BISC209: Lab6

LAB 6: Start the Characterization of Cultured Bacteria by 16srDNA Sequencing and Analysis & Continue Traditional ID Techniques

In addition to isolating genomic DNA from a soil sample, amplifying the 16S rDNA by pcr, inserting the different DNA fragments from the pcr product into a cloning vector, transforming E. coli with your clones, and sending the transformed E. coli off to have the 16s rDNA inserts sequenced in an automatic sequencer so that you can identify a large and, we hope, representative sample of the bacterial flora in the soil community from your habitat, you also have been working, simultaneously, through traditional microbiological culturing techniques, to isolate and identify a few of the culturable bacteria by morphology, physical, and metabolism differentiation. Look how much you have accomplished in these few short weeks!!

By this point you have isolated pure colonies of some soil bacteria on general and enrichment media and you have gotten some preliminary or defining information about the morphologic and metabolic characteristic of the bacteria you have chosen to identify. You will continue learning about how these bacteria are different from one another and how they contribute to their community through research and performing more tests. At the same time we want to identify these bacteria by their 16s rDNA unique sequences. The process will be somewhat simplier this time. We don't have to clone into a vector and transform bacteria. This time we will use a Taq polymerase, rather than a proof-reading DNA polymerase. Taq will not be as accurate as our proof reading polymerase, but it is much less expensive and should be good enough to allow us to get genus and species identification from DNA sequencing.

In schematic the process goes as follows:

To Identify Bacteria from DNA from Isolated Pure Colonies

Choose 4 different isolated bacterial colonies per person

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Lyse Cells by boiling
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PCR amplify 16srDNA with "universal" bacterial primers: 8F and 1492R

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Visualize PCR product by agarose gel electrophoresis ⇓
Prepare PCR product for DNA sequencing
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Submit PCR products for DNA sequencing

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Determine id of cultured and isolated soil organisms with sequence comparisons to database

Part A: Prepare Lysates from pure cultures of 4 Bacteria of Interest

1. Each student will sequence DNA from 4 unique organisms. Use pure colonies from your stock slants and prepare a fresh replacement slant.

2. Label 4 microfuge tubes with unique codes for the bacteria you want to id. (Your initials and a number: Carl Woese CW-1 is a good unique identifier.)

3. Using your P20 micropipet, pipet 20μL of sterile water with 0.05% Non-idet P40 into each of the 4 tubes. Nonidet-P40 is a detergent that keeps hydrophobic domains dispersed and, thus, helps to solubilize membranes. It is similar to Triton-x 100.

4. Touch a colony with a P10 micropipet tip (the tiny ones, not the P20 tips) and resuspend the non-visible bacteria adhering to the tip in the appropriately labeled tube. Resist the urge to pick up too much cell material!! The tinest invisible bit will do and is better than too much, which can inhibit the pcr reaction!

5. Repeat for your other 3 colonies.

6. Boil all 4 samples for 5 minutes. This will lyse the cells and to inactivate bacterial enzymes. You can boil in a heat block or in the thermal cycler if you set a program to boil and you use the smaller pcr tubes. Your instructor will let you know how we will boil the tubes. If you use the heat block, use the caps that prevent the tops from popping off. Be careful when you remove them from the heat block. Point them away from you and ease the lids open but still covering the tops. Do this slowly and carefully with the opening pointed away from you. You don't want them to pop and make an aerosol of your bacteria and you don't want to lose your lysate.

Part B: PCR AMPLIFICATION of 16s rDNA from lysates prepared above

Note: All reagents for the pcr should be kept on ice and the master mix should be thawed on ice. Since Taq can function at room temp, we don't want the reaction to start until all the tubes are in the thermal cycler.

The components below have been aliquoted and prepared for you and are in pcr tubes of your team color. Label 5 pcr tubes carefully with a Sharpie on the top and side of the tube with the unique identifier for each bacterial colony. The other tube is for a neg control.

Master Mix recipe for each reaction: TOTAL VOL 23 microliters
WEAR GLOVES AT ALL TIMES AND DON'T TOUCH THE INSIDE OF THE TUBE CAPS OR YOUR PIPET TIPS--Always use a new tip when going into anything in a pcr reaction. (Contamination is a significant problem in pcr)

REAGENT and VOLUME
Promega Master Mix 2x 12.5 microliters (50 units/ml of Taq DNA polymerase supplied in a proprietary reaction buffer (pH 8.5, 400μM dATP, 400μM dGTP, 400μM dCTP, 400μM dTTP, 3mM MgCl².)
16S_ 27F(15 pmol) primer 2.0 microliter
16S_1492R (15 pmol) 2.0 microliter
nuclease free water 6.5 microliter
23μL TOTAL reaction vol. x the number of reactions

Setting up the PCR Mix
Add 2 microliters of your boiled lysate (containing the template DNA) to 23 microliters of the master mix (containes Taq polymerase, dNPTs, MgCl², and buffers), primers and nuclease free water mixture described above (for a total volume of 25 μL) in clearly labeled pcr tubes of your team color. Make sure you label on both the top and sides of the tube. (The tubes are tiny so you will have to make an identification code and keep the key to the code in your lab notebook and give a copy to your instructor.) Hold the tubes on ice until your instructor tells you the thermal cycler is ready to be loaded. Wipe the outside of the tubes to remove all ice and water before placing them in the thermal cycler.

