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 characterize a few of the culturable bacteria through 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 the Finnzyme Phusion™ polymerase, the proof-reading DNA polymerase. In schematic the process goes as follows:
To Identify Bacteria from DNA from Isolated Pure Colonies
Choose 4 different isolated bacterial colonies per person
Lyse Cells by boiling
PCR amplify 16srDNA with "universal" bacterial primers: 27F and 1492R
Visualize PCR product by agarose gel electrophoresis
Prepare PCR product for DNA sequencing
Submit PCR products for DNA sequencing
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 best plate that looks like a pure culture. DON'T use the stock slants.
2. Get 4 pcr tubes of your team color (0.2ml vol) out of the jar on the instructor bench and label each with a unique code to indicate each isolate you want to id. DO NOT CONTAMINATE THE INSIDE OF THE CAP with your skin flora. Use the schematic we began with the culture independent bacterial id: your sampling habitat id (S, H, or T, including sampling site A or B if appropriate) and a unique set of numbers that your lab instructor will assign each student. Numbers found on the board or in a message to the First Class conference. If you REALLY want to send out more than 4 for id, we probably can do that. See your instructor to get approval to set up a 5th lysate.
3. Using your P20 micropipet, pipet 20μL of sterile water with 0.05% Non-idet P40 into each of the prelabeled pcr 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 well isolated colony from a pure culture with a P10 micropipet tip (the tiny ones, not the P20 tips) or a sterile toothpick and resuspend an invisible amount of bacteria adhering to the tip by swishing the tip around in the appropriately labeled tube with water and detergent. Resist the urge to pick up too much cell material but be aware that some of your isolates, particularly those dry, powdery Actinomycetes, Steptomyces and the violacin producing ones are hard to get any cells to adhere. For those, it is ok to take a barely visible amount. The tinest bit is enough, but make sure there is some part of the colony going into the lysate. Putting in too much can inhibit the pcr reaction.
5. Repeat for your other 3 colonies into separate tubes.
6. Boil all 4 samples for 5 minutes. This will lyse the cells and 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. We will boil the tubes in the thermal cycler today so you need not worry about using the caps that prevent the tops of microfuge tubes from popping off when they are boiled. Be careful when you remove the tubes from the thermal cycler. Point them away from you and ease the lids open while still covering the outside of the tops with your gloved fingers. Do this slowly and carefully with the opening pointed away from you. You don't want the caps 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 a pcr tube for each of your lysates with a fine point Sharpie on the top and side of the tube with the unique identifier for each bacterial isolate. We will set up one tube per lab as a neg control.
Setting up the PCR Mix
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)
Using a P2 or P10 and filter tips (remember that the P2 has two red decimal place volume indicators while the P10 only has 1 red decimal place indicator. MAKE SURE YOU HAVE DIALED IN THE CORRECT VOLUME!), add 2 microliters of your boiled lysate (containing the template DNA) to the prealiquoted 23 microliters of 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.) We need unique code names for your isolates that should continue the scheme we used for the culture-independent bacteria. For example, all isolates from the Durant Camellia house begin with S; Tropical house isolates T begin with T and isolates from around the bamboo in the Hydrophyte house begin with H. If your isolates are from sampling site A vs. B, please add that letter after the S, H, or T. If you are in the Tues lab, your isolates will start with 300 (odd numbers) and if you are in the Wed lab, your isolates start with 400 (even numbers). Each student will be assigned a unique group of numbers within that range to use for coding your isolates as S301 or H490, etc.
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 one person will add 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
|| 5 min.
| Denaturation of template
|| 30 sec.
| Annealing of primers
|| 30 sec.
|| 1.5 min.
| Final Extension
|| 5 min.
The pcr will run for 1.5 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 use the nanodropper or run a gel to assess your success at amplification of the 16S rDNA gene from each of your bacteria.
Master Mix recipe for each reaction: TOTAL VOL 23 microliters
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
Part C: Culturable Bacteria 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
Activity 1 Continue following the protocols for Tests to determine the role of a soil isolate. Assess the tests carried over from LAB 5 by comparing inoculated media to uninoculated.
Start or continue the Antibiotic Production and/or sensitivity test by inoculating on of the plates you prepared last week to test your isolates as producers. Follow the directions for inoculating one plate with a known set of controls and inoculate the other plate with broth cultures of your isolates that you want to test for interactions (sensitivity) with the potential antibiotic producers.
Check on your cellulolytic test plates (with the leaf disks). You may see a lot of "swarming" outside the area of inoculation, making it difficult to use the negative control leaf disk as a comparison or to determine which of the bacteria on the plate are responsible for changes in the leaf tissue. We will continue to watch this reaction for several more weeks but make notes about the condition of the leaf tissue and perhaps take pictures for a record.
Read the OF/Glucose results from the aerobic broth in the loosely capped tube and compare changes in the same media in semi-solid form incubated in low oxygen condition (screw-capped tube). Remember to assess color change in different parts of the tubes since the top portion has been exposed to more oxygen than the middle or lower portions. Use the assessment directions in the OF-GLUCOSE protocol found in Tests to determine the role of a soil isolate. See the directions in Activity 2, NEW TESTS, for setting up further tests for carbohydrate fermentation and/or MR/VP tests.
Refer to the set up and analysis directions in the Protocols section for how to assess your starch digestion tests using iodine as an indicator. Record all your results and observations.
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 functional roles and/or interactions with other microbes in the ecosystem.
Ablilty to ferment sugars: Carbohydrate Fermentation Tests (Find this protocol just before the Enrichment Media Protocol in the Protocols section)
Inoculate any of your isolates that were OF-Glucose positive in both tubes into the other specific sugar fermentation media provided. Each organism is inoculated into one tube of each type of sugar as described in BISC209: Carbohydrate Fermentation Medium. Be sure you label the sugar tubes as you gather them, they look identical.
Inoculate isolates that gave a negative fermentation of glucose result in your OF-Glucose test into MR-VP medium as described in Enzyme tests .
Tests for Motility
Every isolate should be inoculated in a SIM tube. This test gives information about motility and about two other metabolic pathways.
Find the directions in ( Motility Tests and in Enzyme tests ).
If you want additional confirmation of motility, you can perform a wet mount technique for motility and/or the flagella stain. The directions for these tests are found in the Protocols section of the wiki at: BISC209: Motility. If you decide to perform a wet mount analysis of motility, be aware that, unfortunately, when viewing living bacterium in a wet mount at 400x magnification, it is difficult to see unstained, tiny bacteria. If you choose to try to assess motility in a wet mount, ask your instructor for help. Note: You may also use the broth culture that you set up for the cellulose degradation test and to test your isolates ability to resist antibiotics as described in Tests to determine the role of a soil isolate protocol in this wiki can be used to make a wet mount.
Continue or extend other LAB 5 tests if you haven't performed them: : Catalase and Oxidase tests found in Enzyme tests
Although we 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, and many other, 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.
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.
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 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 priniples behind Sanger sequencing, polymerase chain reaction, plasmid cloning, 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., 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: 
Another good source of information is the background information found in this wiki. Be careful about inadventent 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.
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