSA plates – Mannitol Salt Agar. Mannitol makes it differential. Salt makes it selective. It differentiates based on mannitol fermentation. Also has pH indicator → phenol red. In the presence of an acid it turns yellow. Same pH indicator used in TSI slants and Urea agar slants. When it is basic turns red. A positive reaction on MSA is a yellowing of the agar, negative is still pink.
• o It selects especially well for staphs. They can tolerate the high salt level. You can also grow entrococcus on it → positive but did not ferment as well as staph aeurues.
• • M-ENT plates – extremely selective, only grow enterococci. The ingredient that makes this so selective is sodium azide. Similar to cyanide. It also has a pH indicator TTC → doesn’t make it differential just makes the colonies turn a red color when the grow making it more visible.
• • BHI agar – enriched medium. Good for fastidious organisms. Can make blood/chocolate agar with it.
• • Starch agar plates (NA plate with starch in it) – differential. We used them to differentiate organisms ability to break down starch. B. sub could break down starch. E. coli could not break down starch. We are ultimately testing for the presence of amylase → this breaks down starch.
• • TSI slants – highly differential. Three sugars, has iron in it. Three sugars are lactose (10x), sucrose (10x), glucose (1x). Also has iron sulfate which helps with H2S production in some organisms. Has pH indicator Phenol red (yellow with acid, red with bases).
• • 3 things to look for sugar fermentation, gas bubble production, H2S production.
• • Only proteus do iron to H2S
• • Skim Milk Agar plate – differential. NA plate with skim milk to give them some kasin. Testing for presence of a protease or kasinase.
• o B sub – positive (has protease)
• o E. coli
• • Nutrient gelatin stab tubes – differential. Gelatin is a protein matrix – that’s what keeps it solid. If an organism has a protease it will break down that protein matrix. A positive reaction would be a liquid tube. Negative reaction – tube still solid. Used it looking at mostly gram (-)
• • Plate Count Agar – like NA, general purpose
• • Simmons Citrate Agar Slant Tubes – differential. Used for Gram (-) rods. PH indicator is Brohm Thymol. Starts out green – in a presence of a base it turns blue. We are testing for the ability of an organism to utilize citrate as a source of carbon. If they can they will grow. Also present are ammonium salts → if it grows it will use these salts (create ammonia). The ammonia then reacts with the Brohm Thymol and turn it from green to blue (blue is the positive reaction for this one). Used for gram (-)
• • Urea Agar Slant Tubes – used for gram (-). Differential. Testing fro presence of urease. Urea broken down to CO2 and ammonia using a urease. The pH indicator is phenol red. In presence of a base it goes pink. Positive is pink.
• • Mckonkey Agar Plate- just like EMB but with different ingredients. Used for gram (–) organisms. Unlike emb it selects for a more narrow range of organisms. Two of its ingredients make it selective – bio-salts and crystal violet (inhibit gram (+) org). The lactose makes it differential. It has a pH indicator – neutral red. When in the presence of acid it turns pink. Positive reaction is pink meaning lactose fermentation. Negative is clear/see through.
• • MH - Muelerr Hinten Agar – tests for antimicrobial sensitivity. Has beef extract. General purpose.
• • Enterotubes – differential. Results only meaningful if using gram (-) rods.
• • Liquid broth
• • Litmus milk – differential. grow tons of stuff. Looking for three things: lactose fermentation, curd formation (soft or hard), proteolisis (zone of clearing of kasin). Two ingredients = litmus and skim milk.
• • Tryptone broth – used to detect presence of tryptophanase. Tryptone digest of kasin is rich in tryptophan. If an organism makes tryptophanase it breaks it down and results in indole. Put kovacs reagent in and it reacts with indole. If it is positive it will produce a red ring on top (negative- no reaction). Used a lot for gram (-).
• • Nitrate broth – gram (-). It is differential. It is basically beef extract but rich in NO3. NO3→NO2→N2. Positive reaction is the gas bubble. Only one organism was able to make it through and do this → Pseudomonas Auriginosa
• • Rabbit plasma (not as important) – differential. Staph aerues is (+). This means it produces coagulase
• • 35min
• • Lactose broth – differential. Used for water microbiology. Positive reaction is an air bubble. The air bubble in lactose broth comes from lactose fermentation. When you ferment lactose you make acid and a gas byproduct. This test is used to test for gram (-) rods because they will do this.
• • MR-VP broth – what organisms do with glucose. Glucose could be fermented to organic acid which lowered the pH to 5ish. Methyl red is added after incubation - If glucose fermented red appeared in tube. OR . . . VP - glucose is converted into 2,3 butane diole. Barets reagent added to convert 2,3 butane diole into acetoine which would give tube a red color. Good test differentiating gram (-) rods.
• o e. coli → positive for methyl red. Turned glucose into various natural acids
• o e. a. → negative for methyl red
• o Using the OTHER method
• o E.a → positive. Made 2,3butane from glucose
• o E.c. → negative
• • M-9 – chemically defined. E. coli grows on it.