For the negative control use 2 microliters of water (in place of the template DNA). When you have mixed your DNA or water into the pcr mix by tapping VERY LIGHTLY or flicking to be sure that all reagents are mixed and not adhering to the tube wall, take your tubes to the thermal cycler when your instructor says it's ready. Keep them on ice until then, but wipe off the bottom of the tubes before putting them into the machine. Make a template key in your lab notebook as to where in the thermal cycler you put your tubes.

The thermal cycler program is, generally, the same for all pcr reactions, but the annealing temperature (melting) is dependent on the primer pair. When you design primers, the primer melting temp. can be calculated based on the GC content and other factors. Think about which would be harder to denature: GC pairs or AT pairs and why? For 27F and 1492R, a range of 45-55C is ok, although higher temp. may lead to increased specificity that excludes some organisms' DNA from being amplified.

The length of the fragment you are amplifying determines the extension time. A general rule of thumb is to use an extension time of 1kb per minute. Here, we amplify with primers designed for the 27th and 1492th positions in the 16s rDNA gene region. Therefore our fragment is expected to be about 1.5kb long, so we will use an extension time of 1.5 minutes per cycle.

PRC Thermocycler Program= 30 cycles of:

Step	Temp	Time
Initial Denaturation	95C	5 min.
Denaturation of template	95C	30 sec.
Annealing of primers	55C	30 sec.
Extension	72C	1.5 min.
Final Extension	72C	5 min.
Hold	4C	Forever

The pcr will run for 2.5 hours or so. We can leave the pcr reactions in the thermal cycler overnight, as long as we program the last cycle to be HOLD AT 4C. Your instructor will freeze away your pcr products tomorrow so be sure they are labeled clearly. Next week you will clean them up and run a gel to assess your success at amplification of the 16S rDNA from each of your bacteria.

Part C: Culturable Bacteria Identification by Metabolic and Physical Characteristics

By this point, you will know a lot about your bacterial isolates, but you may or may not have sufficient differential test evidence to establish roles for your cultured soil bacteria. You will continue to work to perform more tests, repeat ambiguous tests, or research other tests that may be helpful to characterize your isolates. If a few new interesting isolates are added today, you may set up new tests for them.

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

Activity 1 Continue following the protocols for Tests to determine the role of a soil isolate. Assess the other tests carried over from LAB 5 by comparing inoculated media to uninoculated. Refer to the specific protocols as needed. Record all your results and observations.

Activity 2 Continue or add appropriate new physical, stain, enzyme, metabolic, or other tests that might help characterize your isolate, particularly those tests that provide information suggesting functionals role and/or interactions with other microbes in the ecosystem. The protocol links that follow may help you narrow the tests you select for each isolate.

• Organisms positive in OF-glucose medium: BISC209: Carbohydrate Fermentation Medium
• Organisms negative in OF-glucose medium: methyl red and Voges-Proskauer tests Enzyme tests
• Test for motility (3 options) Inoculate a SIM tube for each organism and a nutrient broth (or the appropriate broth in which your organism will grow) to perform the hanging drop technique for motility. The directions are found in the protocol at: BISC209: Motility.
• Stains:
  Stains (Special): Endospore, Acid fast, Capsule, and Flagella.
• If you are adding new isolates this week, catch up with earlier tests such as Gram Stain, Tests to determine the role of a soil isolate, and catalase and oxidase tests found in Enzyme tests
  
  Although we will should get at least a genus name from our 16S rRNA gene sequencing, microbiologists of previous generations had to do their bacterial identification exclusively from these morphologic, metabolic, and other tests that you have been performing over the last few weeks. If you want to see if you can identify your isolates using the pattern of test results you have so far, give it a try.
  
  The Prokayotes, Bergey's Manual or Reference articles found in the Reference folder on the First Class conference (or those that you have been collecting from other sources) should help you. It's a difficult task to sort out a complex pattern of results and some organisms don't give the usual results. When you get your DNA sequencing information back, that should confirm or clarify ambiguous or conflicting test findings.
• If you think further classical tests are unlikely to aid in the characterization of a particular isolate, omit those tests. If you are in doubt, perform the tests. It is possible one or more of them may supply evidence for a functional role or relationship.

  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 the Sanger method
  
  

  You have already use 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. Wikipedia is a great place to start to find out some of what you need to know for this assignment. There are many sources that will explain the priniple behind Sanger sequencing, polymerase chain reaction, making cells chemically competent for transformation, genomic DNA isolation (which pretty much uses the prinicple of differential solubility of DNA in ethanol), why we picked the 16S rRNA gene for sequencing to differentiate our bacterial species, etc.
  
  

  The users' manuals for the Zero Blunt® TOPO® PCR Cloning Kit will be helpful in getting a better understanding 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. Remember that this summary should be not more than two pages double spaced (or 1.5spacing) and, if you are succinct, 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.
  
  Begin to wind down and finalize the classification of 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