BISC209/S11: Lab6

LAB 6: Finishing the Culture-Independent Identification of Bacteria by 16s rRNA Gene Sequence Analysis

To summarize our culture-independent work, you have extracted genomic DNA from a soil sample, amplified the 16S rDNA by pcr using "universal bacterial primers", inserting the different 16s rDNA fragments from your pcr product into a cloning vector, transformed special genetically modified E. coli with a plasmid in order to separate 16s rRNA genes from different bacterial members of your soil community. Now you are ready to pick transformants that successfully incorporated the plasmid vector genes.

Activity::

Preparing your clones to send away for sequencing analysis of your 16S rRNA gene
When you examine your transformation plates after their initial overnight incubation, there should have been hundreds of well isolated colonies. In theory, each of them should contain the vector plasmid with an insert of the 16s rRNA gene from one of your soil sample bacteria. Since the vector plasmid contains a kanamycin resistance gene, kanamycin resistance is a selectable marker. The genetically engineered strain of E. coli that we transformed is sensitive to kanamycin UNLESS it is expressing the kanamycin resistance gene on the plasmid. E. coli that did not take up a cloning vector plasmid and express its genes do not form colonies on media with kanamycin. Kanamycin is an antimicrobial compound that disrupts bacterial protein synthesis and kills the cells.

We know each of the vector plasmids in the transformed E. coli growing on the plate contains a 16S rRNA gene insert from the genomic DNA isolated from your soil sample for two reasons. First, there is a ccdB gene in the insertion region of the vector plasmid. That gene, ccdB, means "control of cell death". That gene, when not disrupted, expresses the ccdB protein that poisons bacterial DNA gyrase, causing degradation of the host chromosome and cell death. But the presence of your 16S rRNA gene insert has disrupted the ccdB gene and turned off the protein that inhibits DNA gyrase, allowing the cell to live, replicate and form a colony that should appear white, NOT blue. The second reason that we know the white colonies are transformed with the vector plasmid and that the plasmid contains our insert is that there is a lacZ gene positioned next to the ccdB gene in the insert area and when it is disrupted by insertion of your 16s rRNA gene, it turns off expression of the lacZ gene product, beta-galactosidase. Beta-gal is in enzyme that catalyzes the breakdown of several substrates, including lactose and X-gal. X-gal is a colorless substrate that is is cleaved into a blue colored product by beta-galactosidase. Your Luria-Bertoni agar medium contains both kanamycin and Xgal. If you saw blue colonies, those bacteria are daughter cells from a vector transformed E. coli, BUT the vector plasmid probably does not contain the 16s DNA insert we seek. Therefore, you only want to pick "not-blue" colonies to send away for sequencing of the insert. We hope that there are hundreds of these not-blue colonies on your plate (but not so many that they are not well separated from each other). Our goal is to find 16s rRNA gene fragments from DIFFERENT soil bacteria in many transformed clones, but we have no way of detecting right now which colonies contain a 16s rRNA gene from different soil bacterial species because all will be identical looking non-blue colonies on these plates.

Each lab section will be allowed to fill (3) 96 well sterile blocks with their clones. Follow the directions below, carefully, to inoculate each well with a different, well isolated non-blue colony.

Preparing Over-night Cultures to send away for 16s rRNA gene sequencing

1. We need to keep track of which DNA sequences come from which sampling site. Therefore, you should fill the first half (wells A1-D12) or the last half (wells E1-H12), indicating on the template provided in which of the plates (1,2,3,4,5, or 6) and wells your soil sample clones are located. Be sure the plate is labeled Tues. or Wed. lab, Plate # (1-6), and that it is clear which soil sample (A-L) clones are in each well.

2. In the hoods in the lab, you will find 3 prep areas for transferring colonies to the 96 well block.
You will use your P1000 to inoculate 1 ml (1000μL) of LB broth with 50 μg/ml kanamycin (NO X-gal) into each well of your block. We suggest you inoculate one well at a time. First add the medium then add your colony.
3. Find and select well-spaced, white, colonies on your transformation plates.
Use the flat end of a sterile toothpick to pick up a single colony. Be careful NOT to touch any of the area of the plate around the colony with your toothpick! Place the toothpick in a singe well of your 96 well block. Leave the toothpick in the well as added insurance that will know which wells have been inoculated!!!!
4. Once all the wells assigned to you and your partner are filled with toothpicks, carefully pull out each toothpick by wiping it on the edge of the well (to scrape off the organism) on a side of the well that will allow you to discard the toothpick without the chance of dripping this well's contents into another well. BE CAREFUL not to cross-contaminate any wells!!! Discard the toothpicks in the autoclave bag.
Once the block is completely full, apply the sterile sealing mat carefully and label the plate. This label should include your lab section day, team colors, sampling site codes, and the date. Make sure that this identifying information is also on the template. Place full blocks carefully on the platform shaker and tape them down before turning on the shaker. Your block will incubate with constant shaking at 37C overnight. Give your instructor the completed template.