Things to know

• • Gram reaction, shape
• • Do they ferment lactose, do they make spores, do they make pigment
• Ex. Psuedomonas aureiginosa – makes extracellular pyocyanin pigment
• Serratia marcesins – makes intracellular prodigiosin
• ML – makes pigment
• Pseudomonas flourescens – makes pigment
• • Know if they are bacteria, yeast or mold, ect. And any notable reactions.

Lecture 29, 11/26; Infectious Diseases, Micro Terms

Audio for today's lecture is available here.

Quote of the day: “I haven’t slept with anybody. I’m as pure as the driven snow.”

RANDOM
Lab test a week from Monday the 26th
Know the nitrogen cycle for the final
Microbes in the News
5 bonus points for going to a presentation this week about AIDS. Give him single page of what we learned – due next Monday Dec. 3.

• LECTURE
• Quick overview of handout from 11/26 “A STUDY GUIDE FOR MICROBIAL PAHTOGENS”
• • Streptococcus pyogenes – strep throat. Sometimes called acute pharyngitis. Rheumatic heart disease or rheumatic fever. Gram positive cocci in chains. Beta hemalitic
• • Puerperal fever – Semmelweis and Holmes
• • Food Borne and Water Borne
• • Botulism is food borne
• • Shigella dysenteriae is water
• • Vibrio cholerae – water
• • Soil Borne and Arthropod borne
• • Clostridium tetani – spores
• • Borrelia burgdorferi – lyme, tics
• • STD’s
• • Chlamydia
• • Clostridium difficile – pseudo membranous colitis. Overdose of oral antibiotics. All good guys in gut die and this organism takes over
• • VIRAL
• • Rhinoviruses - cold
• • VZV – shingles
• • FUNGAL DISEASES
• • Pneumocystis carinii pneumonia PCP. → It’s a fungus.
• • Other handout from 11/27/07 “Infection and Disease”
• • Incubation period: time between exposure and first symptoms
• o staphylococcal food poisoning has a short incubation period – 1-7hrs
• o long incubation period – leprosy, AIDS
• o streptococcal sore throat – 2-5days
• • commonly reported diseases
• o chlamydia
• o gonorrhea
• o salmonellosis
• o AIDS
• o Pertussis
• o Varicella – chicken pox
• o Lyme
• o Giardiasis
• o Shigellosis
• o TB
• • Infectious diseases summary on page 5 on handout from 11/26 “Infection and Disease” Page labeled → “Summary of Events in the Transmission of Disease”
• • Definitions p 2 of handout 11/26 “Infection and Disease”
• o Bacteremia – bacteria in the blood. Flossing can give you this if you break capillaries with the floss → Strep mutans
• o Carrier – typhoid mary, patient 0 (history of AIDS)
• o Disease – (look up in glossary). Tell diseases apart by symptoms.
• o Endemic – low number but constant. Gonorrhea is an example.
• o Endotoxin – Gram (-) organisms have it. Part of the cell wall.
• o Enteropathogenic – causes problems in the intestine.
• o Enterotoxin – has to do with the gut, intestine → diarrhea
• o Epidemic – high number of cases. Example AIDS in Africa.
• o Etiology – a.k.a. cause. Salmonella typhi → typhoid fever
• o Exotoxin – toxin made inside the cell and then secreted
• • Clostridium tetani – locks your jaw
• • Clistridium botulinum – atoxin
• • Corinii bacterium diphtheria – unique cellular arrangement
• o Infection – something an organism causes. Localized infection (in one place) or systemic infections. Example of opportunistic → Candida albicans (doesn’t get started till something else happens) yeast infection.
• o Nosocomial disease – from the hospital
• o Pathogen – any organisms that can cause disease. Some are extremely virulent (doesn’t take many organisms to cause infection). Some are opportunistic and need many more organisms to cause and problems.
• o Toxemia – toxins in the blood
• o Toxoid – detoxified toxin. Example: used primarily in vaccines.
• • Dpt vaccines; diphtheria and tetanus are both toxoids. They are given to us so that we will form antibodies against those organisms in case we ever see them. (fyi: “p” is for purtussis)

Lecture 28, 11/16 (Maybruck 13); Phosphorus

Here is the audio for today's lecture. It is on ly 16:00 min. long. The rest of the time we watched a video about the pneumonia outbreak in 1918.

