9-2 Identify roles of a clone and vector in maing recombinant DNA Vector= self-replicating DNA used to carry the

e desired gene to a new cell -gene of interest inserted in vitro in vector -vector taken up by a cell where it can multiply -cell grown in culture to form many identical cells each of which carries copies of the vector also known as a clone -must be self-replicating -need to be a size smaller is better -preservation Clone= population of cells arising from one cell; each carries the new gene

9-3 Compare selection and mutation Selection = culture a naturally occurring microbe that produces the desired product Mutation = mutagens cause mutations that might result in a microbe with a desirable trait Site-directed mutagenesis= change a specific DNA code to change a protein Select and culture a microbe with the desired mutation 9-4 Define restriction enzymes and outline how they are used to make recombinant DNA Cut specific sequences of DNA, leaving sticky ends Destroy bacteriophage DNA in bacterial cells Cannot digest host DNA with methylated cytosines Bacterial DNA protected bacterial cell by hydrolyzing phage DNA P.248 9-5 List four properties of vectors Carry new DNA desired to cell Shuttle vectors = can exist in several different species Plasmids and viruses can be used as vectors Self replication Need to be a size smaller is better Preservation= circular form protect dna Viral DNA= accept larger pieces of foreign DNA 9-6 Describe the use of plasmid and viral vectors Plasmid -primarily variant of R factttor plasmids -insert into plasmid Viral vector -accept much larger pieces of foreign DNA than plasmids can -insert corrective genes

9-7 Outline steps in PCR and provide examples of its use 1 each strand of the target DNA will serve as a template for DNA synthesis 2 To this DNA is added a supply of the four nucleotides for assembly into new DNA) and the enzyme for catalyzing the synthesis, DNA polymerase. Short pieces of nucleic acid called primers are also added to help start reaction. The primers are complementary to the ends of the target DNA Will h p.249 9-9 Describe how a genomic library is made Pieces of entire genome stored in plasmids or phages Lyse cells/precipitate dna Dna mass= include organism entire genome Dna digested= fragments combined into phage vector/plasmid Goal to make collection of clones that ensure atleast one clone exist for every gene 9-11 Explain how each of the following is used to locate a clone; antibiotic resistance genes, DNA probes, gene products Antibiotic resistance genes -Bacteria pick up recombinant plasmid where new DNA was inserted into lazZ, this will not hydrolyze lactose and produce white colonies -bacteria receive original plasmid will hydrolyze lacZ to make blue compound (resistant to antiiibiotttiiic ampicillin). DNA probes -short segments of single stranded DNA that are complementary to the desired gene are made - finds match adhere to target gene -labeled with enzyme or fluorescent die (presence detected) -can be used to identify pathogens Gene products -Use E.coli to synthesize gene product -lac operon cloned and this promoter can be attached to clone genes -the amounts of cloned gene product directed by addition of inducer (lac operon) -produce gamma interferon 9-12 List one advantage of modifying each of the fffollllowing E.coli, Saccharomyces cerevisiae, mammalian cells, plant cells E. coli -used because it is easily grown and its genomics are known - not all eukaryotic proteins are processed and folded -need to eliminate endotoxin from products (disadvantage) Saccharomyces cerevisiae

-used because it is earily grown and its genomics are known -may express eukaryotic genes easily Plant cells and whole plants, mammalian cells -better expression of eukaryotic products -harder more expensive Mammalian cells - can be genetically modified to produce diff products -low risk of toxins or allergens -best suited for making protein Plants -sources of valuable products alkaloids -vaccines and antibodies -large scale low cost production using agriculture and low risk of product contamination p. 255 book, 13 of slides,

9-13 List several applications of DNA technology Making human insulin Make somatostatin= another human hormone Artificial blood Treat cancer/Treat viral infections with RNA interference Gene therapy Subunit vaccines Human enzymes and other proteins Gene silencing Shotgun sequencing 9-15 Discuss the value of genome projects Shotgun sequencing = small pieces of a genome are sequenced Metagenomics= study of genetic material taken directly from environmental samples Locate specific genes and functions 9-16 Define the following terms: random shotgun sequencing, bioinformatics, proteomics Random shotgun -blood (female)/sperm from male -only few samples were processed as DNA resources, and the source names are protected so neither donors nor scientist know whose samples were used. Bioinformatics -science of understanding the function of genes through computer-assisted analysis -DNA sequences stored in web Proteomcis -science of determining all of the proteins expressed in a cell

