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 DNA Log 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 line o 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 depleted Stationary 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 phase Calculations CFU mL Colonies per plate Dilution 10 1 Lab 9 Bactericidal Effects of High Temperatures Sterilization 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 denatured and bacteria will be killed Endospores need exposure to very high temperatures for a long time because they are so dense o Hot air oven two hour exposure a 170 degrees C to kill endospores also used for equipment such as glassware and pipettes o 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 gases Decimal 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 Operon Genotype 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 replication o Mutagens induced mutations damage DNA resulting in DNA sequence changes i e chemicals UV X ray Phenotype the expressed characteristics of an organism observable Operon 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 galactosidase permease and transacetylase gal is measured by the action of this enzyme on a chromogenic substrate X gal which serves as an alternate substrate for the enzyme gal cleaves X gal to produce a blue colored end product cells that do not produce gal colonies appear white 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 NaCl Lab 11 Transformation of E coli Transformation the heritable change in the properties of one bacterial strain by acquiring the DNA of another with plasmid DNA 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 screen o 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 s ability to use a particular carbon and energy source selection based on the ability of an organism to 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 gfp Lab 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 transcribe Glucose 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 repression o 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 is regulated by the catabolite activator protein CAP CAP is needed for RNA polymerase to bind to the promoter of the lac operon o The concentration of glucose affects the binding of CAP by regulating