Ill post the notes sometime before Monday.

Lecture 27 11/14 (Dr. Maybruck 12); Biosphere

Audio for todays lecture is available here.

• SLIDE 1 handout 11/14
• • Biosphere (1 giant ecosystem) – region of earth that contains living organisms
• o Living organisms are supported by the interactions that are occurring between abiotic (non living matter) and biotic (living matter) processes.
• • Levels of the Biosphere (ultimately connected together)
• o Lithosphere – the land (and a few miles into the earth)
• o Hydrosphere – aquatic
• o Atmosphere – few miles into the air
• • Ecosystem: Communities of organisms that are interacting with one another and their physical environment
• • Community: populations interacting with one another and their environment
• • Population: group of organisms of all the same species
• • Habitats: location where the populations live
• • Niche: poplulations occurring in the community serve a specific role
• o Example: protozoans consuming bacteria
• SLIDE 2 Components that make an ecosystem work
• • Producers consumers decomposers → different trophic(feeding) levels.
• o Primary – autotrophic organisms. Convert CO2¬ into glucose.
• • Photoautotrphs – use the sun as an energy source to convert CO2 into glucose
• • Chemoautrophic – found in hydrothermal vents. Get nutrients other than the sun
• o Consumers and decomposers
• • Heterotrophs – convert glucose into CO2. Consumers are ultimately responsible for consuming primary producers.
• • Decomposers – consume primary and consumers. Consume them as dead organic matter.
• Example: bacteria, fungi, invertebrates, scavenger animals
• • Diagram on handout illustrating components of ecosystem
• o Energy is lost going from step to step
• o NOTE: energy is not cycled back into the system. Ultimately energy is lost through heat. What IS cycled is carbon and nutrients (elements).
• SLIDE 3
• • Example of energy lost
• o Start with 100 energy
• o Only 10 units of energy to the next level
• o Only 1 unit of energy goes on to the secondary consumers
• o Only .1 unit of energy goes to the final consumer
• • It is important that the primary consumers are sustained – therefore we are sustained
• SLIDE 4
• • Carbon – considered separate from nutrients but still critical
• • Essential Nutrients: nitrogen, phosphurus
• • Liebig’s Law of the minimum – an organisms growth is going to be restricted when the elements (carbon, nitrogen and phosphorus) are not at levels required for the organisms growth.
• • Biogeochemically cycle – ensures all trophic levels receive proper nutrients. The most important part of this cycle is biological processes.
• • Notes on Carbon Cycle
• o CO2 found in high concentrations in aquatic system and aquatic life
• o Photosynthetic organisms that occur on land and have access to this CO2
• o In aquatic life microorganism are the primary producers → algae, bacteria, protozoans
• o Cow consumes glucose of the grass and converts it to CO2
• o Autotrophic organisms break down glucose and produce CO2
• o Limestone erosion: limestone is created from the shells of animals condensing together and being buried. Shells form from the combination of calcium and CO2 → forms calcium carbonate. This makes up the shell of the organism.
• SLIDE 5
• • Nitrogen cycle [how do we get nitrogen to primary producers]
• o Plants prefer to have ammonium rather than nitrate. They would have to break nitrate to ammonium.
• o Ammonium and nitrate are inorganic sources of nitrogen.
• • Animals consume organic matter which is where the nitrogen is.
• • Primary producers get inorganic nitrogen through thee different methods
• o Precipitation – “butloads” of nitrate
• o Erosion of rock (which is weathered by precipitation)
• o Ammonification, nitrification, denitrification, and nitrogen fixation
• SLIDE 6 - ammonification
• o When the heterotrophic organisms is consuming nitrogen and it has excess it will release the extra ammonium into surroundings → ammonium pool
• o Precipitation, weathering and excretion also contribute to the ammonium pool
• Slide 7 nitrification
• o Overall purpose is to turn CO2 into glucose.
• o CO2+H20 → CHO+O2
• o Nitrosomonas (chemoautotrphic)
• • CO2+NH4→CHO+NO2 (nitrite)
• o Nitrobacter
• • CO2+NO2(nitrite) → CHO+NO3 (Nitrate)
• Slide 8 denitrification
• o Looking at heterotrophic bacteria (convert glucose to CO2)
• o These bacteria have an anaerobic metabolism.
• o Animal respiration
• • CHO+O2→CO2+H2O
• o Denitrification (anaerobic process)
• • CHO+NO3(nitrate) → CO2+N2 (nitrogen gas)
• Slide 9 Nitrogen fixation (another example of a anaerobic organism)
• o Nitrogen fixing organism get it from denitrification or from the atmosphere
• o CHO+N2 → CO2+NH4
• o Mutualistic – intimate association with benefits for each organism
• • Rhizobia infects roots of legumes resulting in root nodules. This creates anaerobic environment in root. When the nitrogen gas goes into the root it is converted to ammonium and then the plant can use it. The plant gets ammonium the organism gets nutrients.