Microbial ecology (Ch 9,27 part 1) (p779 =ch 27) What is mutualism? -type of symbiosis in which both organism are benefited What is Agrobacterium and how does it cause disease in plants? -bacterium that has Ti plasmid naturally occurring in it -Ti plasmid causes formation of tumorlike growth called crown gall -T-DNA integrates into genome of infedted plant -T-DNA stimulates cellular growth and causes production of certain products that bacteria use for nutritional carbo and nitrogen -agrobacterium attracted to defensive chemicals made by wounded plant Whati s Rhizobium? How does it benefit plants? How do the bacteria benefit? -Rhizobium infect roots of leguminous plants (beans, peas, clover) -presence leads to formation of nodules -results in fixation of nitrogen for use by plant -get a home

Give examples of the intimate interactions between Rhizobia and their host plant -bacteria fix nitrogen into forms plant can use (NH4) -bacteria cant fix nitrogen in presence of oxygen -plants use nitrogen fixing nodules to provide home for bacteria -Rhizobia carry sym plasmid List 3 similarities and 3 differences between Agrobacteria and Rhizobia and their interactions with plants -Similarities -boith symbiotic -both grow on plant -both pathogen -Differences -Agrobacterium= parasitic bacteria - Rhizobia = mutualistic bacteria -Agrobacterium= cause brown gall (tumors) -Rhizobia= make nodules on host legume plants -Agrobacterium= carry TI plasmid -Rhizobia= carry sym plasmid (form nodules on plants) -Agrobacterium= attracted to defensive chemicals of wounded plant -Rhizobia= attracted to signaling compounds (flavonoids made by roots -Agrobacterium= produce cellulose to anchor plant

-rhizobia= invade root hair What are mycorrhizae? How do they help plants? How do the fungi benefit? -Fungi living in close associate with plant roots -extend surface area and length of roots -protects from disease and toxins Describe two major types of mycorrhizae. How common and important are these associations? -Endomycorrhizae -grow inside plants cells -most important type= arbuscular mycorrhizae -ectomycorrhizae -grow around and between plants cells -importance -endomycorrhiza= arbuscule decomposes releases nutrients for the plant -endomycorrhiza form vesicles (storage structures) -ectomycorrhiza = surrounds tree roots -ectomycorrhizae= degrade complex organic matter ( live as saprotrophs in soil) -ectomycorrhizae= most tree need them to survive (obligate) -ectomycorrhizae= form mycelial mantel Ch 27 part 2 27-5 Outline the carbon cycle, and explain the roles of microorganisms in this cycle -carbon fixation= photosynthesis (respiration=reverse) -Oxygen use up Nitrate and sulfate are good electron acceptors -most co2 produced is microbial -humans burn fossil fuels to contribute -plant roots contribute a lot, microbes, and sea -oxygen>nitrate>sulfate -methanogensis= inhibit by oxygen -nmethane oxidized with oxygen present/electron acceptor when oxygen not present 1. photosynthesis -photoautotrophs such as cyanobacteria, green plants, algae, great purple sulfur bacteria fix (incorporate) carbon dioxide and organ matter -chemmoheterotrophs= animals and protozoa eat autotrophs so carbon dioxide transferred -chemoheterotrophs (animals) use organic molecules -organic molecules released through respiration (start cycle over again) -carbon stay in organism until excrete or die -die= decomposed by bacteria and fungi= organic oxidized and co2 returned

27-6 Outline the nitrogen cycle and explain the roles of microorganisms in this cycle -nitrogen in atmosphere go through fixation, nitrification and dentrification. Nitrates assimilated into plants and animals after nitrification go through decomposition, ammonification, and then nitrification again. -Ammonification -deamination= amino groups of amino acids revomed converted to ammonia -release of ammonia= ammonification -brought about by bacteria and fungi -proteolytic enzymes decompose protein -amino acids put into microbial cells=ammonification occurs -ammonia ions used by bacteria and plants from amino acid synthesis -Nitrification -oxidation of nitrogen in the ammonium ion to produce nitrate -autotrophic bacteria (Nitrosomonas and Nitrobacter) -bacteria obtain energy oxidizing ammonia or nitrite - first stage nitrosomonas oxidize ammonium to nitrites -second stage Nitrobacter oxidize nitrites to nitrates -plants use nitrate as source of nitrogen for protein synthesis -ammonium ions bound to negatively charged clays -Dentrification -nitrogen fully oxidized -nitrogen can be used as electron acceptor absence of oxygen -nitrate becomes nitrogen gas Nitrogen fixation -convert nitrooogggen gas to ammonia -rely on enzyme nitrogenase -free living and symbiotic 27-7 Define ammonification, nitrification, dentrification and nitrogen fixation -Ammonification -release of ammonia from nitrogen-containing organic matter by the action of microorganisms -Nitrification -oxidation of nitrogen in ammonia to produce nitrate -dentrification -reduction of nitrogen in nitrate to nitrite or nitrogen gas -Nitrogen fixation -conversion of nitrogen into ammonia 27-9 Describe how the ecological community can exist without light energy

