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)

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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)

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Figure 3: Typical growth curve

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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

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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)

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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)

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References

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

Week 2 – Hannah, Camille, and Kristine

For TRIM Proteins

Accomplishments:

  • Discovery of TRIM8 and TRIM13v3 gel purified PCR products in the freezer. Check gels confirmed the presence of these TRIMS in the correct place. Strong band of TRIM13v3 was present.
  • pTWIST-TRIM5a digest with NotI & BamHI and observation of TRIM5a at the correct size.

Failures:

  • pGADT7-TRIM5a digest showed TRIM5a in an unexpected spot with a curved band. When we referenced the sequence information for this plasmid, we decided to stop  using this plasmid for now and switched to pTWIST-TRIM5a.

Week 3 “To Do” List

  • PCR of pTWIST-TRIM5a
  • Gel pure of a TRIM8 band for use as template DNA for PCR.
  • Recombination of TRIM13v3 into pGADT7, pEGFP-C1, mAzurite, and pGBKT7
  • Bacterial Transformations of TRIM13v3 into pGADT7, pEGFP-C1, mAzurite, and GBKT7.
  • Finish up Glycerol Stock Archive Maps.

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.

New Antiviral Defense Systems Discovered

A one-minute summary of “Systematic discovery of antiphage defense systems in the microbial pangenome” by Doron et. al

Some of the most useful tools in the molecular biologists toolkit come from using defense systems of prokaryotes (bacteria and archaea). These defenses protect the prokaryotes from bacteriophages (aka “phages” – viruses that infect bacteria).

A new study in the journal Science identified several (9) novel bacterial defense systems. Here’s how they did it:

  1. Look through tons of bacterial genome DNA
  2. Find lots of known antiviral gene clusters
  3. Take note of gene clusters found nearby and conclude “guilt by association”
  4. Copy and paste the guilty-looking gene clusters into model bacteria
  5. Infect the model bacteria with a variety of phages
  6. Look to see if the success rate of the bacteria was reduced (a reduction in “efficiency of plating”)

Conclusion: The nine new defense systems were named after mythological gods, goddesses, and spirits that served protective roles.

Link to paper: http://science.sciencemag.org/content/early/2018/01/29/science.aar4120

Lab Meeting Notes – May 7, 2018

Lab Meeting

May 7, 2018

 

Sequencing

-getting plasmid-sequencing primer- ship through the mail

-analyze sequences (bioinformatics)

 

Archiving/Organizing

-glycerol stocks for all clones

-tables (charts/map for everything and they should be in their place)

-stockpiles of miniprep plasmids

Progress Report

wins for the team

-condensed summary for the week

-what you’re doing

 

Outreach, program development

– “fluff pieces”

-mini resumes

-techniques they have done

-youtube videos explaining cloning strategies

 

POLS videos (Riley)

-Prep videos through characterization of different viruses.

TRIM Review

-Write a review paper summary on the different TRIMS

-Literature Reviews about the TRIMs you are studying

-be able to talk about those papers and tell everyone about it.

 

Tasks for Team Leaders

  1. Perform literature searches on the TRIMs and MAGEs that you are studying.

-Come up with a computer file of different sequences, look at database, find splice variants, come up with cloning strategies to get rid of the extra sequences.

  1. Determine how you will keep track of hours worked and lab book pages logged.
  2. Look to see what PCR primer sets you have for your TRIMs/MAGEs. Make sure that there are primers designed for cloning by recombination into pGADT7 and pEGFP/mAzurite
  3. Start performing PCRs for cloning.  Then gel purify.  Then check gel.  Then recombination reactions.  Then transformations.  Then screening for inserts.
  4. Map out the splice variants for your TRIMs/MAGEs and design both general and variant-specific primers for qPCR.  Also design variant cloning strategies.

Research training opportunity

Greetings Montana INBRE Project Leaders, Mentors and former and current students:

If you know an undergraduate who’s interested in gaining High Performance Computing skills, South Dakota State University is now accepting applications for its 2018 High Performance Computing (HPC) Research Experience for Undergraduates.

This is an intensive 10-week program for HPC and data analysis research in areas such as computational and applied mathematics, statistics, engineering and technology, and biological sciences.

Deadline: February 28th

Details: https://www.sdstate.edu/mechanical-engineering/research-experience-undergraduates

Contacts: Dr. Stephen Gent (SDSU HPC REU Program Director)

605-688-5337

Dr. Jung-Han Kimn (SDSU HPC REU Associate Program Director)

605-688-5842