Lecture 26, 11/12 (Maybruck 11); Viruses

Audio for lecture on 11/12 is available here.

• • Slide 1 handout from 11/9
• o Regulation of gene expression at the level of translation: Inhibit gene expression of a dominant protein. This is done by regulating translation. Bacteria do it using antisense RNA. Antisense RNA will complimentary bind its sequence to a specific mRNA sequence. This forms an RNA double stranded. RNA must be single stranded in order for it to translate. We use antisense DNA because it is easier to manufacture. Antisense DNA makes its way into the nucleus of the cell where it binds to a specific sequence and translation can no longer occur (double stranded → doesn’t work AND the double stranded nucleic acid doesn’t leave the nucleus. Translation usually occurs in the ER)
• o Another therapy is triplet DNA therapy regulation at transcription level: DNA strand that is complimentary to template strand. This is important because in transcription it only reads off the template strand DNA. This prevents DNA polymerase from attaching, binding and reading.
• • This process in its early stages – trouble getting DNA into nucleus
• • Can be used for cancer
• • Slide 1 11/12 VIRUSES
• o Ways to tell a virus is alive: growth, heredity, metabolism, protective mechanisms, transport of substances
• o A virus cant do this stuff independently → it can do it once it infects a cell. Virus should be considered as infectious particle.
• • Slide 2 Virus size
• o Virus size are less than .2 um → VERY small
• • Slide 3 virus structure
• o Virion – completed virus particle which has two parts
• • Outer covering – consists of a capsid and an envelope
•  Capsid (made with same protein subunit called capsomer) comes in two different forms: helical and Icosahedron.
• • Helical form - Capsids bind to one another and extend in a condensed helix. It manifests itself in a rod → called a naked virus.
• • Icosahedron – a polyhedron (3-d structure that contains sides that are polygons – a polygon has three straight sides and three angles). Icosahedron is made up of twenty polygons. This formation is called a facet. The facets are made up of 20 capsomers. As a result of this geometric formation there are 12 equally spaced corners. At each corner is 5 capsomers called penton center.
•  Envolope
• • Taken from the cell host. The envolope provides additional protection for the genetic material AND aids in release of genetic material into cell host.
• • Central core – consists of genetic material and enzymes
• o All viruses will have a capsid and genetic material meaning they also must have an outer covering and central core.
• • Slide 4 genetic material of viruses
• o Viruses contain either DNA or RNA (not both)
• o Viruses also use single stranded nucleic acids as their genetic code.
• o Because viruses are very small their genetic material must be concise. Hepatitis B has 4 genes (Human eukaryote has tens of thousands of genes). Those genes in a virus must code for synthesis of the viral capsid and genetic material, produce protein products that will regulate the actions of their cell host, they need to code for genes that will help package the virus (virion).
• • Slide 5 viral multiplication
• o 1st step – absorption
• o viruses attack specific cells. They determine it based on receptors of the cell host. This can help us characterize viruses into these categories:
• • restricted: example hep B → only infects liver cells
• • intermediate: example polio virus → infects intestinal cells and nerve cells
• • broad: example rabies virus → infects a bunch of eukaryotic cells
• • slide 6
• o it needs to release its genetic material into the cell host. This can be done by endocytosis (similar to how an amoeba eats food). Food vacuole is formed. Food vacuole is bombarded with enzymes and breaks down the capsid. As a result genetic material is released into the cell.
• o It can also transfer its DNA by merging. As it merges it will spread open and genetic material is released.
• • Slide 7 synthesis and assembly
• o Double stranded DNA virus can recombine into our genome and creates potential for cancer.
• o RNA polymerase transcribes viral DNA → it then moves to the ER where it is translated → proteins are then brought back in to the cytoplasm.
• • Slide 8 viral release
• o There will either be cell lyses or exocytosis.
• o Cell membrane is critical to life of cell

