BIO 326R 1st EditionFinal Study Guide Lectures: 1 – 35Tips: For Answering questions for designing experiments:- know how to make a plasmido use PCR and restriction enzymes along with necessary components of plasmid- know how and when to use a transposon versus targeted mutagenesiso transposons randomly insert themselves, so they can be used to find a gene of an unknown locationo targeted mutagenesis can be used when the gene and its location is known but you want to test its function or effect when mutated- always include controls- remember to use antibiotic resistance or reporters like GFP, lux, or beta gal to determine the presence or location of a gene- microarray is a method for using RNA sequencing to determine the function of genesMain focus of Exam 1: - Earth history- Characteristics of gram positive and gram negative- metabolism of the cell—respiration and fermentationMain focus of Exam 2:- replication, transcription, translation, cell divisiono differentiate between all of these processes know the goal of each and the required components involved- plasmidsMain focus of Exam 3:- gene transfero differentiate between the processes of gene transfer with plasmids, transposons, transduction- sequencing methodso use this methods to answer free response questions- quarum sensingMain focus of Exam4:- biofilms- endospores, siderophores, heterocyteso differentiate between these - archaea and methanogens- motility and stress responseo know different types of motlitiy and stress responses and know which cell components are used for each- antibiotics and virusesMain focus of Exam 5:- normal florao what parts of body are sterile- immunityo differentiate between innate and adaptive and the different processes and components included in each- vaccineso passive vs active and adjuvantsThings to know from each exam’s material:Exam 1Earth History- the 6 main events and their approximate dateHistory of Microbiology- discovery of microorganisms and the microcope- in 55 BC someone though that microbes made you sick- Leuwenhoek- Pasteuro Swan Necked Flasko Spontaneous generation- Redi Phylogenetic Tree- 3 Domains of Lifeo Bacteria, archae, eukaryotes (see Bb slide for more info)o Know that not all eukaryotes are multicellular (ex: fungi)- Bacteria Compositiono Know average size, volume, protein composition, shape, surface area and its effects, and doubling time- Bacteria Structureo Know composition of cytoplasm and cell membraneo Viscosityo Sterol-like molecules (hopenoids) Control fluidity (know relationship between temperature and fluidity)- osmotic pressure and how it is stabilizingGram positive- know examples!- Peptidoglycano Know components- Teichoic acids and lipoteichoic acidsGram negative- know examples!- outermembraneo Inner and outer leaflet of outermembrane- Outer leaflet of outermembrane—LPSo Know composition of Lipid A, core, and o-antigeno Know conformation for toxicity- periplasmo gel-like, diffusion is slower than cytoplasm, oxidizing environment, know protein types and volume make-upGrowth of Bacterial Cell- measuring growtho absorption, viable counts, direct counting, dry count know problems with each methodGrowth in a closed system- growth curves (know stages and what each means)- doubling time (defined and complex)o formulas and how to use them- closed system vs continuous culture- persister cells and VBNCEnvironmental Factors on Growth- temperatureo psychrophiles, mesophiles, thermophiles- osmotic pressureo H2O availability and turgor pressure- Atmosphereo Anaerobic, aerobic, facultative anaerobes, microanaerobesMetabolism- catabolic vs. anabolic- energy currency (ATP and NADH/NADPHCatabolic Reactions- energy yielding, break things down, oxidative, exergonic- know definition of reduction potential and standard Gibb’s free energyAnabolic Reactions- energy using, build things, reductive, endergonic- building blocks: amino acids to protein, sugars to PG, and nucleotides to DNA- coupled reactionsEnzymes- substrate to enzyme to products- ways to regulateo allosteric and competitive inhibitorsEnergy Sources in the cell- ATP and transmembrane ion gradients (pmf)Electron sources- oxidative reactions- H atoms carrying a proton and an electron are transferred to coenzymes that can store theH; store the electron—the reductive poise of the cello Know the difference between NAD+ , NADH, NAD(H) Used for energy generation and growth—where the electron is stored when things are broken down and energy is releasedo Know the difference between NADP+, NADPH, NADP(H)o NAD vs NADP Specificity and function- NAD to break things down and NADP to build things Know ratioHow do bacteria make ATP- pmf and substrate level phosphorylation (fermentation)Substrate level phosphorylation- convert ADP to ATP- know the 4 steps is follows- glycolysis (follows the 4 steps twice)o net 2ATP, 2NADP, 2 pyruvateso know the problem with glycolysis- fermentation (know how it works and the problem)o after glycolysis, NADH is dumped back on pyruvate to make lactate- respiration o ~30 ATP per glucoseo Pmf used to generate ATP along with substrate level phosphorylationFermentation- When is it usedRespiration- when it is used- terminal electron acceptorso oxygen, nitrate, sulfate know which is used by anaerobes and which by aerobes- anaerobic and aerobic growth both use fermentation AND respirationProton motive force- know uses: synthesize ATP, transport, motility, maintain pHo know how the gradient is used to do eacho know how respiration uses pmf to synthesize ATPo know how respiration and fermentation use pmf to transport molecules/do workExam 2DNA Replication History- know what Roslind Franklin and Linus Pauling did- DNA discovery experiments were done on bacteria in 1952Steps to Replication (bacteria)- find origin— know function of OriC and DnaA- unwind DNA—know function of helicase (DnaB, uses ATP) and DnaCo unwinging at OriC—A/T richo replication bubble—single strand binding protein- assemble replisome—DNA polymerase I (RNA primer removal) and DNA polymerase III (DNA replication, bidirectional)Initiation of Synthesis- know function of RNA primase and RNA primer- continuous vs. discontinuouso leading vs. laggingControlling Initiation- DnaA doesn’t bind to hemimethylated DNA well (old strand is methylated)o seqA= binds hemimethylated OriC and prevents DnaA binding- Cell size—understand how concentration of DnaA affects