BISC209: Lab6

LAB 6: Start the ID 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 person will be able to 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 (NP40)into each of the 4 tubes. NP40 is a detergent that keeps hydrophobic domains dispersed and, thus, helps to solubilize membranes.

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 wait a little while before opening the lids slowly and carefully. You don't want them to pop and making an aerosol of your bacteria. BR>

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
16S_ 8F(15 pmol) primer 2.0 microliter
16S_149R (15 pmol) 2.0 microliter
nuclease free water 6.5 microliter
23μL TOTAL reaction vol. x the number of reactions

Add 2 microliters of your boiled lysate (containing the template DNA) to 23 microliters of the master mix described above for a total volume of 25 μL in properly labeled pcr tubes of your team color. Hold them 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 master mixture 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 8F 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 8th 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	46C	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 may or may not have sufficient differential test evidence to begin to identify one or more of your cultured soil bacteria. You will continue to work to perform more tests, repeat ambiguous tests, or research other tests that may be helpful in your organism id.

If you set up OF-glucose tests, compare the tubes to an uninoculated OF-glucose tube and record your results.
If you set up Starch plates, follow the protocol to read the Starch plates and record your results.

Continue with the following new tests, IF you think that they are appropriate your organisms. If you don't think some or all of them unlikely to aid in the identification of a particular bacterial strain, omit those tests. If you are in doubt, perform the tests. It is possible one or more may help as you approach identification at the species level.

Select all the organisms that produced acidic products from your results in the OF-Glucose test. Inoculate the positives into the following carbohydrate fermentation tubes: Lactose and Sucrose.

Test for non-acid products such as acetoin, using the methyl red and Voges-Proskauer tests (MRVP). These tests are performed in one tube/ organisms. Follow the directions in the Protocol section carefully, as it is easy to get confused.

Testing for Motility
You will inoculate a SIM tube for each organism and a broth tube to perform the hanging drop technique for motility next week.

Perform any other stains that you think might be useful to determine morphology.
Observe and continue to incubate the leaf cellulose digestion plates
Continue the antibiotic test protocol.

Activity
Actively begin to research and develop a Student Plan for Identification, based on the guidance of The Prokaryotes and Bergey's Manual.


Do the results you have so far seem to lead to an ID or do some of them confuse the identification? If you feel confused, research and plan future tests that might clear up ambiguities or descrepancies. 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. Don't get frustrated! 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 help things.

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