Lecture 25 11/9 (Maybruck 10)

I was absent for this lecture. If anyone has notes/audio that they would want to post - let me know via a "comment".

Thanks

Micro Extra Credit 3

LECTURE 10/31 is UP!

AND . . .

Here is a possible extra credit article "Fuel Without the Fossil" from the New York Times.

Lecture 24, 11/7 (Dr. Maybruck 9); Bacterial Genetics

Audio for today's lecture is available here.

• • Slide 1 (handout from 11/5) tools and techniques for biotechnology applications: Plate hybridization
• Plate hybridization - Helps identify specific DNA sequence associated with an organism
• You use plate hybridization in order to determine if an organism is present and is harmful by looking at the DNA.
• Take your food sample and streak a plate –-> incubate plate for a day → bacteria grow. From this growth we do gene analysis.
• Place nitrocellulose on top of the colonies (so that they touch) that have grown. Pull the membrane off and it contains smears of the colonies that had grown on the plate. Next you lyse the cells and denature the DNA. Denaturing the DNA causes it to separate into single strands. You then expose the membrane to a gene probe. The gene probe is a sequence of DNA that will complimentary bind to the gene of interest (the one that has the toxins). If there is no harmful gene then the gene probe will not bind. The gene probe has a radioactive label that emits energy. The photographic film picks up the radioactive label (if it is bound to the DNA in one of the colonies) and a mark appears on the photographic film. You can then take the photographic film, lay it over the original colony again, and determine which colonies have the genes of interest.
• • Slide 2 – PCR
• Polymerase chain reaction allows us to amplify specific sequences of DNA.
• Components of a test tube:
• First thing you need is your DNA fragment that you wish to amplify. Then you need DNA polymerase which is responsible for synthesizing the new complimentary strand of DNA (semiconservitive replication). DNA polymerase will not add complimentary bases to make a new strand of DNA without primers. When making a complimentary new strand of DNA you need nucleotides.
• The above components will be put into a thermocycler (instrument that cycles through temperatures).
• 3 steps occurring in one cycle of PCR (the more cycles the more DNA molecules)
• • denaturization – heat can be used to separate the DNA strands to separate at 94ºC
• • priming – temperature is cooled to 50-65ºC. this causes the primers to bind were they need to. This is important because DNA polymerase wont start adding new nucleotides until there is a primer there.
• • Extension – temp is increased to about 70º which causes DNA polymerase to come in and start making new strand of DNA.
• Thermos aquaticus – thermophile. Organism able to survive in extreme heat.
• • Taq polymerase – used often in PCR because its polymerase is used to being at hot temperatures.
• Video explanation titled “Polymerase Chain Reaction” – available on blackboard
• • Slide 3 DNA sequencing
• DNA sequencing: Gives us an idea of what we are dealing with
• Sanger dideoxy method of DNA sequencing
• • Dideoxy nucleotide is missing an oxygen from its 3 carbon sugar.
• In a test tube you have: gene, primers, DNA polymerase, 4 nucleotides (G,C,A,T), 1 modified nucleotide (this is the dideoxy nucleotid missing oxygen from its 3 carbon sugar)
• Once the modified nucleotide is added to the chain then nothing will add on afterwards.
• Video titled “SANGER SEQUENCING” available on blackboard
• • Slide 1 (handout 11/7)
• Recombinant DNA is taking the genetic material of one organism and incorporating it into the DNA of another
• This is a natural occurrence among bacteria. It is called transformation.
• We can use this to our benefit as we can make organisms make proteins, Make transgenic organisms and amplify DNA.

Lecture 23, 11/5 (Maybruck 8); Operons + Bacterial Genetics

You can get the audio for lecture on 11/5 here.