-thermal vents -hydrogen sulfide coming out of vents (energy source( -cold oxygen (electron acceptor) sea water - mutualist bacteria use energy (H2S) to fix carbon dioxide = chemoautotroph -bacterial embiosymbioant -primary producers are chemoautotrophic bacteria What types of microbes are involved in each step of the C and N cycles? Bacteria, Archae or Eukaryotes (fungi, algae)? Aerobic or anaerobic? Strict or facultative? Phototrophs or chemotrops? Autotrophs or heterotrophs? How are microbes involved in the production of greenhouse fases? -anaerobic -cyanobacteria carry nitrogenase enzymes in heterocysts that provide anaerobic conditions for fixation

Ch 12 Euk 12-1 List the defining characteristics of fungi Domain -Eukarya Nutritional Type -Chemoheterotroph Multicellularity -All, except yeasts Cellular Arrangement -Unicellular, filamentous, fleshy Food acquisition method -Absorptive Characteristic features Sexual and asexual spores 12-2 Differentiate asexual from sexual reproduction, and describe each of these processes in fungi 12-4 Identify two beneficial and two harmful effects of fungi Beneficial -expand root systems mychorrizal relation helps plants take up nutrient -carbon and nitrogen cycle break down organic compounds Harmful -coccidiomycocus flu like (get into lungs)

-can destroy food, crops 12-7 List the defining characteristics of algae Domain -Eukarya Nutritional type -photoautotroph Multicellularity -some Cellular arrangement -unicellular, colonial, filamentous, tissues Food acquisition method -diffusion Characteristic features -pigments 12-8 List the outstanding characteristics of diatoms and dinoflagellates (algae) Diatoms -silica cell walls -produce domoic acid -neurotoxin cause diarrhea accumulate in shell fish -asexually= get too small then they go to sexual reproduction with gametes Dinoflagellates -have two flagella -Endosymbionts in corals -zooxanthellae -cause red tides (negative) -neutrotoxins cause paralytic shellfish poisoning -Alexandrium 12-9 Identify one beneficial and one harmful effect of algae Beneficial -base of food chains -photosynthesizer half photosynthesis from ocean -kelp Harmful -look at characteristics of diatoms and dinoflagellates 12-10 List the defining characteristics of protozoa Domain -eukarya Nutritional type -chemoheterotroph

Multicellularity -none Cellular arrangement -unicellular Food acquisition -absorptive; ingestive Characteristic features -motility;some form cysts 12-12 Differentiate an intermediate host from a definitive host Intermediate host -axesual reproduction -sporo = asexual reproduction Definitive host -sexual reproduction Know at least three examples of disease-causing protozoa -giardi lambila (diahrrhea) -naegleria fowleri (eat brain) -plasmodium vivax -first is mosquito Second is humans (asexual reproduction) -definitive host/intermediate host (humans -cause malaria -taxoplasmosis -kitty 12-13 Compare and contrast cellular slime molds and plasmodial slime molds Cellular slime molds -eukaryotic cells -ingest fungi and bacteria by phagocytosis -conditions unfavorable large number of ameboid cells aggregate to form a single structure -individual amebae produce cyclic amp -some form stalk other form spore cap -under favorable conditions spores germinate to form single amebae -happens asexually Plasmodial slime molds -engulfs bacteria and debris -protoplasm with many nuclei= plasmodium -mass of protoplasm is plasmodium -lab=cytoplasmic streaming

-short supply= plasmodium separates into protoplasm form stalker sporangium= haploid spores develop -conditions= spores germinate fuse to form diploid cells -can happen sexually and asexually Test questions 1. Self-replicating DNA used to transmit a gene from one organism to another is a A. library B. clone C. vector D. Southern blot E. PCR 2) In the figure above the marker genes iused for selecting recombinant DNA are A.= Hind III, BamHI, EcoRI= restriction enzymes, cut DNA in specific, leavy sticky ends =insert gene of interest B = . lacZ/ampR= gene of interest= blue/white C = ori = origin of replication=replic ate plasmid 3) A source of heat stable DNA polymerase is A= agrobacterium tumefaciens= makes tumors B=Thermus aquaticus C=saccharomyces cerevisiae= brewers yeast, baking bread beer wine D= bacillus thuringiensis= produces toxin kills caterpillars E=pseudomonas 4. Which of the following microbes fixes atmospheric N2 for its host plant? A. Agrobacterium B. Arbuscular Mycorrhizae (endomycorrhizae) C. Ectomycorrhizae D. Rhizobium E. All of the above