Preparing Glycerol Stocks from your Overnight Cultures(Your instructor will do this part for you so make sure that your plate and wells are clearly identified!)

1. Pipet 50 μL of 50% glycerol in each well a 96 well Costar round bottom plate.
2. Mix each overnight culture from the 96 well block by pipetting up and down and transfer 50 μL of each culture into a separate well of Costar plate. Mix well.
3. Seal the plate with an aluminum foil special seal and label the plate clearly: Wellesley College, BISC209, Tues or Wed (for lab day), (for sampling site codes) and the date.
4. Freeze at -80C and send away for sequencing on dry ice.
The sequences should come back in a week or two.

Culture-Dependent Analyses

Cultured Bacterial Isolate Characterization by Metabolic and Physical Tests

By this point, you are beginning to learn a lot about your bacterial isolates, but you may or may not have sufficient differential test evidence to establish roles and metabolic or physical characteristics for your cultured soil bacteria. You will continue to work to perform more tests or to repeat ambiguous tests that may be helpful to characterize your isolates.

Actively begin/continue to research and develop your evidence. Use The Prokaryotes and Bergey's Manual. Link to the electronic edition of | The Prokaryotesthrough Springer ebooks.
Link to the electronic edition of | Bergey's Manualsthrough Springer ebooks

Activities: Continue, start, or repeat appropriate tests or stains that might help characterize your isolates.
Testing for Antibiotic Production and/ or Sensitivity

Start the Testing for Antibiotic Production and Sensitivity test. Use the control Micrococcus, Staphylococcus, and E. coli cultures.

Record all your results and observations.

Tests for Motility & Metabolic Capabilities
Every isolate should be inoculated into a SIM tube. This test gives information about motility and about two other metabolic capabilities(hydrogen sulfide production and production of the enzyme tryptophanase).
Find the directions in ( Motility Tests ).
You can obtain additional confirmation of motility, by performing a Hanging Drop Motility test and, if positive, you could try the flagella stain. The directions for the hanging drop test and flagella stain are found in the Protocols section of the wiki. You should have time to do these tests in Lab 8.

Special Stains:
Perform appropriate special stains as indicated. All isolates growing from your dried soil extract on Glyerol Yeast Extract Agar (GYEA) medium that are potential spore formers should be stained for endospores. Only slimy or mucoid colonies should be stained with the capsule stain and only motility positive isolates on SIM medium should be tested by hanging drop or the flagella stain.
Directions for stains are found in the Protocols section of the wiki.
Stains : Simple, Gram, Endospore, Capsule, and Motility Tests

Although we should get at least a genus name for many of our soil community bacterial from our 16S rRNA gene sequencing, microbiologists of previous generations had to do their bacterial identification exclusively from these physical and metabolic tests. The ones we are performing are a tiny subset of all the morphologic, metabolic, and other tests that you could perform on your isolates to try to identify them through a battery of positive and negative tests for different metabolic capabilities and characteristics. Be very glad that you are training as a microbiologist in the era of genomics!

CLEAN UP

1. All culture plates that you are finished with should be discarded in the big orange autoclave bag near the sink next to the instructor table. Ask your instructor whether or not to save stock cultures and plates with organisms that are provided.

2. Culture plates, stocks, etc. that you are not finished with should be labeled on a piece of your your team color tape. Place the labeled cultures in your lab section's designated area in the incubator, the walk-in cold room, or at room temp. in a labeled rack. If you have a stack of plates, wrap a piece of your team color tape around the whole stack.

3. Remove tape from all liquid cultures in glass tubes. Then place the glass tubes with caps in racks by the sink near the instructor's table. Do not discard the contents of the tubes.

4. Glass slides or disposable glass tubes can be discarded in the glass disposal box.

5. Make sure all contaminated, plastic, disposable, serologic pipets and used contaminated micropipet tips are in the small orange autoclave bag sitting in the plastic container on your bench.