• • Review of last class - check out the animations on blackboard.
  
• • Slide 1 (handout from 10/26) Lactose operon: inducible operon
• Operons (structures found in bacteria (prokaryotes)) – grouping of genes that are adjacent to each other and are involved in coding for one particular phenotype
• Their transcription will be regulated together
• Lac operon (inducible operon) – the only way that transcription is going to occur is if lactose is present.
• Promoter region – where RNA polymerase is binding. Once it binds there it will start transcribing (3’-5’ direction). This is what operons are regulating – with the help of an operater region. This is found “downstream” of the promotor. (downstream indicates that it is in the direction that transcription occurs – 3’-5’ . . . upstream is the opposite.)
• If you want transcription to happen in lac operon you must get rid of repressor protein. The repressor protein has two binding sites one binds to the operating region. Once bound it prevents RNA polymerase from transcribing. The other binding site will bind to lactose. Lactose is the inducer of this operon. Lactose binds to repressor protein and causes it to change protein form. This protein can no longer bind. This allows rna polymerase to begin transcribing. The only time this operon will transcribe is when lactose is present.
• What is being transcribed in this operon?
• • Beta-galactosidase : this enzyme is responsible for breaking down lactose into galactose and glucose.
• • Slide 2 arg operon: repressible operon
• Arg operon – responsible for a group of genes that are responsible for building the amino acid argenine. RNA polymerase comes along and transcribes Arg operon. Argenine acts as a repressor. It associates with a repressor protein (which has two binding sites like the other one). One site is for argenine the other is for the operator region of that operon. When argenine is no longer available to bind to the repressor protein the repressor protein will move away from operater region and allows transcription to occur once more.
• • Slide 1 Application of bacterial genetics (handout11/5/07)
• Biotechnology – manipulating the biochemical processes of an organism to benefit humanity.
• • Example: insulin → there are bacteria that make our human insulin
• • It can also help us identify different bacteria
• • Slide 2 restriction endonucleases
• Restriction endonucleases - Internal cutting of DNA
• The restriction endonucleases are bacteria’s immune system. They try to stifle viruses attempt to inject their DNA into the bacteria.
• They always cut palindrome sequences.
• Sticky ends and blunt edges
• • Sticky ends created by restriction endonucleases. They occur when the DNA is split. These sticky ends go on and bind to other complimentary pairs.
• • Blunt ends are cut clean
• Sticky ends are used in a process called recombinant DNA. Recombinant DNA splices foreign DNA.
• Restriction fragment length polymorphism (RFLP)– a piece of DNA that has been cut by a restriction enzyme. We can use the RFLP to distinguish between different organisms.
• • Slide 3 analysis of DNA + getl electrophoresis
• Polymorphisms – the subtle differences that are occurring between the different nucleotide sequences of the organisms.
• EcorI cuts up certain base pair sequences consistently for different organisms. The cuts leave certain lengths which cause it to be identifiable.
• Gel electrophoresis helps identify cut pieces
• • Add the samples (cut pieces) to gel wells.
• • The gel is exposed to an electrical current which creates a negative charge at one end and a positive charge at the other end. The positive charge is always away from the wells. DNA has an overall negative charge to it. This means that it will want to move away from the negatively charged wells.
• • The fragments then spread out through the gel. Smaller fragments migrate faster. Large fragments move slower.
• • From there you can compare electrophoresis tests to compare and classify different organisms.

Lecture 21, 10/31 (Maybruck 6); Bacterial Genetics

Here is the audio.

