Why work with Escherichia coli (E. coli)?

For Discover Biology Lab (Background Information)

It would probably be difficult to find a molecular biology laboratory that didn’t have a freezer full of E. coli.  Species that are commonly used in biology labs are called model organisms.  You will find that there are people that can cite numerous reasons why one particular organism is a “bad” model organism but, in practice, there is no perfect model organism.  This essay will describe some of the reasons why scientists use E. coli.

Ease and cost are two factors that should be a considered when working in a lab.  E. coli excels at both of these.  The first two talking points (figure 1) are the facts that, like all bacteria, they reproduce by binary fission and, unlike all bacteria, they can do so very rapidly.  Under optimal conditions, E. coli can replicate about once every 20 minutes.  In contrast, Mycobacterium tuberculosis has a much slower optimal growth rate (1).

The final talking point in this section is that the medium (typically Luria-Bertani broth or “LB” broth) is both easy to make and inexpensive.  The ingredients are 10 grams tryptone, 10 grams sodium chloride (table salt), and 5 grams yeast extract per liter of water (2).  Add some E. coli to LB broth, heat it up to 37 degrees Celsius (aka body temperature or 98.6 degrees Fahrenheit) and shake it at about 200-220 rpm to aerate the sample and the bacteria will multiply for you.

Figure 1: Benefits of working with E. coli (part 1)


Bacteria replicate by  binary fission (figure 2).  This means that one “parent” cell will divide into identical “daughter” cells.  As the bacteria continue to replicate, their population size will repeatedly double.  A generic growth curve is shown in in figure 3. At first the doubling rate is slow and this represents the “lag” period of growth.  Next, the replication is optimal and this growth phase is referred to logarithmic (or “log”) growth.  As nutrients become limiting, the growth rate slows to a standstill (stationary phase).  After this, the bacteria will begin dying faster than replicating and the population will crash.

Figure 2: Replication by binary fission (one becomes two)


Figure 3: Typical growth curve


When certain species of bacteria are placed in a stressful environment, such as higher than normal temperatures, they can increase their activity of taking up DNA from the environment through a process called transformation.  E. coli can be grown in such a manner as to increase the competence of taking up DNA in response to stress.  In fact, we follow a protocol for making competent bacteria to create an inexpensive source of these in my lab.  The extracellular DNA that is picked up from the environment can be in the form of plasmid DNA (figure 4).

Figure 4: Chromosomal (genomic) and extrachromasomal (plasmid) DNA in E. coli


Like genomic DNA chromosomes, plasmid DNA is circular.  Each plasmid has an origin of replication (aka “ori”) that will recruit the same DNA replication machinery used in replication of genomic DNA.  Depending on the sequence in the origin of replication, the plasmids will be found at varying concentrations in each bacteria.

Many cellular and molecular biology research tools are built from plasmids that were created and modified for a particular purpose.  Common and useful genetic elements in a variety of plasmids will be covered in a future post.

Figure 5: Benefits of working with E. coli (part 2)


In conclusion, it’s the ability of E. coli to make copies of plasmids is what makes them such a welcomed addition to most biology laboratories.  The last aspect to discuss in this post is the ability to easily and cheaply store these plasmids and plasmid makers (figure 6).  Monocultures of unique strains of bacteria that copy a specific plasmid can be stored in a 50-50 glycerol/growth medium mix at very cold temperatures (typically -80C).  Amazingly, these “frozen” cultures can be stored for years to decades in this state.

Alternatively, cultures of bacteria can be busted open and, through a plasmid preparation procedure, the plasmid DNA can be purified.  The plasmid DNA is usually stored in water (or Tris-EDTA buffer) at -20C for months to years.

Finally, if you’d like to ship some plasmid DNA through the mail to a collaborator, just drop some plasmid solution onto heavy weight paper and let it dry.  If you circle where the plasmid dried, your collaborator can cut out that area of the paper and suspend the DNA in some water.  A quick bacterial transformation will produce plasmid makers at the destination site.

Figure 6: Benefits of working with E. coli (part 3)




  1. http://mmbr.asm.org/content/72/1/126.full
  2. http://cshprotocols.cshlp.org/content/2006/1/pdb.rec8141.full?text_only=true

My cloning attempt into a plasmid yielded colonies following transformation. Now what?

For Standard operating procedures

Congratulations!  But…before celebrating too much, make sure to check that your insert is what you think it is.  Here are things you should work on now:

  1. Pick a colony or two from the plate and spot some of the bacteria onto another plate (aka masterplate) and shake the rest of the bacteria off into 5mL LB broth.
    • Make sure the plate and broth is supplemented with the correct antibiotic.
  2. Incubate plate and liquid culture at 37C overnight.
    • The liquid culture should shake at ~220rpm
    • Store the masterplate at 4C
    • Purify the plasmid from the bacteria in the liquid culture using a plasmid miniprep kit
  3. Digest the vector with cut 800-1000ng of plasmid in a 10uL reaction containing your gene
    • Cut 800-1000ng of plasmid in a 10uL reaction
    • Use 0.5 ul of each restriction enzyme
    • Incubate for at least 2 hours at 37C (unless the recommended temperature for the enzyme is 25C!)
  4. Run the sample on at check that the insert is the correct size.  If your gene is cut into smaller pieces by either the restriction sites that flank your gene (for example: EcoRI and BamHI in pGBKT7), do the following:
    • Copy and paste the gene sequence into NEBcutter V2.0 and run the program
    • Look for the restriction enzyme you used to be listed in the 0 cutter list.  If it’s not there, look in the “1 cutter” and “2 cutter” lists.
    • Calculate the sizes of fragments you would expect from the restriction digests and compare these expectations with your gel image.
    • Note that small fragments of 200bp or less may be difficult/impossible to detect.
  5. If the fragment(s) is the correct length, make a glycerol stock
    • Pick bacteria from the appropriate colony on the masterplate
    • Inoculate and grow a 5mL culture as described above.
    • Pellet the bacteria and suspend in 400uL of bacterial freezing media (normal LB broth with 50% (v/v) glycerol)
    • Transfer to a well-labeled 1.5mL tube
    • Store in -80C glycerol stocks
    • Enter the information for this new construct into the log sheet for glycerol stocks (the sheet should be in a oligo information 3-ring binder)
  6. Sequence the entire length of the gene that is inserted into the vector.  We are set up to use the SimpleSeq DNA Sequencing Service from Eurofins.