5. Which of the following is NOT true of both Agrobacterium and Rhizobium? A. both are Alpha-Proteobacteria B. both form associations with plants C. both carry plasmids that are necessary for their interactions with plants D. both transform the hosts DNA (only agrobacterium does) E. all of the above are true 6. Mycorrhizae are associations between plants and A. mutualistic archaea B. mutualistic bacteria ( rhizobia) C. parasitic bacteria (Agrobacterium) D. mutualistic fungi E. parasitic fungi

7. Certain methanogens use hydrogen gas (H2) as their energy source and carbon dioxide (CO2) as their electron acceptor. Based on their metabolism they can be characterized as inorganic as energy source = autotrophs C02 electron acceptor= anaerobic Organic energy source (glucose, acetate) = heterotrophs A. aerobic chemoautotrophs B. anaerobic chemoautotrophs C. aerobic chemoheterotrophs D. anaerobic chemoheterotrophs E. anaerobic photoautotrophs Bio211, Practice Exam #2 Key

8. Denitrifying microbes start to use nitrate (NO3 -) as a terminal electron acceptor when oxygen becomes scarce. Therefore they are: A. obligate aerobes

B. microaerophiles C. facultative anaerobes D. aerotolerant anaerobes E. obligate anaerobes 9. Which of the following compounds is a greenhouse gas produced by microbial metabolism? A. carbon dioxide (CO2) B. methane (CH4) C. nitrous oxide (N2O) D. all of the above E. none of the above 10. The largest microbial population associated with the human body is in/on the A. genitals B. large intestine C. mouth D. skin E. stomach

Bio211, Practice Exam #2 Key 11. The two most abundant bacterial phyla in the human gut are A. Actinobacteria and Bacteroidetes (all oral cavity) B. Actinobacteria and Firmicutes C. Bacteroidetes and Firmicutes D. Bacteroidetes and Proteobacteria (oral cavity) E. Firmicutes and Proteobacteria (oral cavity) 12. Which of the following pairs of individuals will likely have the most similar bacterial communities in their intestines? A. mother and child B. father and child C. husband and wife D. all of the above pairs have highly similar communities E. impossible to tell from this information 13. Which of the following is NOT true about microbial communities living in the human or mouse gut? A. they contribute to digestion of complex polysaccharides B. they can influence weight gain or loss of their host C. they can protect the host from bacterial infection D. they can influence the metabolism of medicines taken by the host E. all of the above are true (none are false) 14. In the mouse model of autism, what are the effects of probiotic treatment with Bacteroides fragilis? A. decreased leakage of a key metabolite from the gut, improvement in behavioral symptoms B. increased leakage of a key metabolite from the gut, worsening of behavioral symptoms C. bacterial production of substance that improves the behavior of mice D. weight gain in mice E. weight loss in mice

Bio211, Practice Exam #2 Key

15. The above figure shows a principle component analysis of the microbial communities from feces of healthy individuals and in patients with inflammatory bowel disease (either ulcerative colitis or Crohns disease). What conclusion can be drawn from this graph? A. The microbial communities from the three groups are different B. The microbial communities from the three groups are not statistically different C. Differences in gut microbial communities cause inflammatory bowel disease D. Inflammatory bowel disease causes changes in gut microbial communities E. None of the conclusions above can be made from these data 16. In fungi, which of the following statements about sexual spores is true? A. they are produced by karyogamy followed by meiosis B. they are produced by conidiophores C. they are produced by fragmentation of hyphae D. all the spores produced by an individual are genetically identical E. all of the above are true 17. Which of the following is an example of a disease caused by a fungus? A. Coccidioidomycosis B. Giardia C. Malaria D. Toxoplasma E. Paralytic Shellfish Poisoning 18. Which of the following microbes causes red tides? A. Coccidioides B. Diatoms C. Dinoflagellates D. Giardia E. Toxoplasma

Bio211, Practice Exam #2 Key 19. In the life cycle of Plasmodium, the organism that causes malaria, A. Humans are the definitive host B. Mosquitos are the definitive host C. Mosquitos are the intermediate host D. the sexual stage occurs within human liver cells E. the sexual stage occurs within human blood cells 20. Which of the following statements is NOT true for both plasmodial and cellular slime molds? A. both are eukaryotic B. both are chemoheterotrophic C. both have single celled life stages D. in both, single cells can aggregate together to form a multicellular slug E. both produce spores