• • Slide 1 of handout from 10/29 – bacterial genetics replication, transcription, and translation
• o The study of bacterial heredity discusses passing of traits to subsequent generations and evolution of genetic material (more on slide one of handout from 10/29)
• • Slide 2 levels of structure and terminology
• o Genome – total amount of genetic material in a cell
• • In bacteria and eukaryotes and viruses the genetic material is DNA. In a retrovirus it would be RNA (ex. HIV)
• o Chromosome – includes genes that are critical for the survival of the bacteria/cell.
• • We have diploid chromosome sets (two copies). 23 chromosomes 2 copies . . . 46 total
• • Haploid have only one copy.
• • Chromosome is supercoiled to save space. For it to be copied it will be unwound by a DNA gyrase.
• o Plasmids – another type of genetic material. Plasmids help but are not essential. They allow the bacteria to adapt to a certain situation
• o Gene – a specific sequence of nucleotides. Found within the chromosome they are a specific sequence of nucleotides that will code for a protein.
• o Genotype – genetic makeup of a gene. Genotype is a specific nucleotide sequence of that gene.
• o Phenotype - The protein that was produced by the gene produces a trait and that is called the phenotype
• • Slide 3 DNA
• o Basic unit of DNA is a nucleotide.
• o Nucleotide – includes nitrogenous base which defines the type of nucleotide we have, They have a phosphate group and a sugar group.
• o Hydroxyl group lacking on the ribose sugar of DNA. If you see a hydroxyl group you know you have an RNA.
• o Each separate strand of the DNA (nucleotide) is covalently bound – unequally shared electrons.
• o The phosphate group is covalently bound to an adjacent nucleotide at the #3 carbon.
• o sugar phosphate linkage occurs on outside of helix
• o you ultimately get the double helix
• • slide 4 DNA
• o purines: adenine and guanine
• o pyrmidines: thymine and cytosine
• o two colons two hydrogen bonds 3 colons three hydrogen bonds. A::T G:::C
• • slide 5+6 DNA replication in bacteria: a semiconservative process
• o semiconservative - formation of a new DNA molecule from old DNA strands
• o STEP 1
• • uncoiling the DNA using DNA gyrase
• • separating the DNA molecule into 2 strands – helped by the enzyme helicase (which goes to “A” “T” rich area which is the origin of replication) and single stranded binding proteins (without these the hydrogen bonds would reattach). The A::T rich sight is the origin of replication – it is easier for the helicase to break down the bonds here.
• • The area where the strands are being split is called the replication fork.
• • Slide 7 DNA replication in bacteria: a semiconservative process
• o In order for the synthesis of new nucleotides to be added to the old DNA strand to form a new DNA strand you need RNA primase and DNA polymerase III.
• o DNA polymerase III – before it can add new nucleotides it needs a primer (RNA primase). It works down the strand adding nucleotides according to what it reads on the old strand. It can only read DNA strand in 3’-5’ direction. SO in order for it to replicate 5’-3’ direction it waits for helicase to open up the two DNA strands wide enough so that an RNA primer can get in. The DNA polymerase III uses it to replicate in the opposite direction.
• o RNA primase – adds complimentary RNA nucleotides
• o Processing of the lagging strand creates okazaki fragments.
• o DNA polymerase I (repair polymerase) - removes all of RNA primers and replace it with the correct complementary nucleotides in the newly synthesized strand.
• • Slide 8
• o DNA ligase – connects okazaki fragments
• o Freesciencelectures.com video DNA replication process
  
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• • Slide 9 transcription and translation overview
• o Gene has a specific sequence of nucleotides that’s going to code for a protein. That DNA within the gene has nucleotides that can be grouped into 3’s (triplets). Each of those groups will code for a specific amino acid. We get a protein that rolls over the DNA and creates an exact copy of the gene in RNA (mRNA). RNA’s grouped together as three is known as a codon (in DNA it is a triplet) which codes for a specific amino acid which is what proteins are made of.
• o DNA → RNA = transcription
• o mRNA → amino acids = translation
• • Slide 10 RNA
• o Look at diagram on the handout.
• o RNA uses uracil (in pyrimidine group) instead of thymine
• • Slide 11 RNA (1 strand of covalently bound nucleotides)
• o mRNA – RNA that is involve in coding for a protein. Provides an exact copy for a gene.
• o tRNA – parts of tRNA that complimentary bind to one another making it look like it has two strands. This creates hairpin structures. On one of the hairpin structures there is a triplet nucleotide sequence. That nucleotide sequence will bind to a specific sequence of mRNA. At the other end of the tRNA a specific amino acid will be attached.
• • tRNA brings the specific amino acid of the mRNA to the codon. It knows because of the anticodon which complimentary binds to the codon.

Lecture 22, 11/2 (Maybruck 7); Translation + Transcription regulation

Audio for lecture on 11/2 can be heard here.

