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BSCI223 Lab Final Study Guide-Lab 8- Bacterial Growth*Growth of bacteria does not reflect the increase in size of an individual bacterium, but an increase in the total number of bacteria within a population.Lag Phase:- The number of cells in the bacterial population does not increase- Individual bacteria are preparing for division by synthesizing enzymes and DNALog Phase:- Cell division occurs through the process of binary fission (gives two equal daughter cells)- Number of bacteria in the population increases logarithmically/exponentially- Graph: Log of Cells/mL vs. Time will give a straight lineo The slope will represent the rate at which the population doubles (Population Doubling Time)- Towards the end of the log phase, toxic products can accumulate and essential nutrients are depletedStationary Phase: - Cell concentration ceases to increase; there is no net increase in cell number- Cells may continue some cell functions including energy metabolism and biosynthesis- Secondary metabolites (including antibiotics) may be synthesizesd- With time, cells cease to replicate and die…Death Phase:- Number of cells dying > Number of cells dividing- Number of viable cells within the population decrease exponentially- Death rate is generally slower than the rate of the log phaseCalculations:CFU/mL= (Colonies per plate/Dilution)/(10-1)Lab 9- Bactericidal Effects of High TemperaturesSterilization- the process used to kill all bacteria in a defined area - Exposure to high temperatures is a bactericidal treatment- At temperatures above the maximal growth temperature, cellular enzymes and proteins will be denaturedand bacteria will be killed- Endospores need exposure to very high temperatures for a long time because they are so denseo Hot air oven- two hour exposure a 170 degrees C to kill endospores; also used for equipment such as glassware and pipetteso Autoclave- steam under pressure rises to a temperature of 121 degrees C; moist heat penetrates biological tissues, endospores killed in 15-20 minutes; bacterial media, rubber, and other material sensitive to dry heat are commonly sterilized in the autoclave- Other sterilization techniques: filtration, UV radiation, and exposure to lethal gasesDecimal Reduction Time (D): used to reflect the effectiveness of heat killing at a particular temperature- The value of D is defined as the time required for one tenfold reduction in the population (determined from a survival plot)Lab 10- Phenotype and Genotype: The Lac OperonGenotype- genetic make-up of an organism- Change in genotype=change in phenotype (usually due to carbon source availability)- When a change occurs, it is stable; inherited by daughter cells-Mutations:o Spontaneous mutations- occurs as a result of mistakes in DNA replicationo Mutagens (induced mutations)- damage DNA resulting in DNA sequence changes (i.e. chemicals, UV, X-ray)Phenotype- the expressed characteristics of an organism; observableOperon- a set of genes that are regulated through the same promoter- The lac operon codes for proteins that act in the same metabolic pathway, which allows for the metabolism of lactose- Grouping genes whose products are needed by the cell at the same time, allows the cell to coordinate regulation of the expression of those genes (i.e. if lactose is unavailable, efficient to shut off pathway)- When lactose is available, the cell can induce the transcription of the genes and “turn on” the metabolic pathway- Genes that make up the lac operon are those that code for β-galactosidase, permease, and transacetylase- β-gal is measured by the action of this enzyme on a chromogenic substrate, X-gal, which serves as an alternate substrate for the enzyme- β-gal cleaves X-gal to produce a blue-colored end product (cells that do not produce β-gal, colonies appear white)- β-gal production reflects the presence or absence of lactose in the media- To control the carbon source usage of E.coli, assays for utilization of lactose are performed in defined media with a minimal number of constituents (minimal media)o The medium used in lab (“M9”) contains lactose or glucose and salts such as phosphate/NaClLab 11- Transformation of E.coli with plasmid DNA Transformation- the heritable change in the properties of one bacterial strain by acquiring the DNA of another- Allows for gene transfer and genotypic variation- Only competent cells can take up naked DNA from the environment- When the transferred gene(s) is expressed in the recipient bacterium, the phenotype of the recipient is also altered- In lab, researchers use two tools to detect changes in an organism’s genotype: selection and screeno Selection- preventing a subpopulation of cells from growing by exposing them to conditions that forbid their growth (i.e. selection based on antibiotic resistance, selection based on an organism’sability to use a particular carbon and energy source, selection based on the ability of an organismto make essential growth factors such as amino acids, nucleic acids or vitamins) Resistant organisms grow in presence of antibiotics Lac+ media allows for the growth of only Lac+ cells Organisms that can synthesis all 20 amino acids will grow on minimal media (prototrophs)o Screening- allows organisms of different genotypes to survive, but observation of phenotype is regarded as evidence of different phenotypes Changes in size, shape, or structure of colony MacConkey’s Agar: cells that can use lactose are pink, those that cannot are white/clear- In lab, we selected for transformants using media containing antibiotics. Then, screened the organisms for the ability to use lactose, or if they glow when exposed to UV light. o X-gal was used as an indicator of lacZ expression and UV lights were used to screen for expression of gfpLab 12- Enzyme Induction*there is an additional regulatory pathway that affects the induction of the lac operon and utilization of lactose*Promoter, Operator, lacZ, lacY, lacA*Repressor binds to operator, lactose binds to repressor- removes it and RNA polymerase can transcribeGlucose Effect- Glucose is the favored carbon source for E. coli, it will always be used before another carbon source (lactose)- The mechanism of control is called catabolite repressiono Diauxic growth- glucose will be used first, but when it is depleted the growth will cease (lag). Once the pathways using the second carbon source are induced, growth will begin.- Repression of the utilization of lactose

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UMD BSCI 223 - Lab Final Study Guide

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