Lecture 3 Microorganism, Growth Curve Flashcards
What can microorganisms be used for in molecular biology?
- Express protein and study protein structure & function
- Study gene structure, gene expression, etc.
- Mutate genes and study function
- Create new genetically engineered microorganisms
- Used in forensic applications
- Human gene therapy
What is Esherichia coli (E.coli)?
- Gram (-) rod shaped bacterium
- Encodes at least 4000 genes
- The K12 (wildtype) strain has been maintained as a pure strain in laboratory stocks for the last 100 years
- Molecular biology strains are derived fro K12 via mutations
- Virulence factors (cause diseases) removed and built in mutations that prevent growth outside of the lab
- Very difficult to grow outside of the lab
- Can support the replication of DNA plasmids, which can encode antibiotic-resistance genes
- Restriction modification systems have been removed from E.coli K12 strain because they interfere with replication of foreign DNA in bacterial cells
- In wildtype, the bacteria will degrade foreign DNA as a defense mechanism
What are the advantages of using microorganisms?
- Biology/Genetic well studied
- Well-defined genetic system
- Mutation isolation relatively straight forward - Genome sequencing complete (exact genetic content)
- Easy to grow/rapid (ideal conditions t=20 mins)
- Dispersed single celled
- Grows in liquid and solid media
- Can grow in bulk
- Form discreet colonies (single individual colonies) - Easy to manipulate
- Can store frozen stock for decades
- Highly versatile DNA transformation system
- Can be used to store via transformation foreign DNA
- Can be used to express foreign gene product
What are recombinant DNA methods from E.coli?
Genetic variants of E.coli K12 have led to improved strains for recombinant DNA methods:
- Removed bacterial restriction modifications systems
- DNA recombination systems are modified to prevent rearrangement
- Endonuclease activity (cut DNA) has been mutated to increase plasmid yields
What is Saccharomyces cerevisiae?
- Ideal eukaryotic microorganism for research
- Approx. 8-10 um diameter spheroids/ellipsoid
- Greater genetic complexity than bacteria, containing 3.5x more DNA that E.coli
- Type of nonpathogenic yeast
- Strains have both a stable haploid and diploid state
- Many well characterized strains (temp-sensitive, nutrient sensitive)
- Normal laboratory haploid strains have a doubling time of 90 mins in YPD medium and 140 mins in synthetic media during growth phase at 30C (opt temp)
What are the differences between E.coli and yeast? What are their growth patterns?
E.coli (prokaryote)
Saccharomyces cerevisiae (eukaryote)
Yeast has more complex system than E.coli
Antibiotics only work for bacteria (E.coli)
Bacteria is usually reproduced by binary fission (cells enlarge and divide to yield two progeny of approx equal size)
S. cerevisiae grows by budding (asexual), daughter cell buds from mother cell
What are factors that affect growth?
- Chemical and physical nature of their surroundings (controlling these can opt growth rate)
- Genotype: different genus/species/strains can grow at diff rates
- E.coli t=20 mins at 37-39C opt temp and nutrients
- S.cerevisiae t=90 mins at 30C - Chemical factors in the medium
- Nutrients available (enriched vs minimal media) - Physical factors
- Temp: usually 37C for e.coli
- pH: role of buffer in media, resist pH changes
- Solutes/water availability: osmotic concentration of media
- Oxygen levels: e.coli and s.cerevisiae are facultative anaerobe but can switch respiration
What does growth affect?
Increase in cellular constituents
May result increased organism size, population or both
- Growth rate (doubling/generation time)
- time it take population to double, calculated during log phase - Biomass obtained (total cell # and size)
- Metabolic pathways (depending on the media)
What are the phases of growth?
Lag - no increase in cell number
Exponential/Growth - max rate of growth
Stationary - no net increase in cell number
Death - cell death exceeds division
What are methods of measuring growth?
Viable counts/plate count - measure cells that are alive, requires overnight incubation, during log phase, in solid media
Direct counts - total cell number (both viable and non-viable) microscopic count using Petroff Hauser or electronic counters
Turbidity - cell number/unit volume, estimate of cell concentration, more scatter = more cells, during log it is a good indicator of growth rate
What are the plots for exponential growth?
Arithmetic scale
Semilog scale - can be used to determine generation time
What is generation time and how is it calculated?
Time it takes for population to double
Find two points where the cell count is double and then calculate the difference of the time
How does turbidity and growth curves relate? Specificity?
Ex. log of turbidity vs time or log viable cells vs time
- The growth curve constructed is specific for the strain, media, and temperature
- Diff strains can be different sizes and size varies with physiological conditions (media)
- Size of cell refractive index of cell can affect turbidity readings
What is the streak plate method?
- Involve spreading a mixture of cells on an agar surface so that individual cells are well separated from each other
- Each cell can reproduce to form a separate colony (visible growth or cluster of microorganisms)
- A separate colony (colony forming unit) reflects a pure culture
How to calculate CFU?
Individual colonies of E.coli -> individuals cells give rise to a colony (CFU)
CFU = (# of colonies x dilution factor)/volume plated (ml)
TMTC = 300
TFTC = 20