• • Slide 1 (handout 10/29/07)
• RNA are the work horse of the DNA, they are responsible for construction of proteins. The mRNA has the blueprint of the nucleotide sequence associated with the gene. (main difference between DNA and RNA is uracil (in RNA) instead of thymine which is found in DNA)
• tRNA (transfer RNA) – single stranded nucleotide sequence (characteristic of all RNA). It has specific areas that are complementary to each other where it folds on itself forming hairpin loops. On a hairpin loop there is a span RNA nucleotides (3) that are referred to as anticodon. This is the compliment to the codon.
• On a gene there are 3 consecutive bases that code for a specific amino acid. The gene is coding for a protein. That protein consists of a bunch of amino acids all linked together. With every three nucleotides in DNA will code for a protein. The three nucleotides link together is referred to as a triplet. In RNA this is called a codon. . . . You have a triplet that is coding for a specific amino acid within mRNA that grouping of three nucleotides is referred to as a codon.
• Anticodon also codes for a specific amino acid. The tRNA knows which amino acid to bring over due to complimentary binding between the codon and anticodon.
• The function of the tRNA is to bring the amino acid over to the mRNA which is within the ribosome.
• • Slide 2
• Ribosomal RNA – involved in being part of protein synthesis
• • Slide 3 bacterial genetics: Transcription
• RNA polymerase only reads one strand which is the 3’ to 5’ strand. The strand that it reads is referred to as the template strand.
• RNA polymerase begins transcribing when it sees a certain pair of sequences. RNA polymerase binds to promotor region (which is made up of a couple different nucleotide sequences) and begins transcribing.
• Continues to transcribe until it gets to the termination DNA sequence → end transcription.
• • slide 1 of bacterial genetics: details of translation and transcription regulation (handout from 11/2/07)
• • slide 2
• you want genes to be selectively expressed. If all genes were expressed at the same time there would be energy lost by the bacteria.
• You can regulate gene expression during replication, transcription and translation.
• mRNA codons specifying for amino acids
• genetic code – a way of translating the nucleotide sequence of a gene into the correct amino acids (amino acids make protein and in turn exhibit a trait).
• 20 amino acids and 64 codons
• • this means codon usage will be redundant in formation of a particular amino acids
• • slide 3 translation – protein synthesis
• translation is happening on a ribosome
• in prokaryotes it is referred to as a 70s ribosome. The “s” stands for Svedberg units. “S” units measures the sedimentation rate of a particle during centrifugation. The larger the particles the larger their “S” value.
• Characteristic of a ribosome – there are spots in it for tRNA (settles within the 50s) and mRNA (settles within the 30s)
• There is an “E” site an “A” site and a “P” site
• 3 stages in translation – initiation, elongation, termination.
• • slide 4 translation
• initiation (formation of a protein – the adding together of amino acids) – starts with one codon – methionine (always first amino acid that is translated).
• There are different tRNA’s with different amino acids that try to get into the slots. The only ones that get in are the ones with anticodons that will bind to the codons. For example an AUG looks to bind with UAC.
• CUG (codes for lucine) looking to bind with something that has an anticodon for lucine.
• Ribosome is mixture of protein and RNA
• • slide 5 translation
• elongation
• dipeptide strand - peptide strand of two amino acids bound together (the beginning of the building of the protein)
• the ribosome moves down the mRNA and moves in 5’ to 3’ direction.
• As it (ribosome) is moving in 5’ to 3’ direction (elongation) the dipeptide moves from both tRNA’s to just one tRNA. It is moving to the one located in the “A site” of the ribosome. The tRNA in the “A site” then moves to the “p site”. The one in the “a site” (bare of amino acids) moves to “e site”. Once in “e site” it is released into the surrounding. Now tRNA that was in “a site” is in the “p site” and has two amino acids associated with it (a dipeptide).
• this process made a polypeptide strand that has two amino acids in it.
• A site – where the new tRNA’s with amino acids are going to be added
• P site is where elongation is occurring
• E site is where tRNA is being discarded
• Protein synthesis: termination
• Stop codon stops translation
• • slide 6 regulation of protein synthesis at the level of transcription
• operon – grouping of genes right next to each other. Also are working towards expressing one particular phenotype
• Transcription regulation in the operon is handled by two general processes: induction or repression
• • inducible regulated operon – regulates transcription – transciption is off. This means that transcription must be induced to get going.
• • repressible regulated operon – (always on) to turn this off it needs a molecule to repress it.
• Examples
• • Catabolic (breaking something down) operons – are usually inducible. Example → glucose.
• • anabolic operons – associated with repressible regulated operons. molecule has to be present to turn transcription off.