Escherichia coli (E. coli) Bacterium

Escherichia coli (E. coli)

• Gram-negative, rod-shaped, and facultative anaerobic bacterium
• Discovered in 1885 by Theodor Escherich
• Can be a major foodborne pathogen causing severe disease in humans
• Healthy cattle carry E. coli in their intestinal tracts
• Can be found in undercooked bovine food products
• Runoff from farm fields that fertilize with cow manure may contain it
• Most strains are harmless and colonize the GI tract
• Certain strains can become pathogenic through plasmids and phage
• Pathogenic strains causes abdominal cramping, diarrhea, vomiting, fatigue, and fever
• Thoroughly cooking burgers can prevent infection

E. coli in Research

• Easy and inexpensive to grow and culture in the lab
• A widely studied prokaryotic model organism, important for microbiology and biotechnology
• Used as a host organism for most work with recombinant DNA
• K12 strains are well-adapted to lab conditions
• K12 strains lost their ability to thrive in the intestine
• K12 - BL21 strain is an expression strain
• K12 - JM109 strain is a cloning strain

Recombinant DNA

• DNA molecules created in the lab by bringing together genetic material from multiple sources
• Creates sequences not typically found in the genome
• Can be created from two different species
• DNA is the same molecule between all organisms
• Nucleotide sequence is what differentiates organisms
• Restriction enzymes cut DNA at specific sequences, acting as scissors
• Ligase enzymes seal cut DNA together, acting as glue

Cloning

• Molecular cloning involves the assembly of recombinant DNA and replication with host organisms
• Replicates one DNA molecule to produce a population of cells with identical DNA molecules
• Plasmids are extra-chromosomal DNA in bacteria
• Plasmids are small circular DNA molecules
• Gene cloning involves cutting DNA and ligating it into a new DNA sequence within a plasmid
• Then inject the recombinant Plasmid DNA into the host organism (Bacteria)
• Grow Bacteria = Increase in Bacteria = Increase in Plasmids

E. coli & Biotechnology

• Cloning is commonly performed in E. coli
• Transformation is the process of inserting the cloning vector (recombinant plasmid) into a host (E. coli)
• Two methods to achieve transformation
• Heat Shock: Cold conditions followed by a heat pulse with divalent cations (CaCl2)
• Makes the cell temporarily permeable to the plasmid DNA
• Electroporation: Briefly shock the cells in an electric field
• Creates temporary holes in the cell membrane
• Once inserted, E. coli is grown in incubators at 37ºC
• The plasmid replicates every time the E. coli replicates (20 min doubling)
• Once enough DNA is produced, it's extracted and purified

Purification of DNA from E. coli

• The key steps in DNA extraction for the semester include:
• Preparing growth media for the E. coli
• Sterilizing media in an autoclave
• Pouring nutrient agar plates
• Inoculating plates with E. coli and growing them in a 37ºC incubator
• Preparing TE buffer and harvesting E. coli
• Collecting cells by centrifugation
• Lysing (breaking open) E. coli
• Removing proteins by chloroform extraction
• Precipitating and collecting purified DNA
• Quantifying the amount of DNA by spectrophotometry
• Running DNA on an agarose gel

Liquid Nutrient Media

• LB (Lysogeny Broth) Media is standard for E. coli growth
• Components of LB Media
• Tryptone: source of amino acids
• Yeast extract: amino acids, vitamins, nucleotides, ions, glucose
• LB Broth is a liquid media that supports bacterial growth in suspension

Solid Nutrient Media

• Agar is a gelatinous substance from red seaweed
• Agar won't dissolve in water unless heated to 100ºC
• Agar dissolves and remains a liquid until cooled to 40ºC
• Once solidified, agar remains a solid until heated to 100ºC
• LB Agar is solid media, containing LB Broth + Agar
• Supports bacterial growth on a solid surface

Autoclave

• Bacteria are everywhere, but the goal is to grow only E. coli
• Some microorganisms can survive high temperatures
• Sterilization of media is achieved through the autoclave
• uses steam under pressure to kill harmful bacteria
  • 121ºC at 15 psi for 20 minutes
• Used in the lab to
• Kills all bacteria in media
• Heat to dissolve the Agar in the LB

Preparation of Plates

• Use Plate Count Agar or Nutrient Agar, which contains all components of LB Agar
• Prepare 200 mL in an Erlenmeyer flask, enough media for 8 plates (25 mL each)
• Use 2.3% (w/v) Nutrient Agar (2.3 g / 100 mL)
• Cover the flask with a foam plug and foil
• Add autoclave tape, which has stripes that turn black when autoclaved
• Avoid using autoclave tape to label due to expense
• Autoclave for 20 minutes (cycle takes about 1 hour)
• After autoclaving, the agar will be at the bottom of the flask
• Gently swirl the media to mix using a heat glove, and avoid creating bubbles
• Allow the media to cool until the flask is cool enough to hold
• Label Petri dishes on the bottom to avoid mislabeling if lids are swapped
• Pour the media into a Petri dish in a single pour
• The bottom plate should be around ½ full
• Let the plate cool (clear becomes opaque)

Inoculating with E. coli

• Once plates are cooled and solidified, add E. coli using the "Hockey Stick" method
• Cover the entire plate: Dip "Hockey Stick" into alcohol, then light on fire to sterilize
• the aim is to burn off bacteria, not to heat the glass
• do not dip flaming glass back into the ethanol beaker
• Add a small volume of the cell suspension to the plate
• Use the sterile “Hockey Stick” to spread bacteria over the surface
• The Streak method is used to obtain a single colony
• Flame sterilize the inoculating loop in a Bunsen burner
• the goal is to burn off bacteria, not to heat the loop
• allow the loop to cool
• do not dip flaming glass back into the ethanol beaker
• Dip the sterile loop in the cell suspension and remove it
• Open the plate and gently glide the loop back and forth
• do this over a small section of the plate, and do not scratch the agar with the loop
• Sterilize loop and drag through streak #1 on the plate, then streak it over a different section of the plate
• Sterilize loop and drag through streak #2 on the plate, then streak it over a different section of the plate

Incubation of Plates

• Place the inoculated plates into the 37ºC incubator
• Plates should be incubated face down (agar side up)
• This helps reduce the accumulation of water condensation on the lid
• Could disturb or compromise the culture

Summary of E. coli Plating

• E. coli is used in biotechnology as a model organism
• Bacterial plasmid DNA can be altered into recombinant DNA
• Plasmid DNA can be inserted into E. coli after editing
• Growing more copies of E. coli will make more DNA copies
• E. coli can be grown in a liquid or on a solid in the lab
• Media must be autoclaved to ensure sterility
• Agar must be heated to be dissolved
• E. coli is grown at 37°C

Next Steps

• Complete the plating bacteria assignment to prepare for the quiz during Lab #9
• Also review the following videos to understand the concepts
• Making LB Agar: https://www.youtube.com/watch?v=uQ_mqv7zngk
• Streaking LB Agar: https://www.youtube.com/watch?v=us2QbN5dxXw
• Creating Bacterial Lawn on LB Agar: https://www.youtube.com/watch?v=KFZYeApJ1Ls