6. If you used the microscope, clean the lenses of the microscope with lens paper, being very careful NOT to get oil residue on any of the objectives other than the oil immersion 100x objective. Move the lowest power objective into the locked viewing position, turn off the light source, wind the power cord, and cover the microscope with its dust cover before replacing the microscope in the cabinet.

7. If you used it, rinse your staining tray and leave it upside down on paper towels next to your sink.

8. Turn off the gas and remove the tube from the nozzle. Place your bunsen burner and tube in your large drawer.

9. Place all your equipment (loop, striker, sharpie, etc) including your microfuge rack, your micropipets and your micropipet tips in your small or large drawer.

10. Move your notebook and lab manual so that you can disinfect your bench thoroughly.

11. Take off your lab coat and store it in the blue cabinet with your microscope.

12. Wash your hands.

Assignment

Write a brief summary of the theory behind the following techniques that we used to identify our bacterial species by molecular tools:
Genomic DNA isolation,
Polymerase chain amplification of part of the 16s rRNA gene,
Use of the Zero Blunt® TOPO® PCR Cloning Kit to create a library of unique plasmid vectors with different bacterial 16S rRNA gene inserts,
Transformation and selection of One Shot® TOP10 Competent E. coli Cells that allowed us to select and separate our 16S rRNA genes for sequencing,
DNA sequencing by chain termination, sometimes called Sanger sequencing, (not 454 pyrosequencing)

You have already used each of these molecular tools and written about all of them as a Material and Methods section, but you haven't yet been required to explain the theory behind how each of them accomplishes each of crucial steps toward our goal of identifying unknown bacteria by genus and species name from DNA sequencing. One of the problems in using sophisticated molecular tools is that you can have a very successful lab day, yet it can be mostly "hands on, brain off". Since much of what you have been doing is pipeting, mixing, and incubating of miniscule quantities of liquid reagents that come in kits, it is easy to lose sight of what is actually happening in those tubes or spin columns at each stage. The problem of "doing without knowing" is exacerbated by kit manufacturers who make their reagents "proprietary". That prevents us from knowing exactly what's in them, making it even harder to follow the chemical or physical reactions.

Despite our use of such proprietary kits, it is possible to understand how it all works. All of these tools were discovered by scientists who published their findings. You don't, however, probably need to go to primary literature (Sanger's original paper, for example) to find out how Sanger sequencing works. There are good animations of Sanger sequencing, transformation, pcr, etc. prepared by the Dolan DNA center at [| http://www.dnalc.org/resources/animations/]. Pay particular attention to the difference between a polymerase chain reaction and the Sanger sequencing reactions described. Note that the type of cloning described in the Dolan animations is organismal cloning---not what we are doing. We are doing molecular cloning. A good animation that describes our type of plasmid cloning is found at : | http://www.sumanasinc.com/webcontent/animations/content/plasmidcloning.html. Wikipedia is also a great place to start to find out some of what you need to know for this assignment. Although it won't be difficult to find out the principles behind Sanger sequencing, polymerase chain reaction, plasmid cloning, making cells chemically competent for transformation, genomic DNA isolation (which pretty much uses the principle of differential solubility of DNA in ethanol), why we picked the 16S rRNA gene for sequencing to differentiate our bacterial species, etc., it will be challenging to condense each tool to essentials in your summary. Being able to distill and write a broad outline, while understanding the specifics, will be important when you describe your experimental design in your final paper.

The users' manuals for the Zero Blunt® TOPO® PCR Cloning Kit might be helpful in getting a better understanding of the specifics of our cloning. You can download it as a pdf file from the manufacturer, Invitrogen's web site at: [1]

Another good source of information is the background information found in this wiki. Be careful about inadvertent plagarism.

Remember that this summary should be not more than a couple of pages double spaced (or 1.5spacing). If you are really good at picking out essentials and being concise, you might be able to adequately explain these molecular tools in a page.

The goal of this assignment is to make sure that you have a clear understanding of the biological and chemical basis of these common molecular tools and an appreciation of the complexity of the genetic engineering that went into the creation of our cloning vector and the genetically modified strain of E. coli we transformed.

Continue to characterize your culturable isolates.

Links to Labs

Lab 1
Lab 2
Lab 3
Lab 4
Lab 5
Lab 6
Lab 7
Lab 8
Lab 9
Lab 10
Lab11
Lab 12