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. These E coli clones should grow on media with kannamycin and be white rather than blue on LB with X-gal. Your task today is to pick cells from a random set off "not-blue" colonies from your transformation plates and subculture these bacteria in Lura-Bertoni broth in deep-well 96 well plates, keeping tract of which plate and which wells contain your soil sample's bacterial genes. We will grow the sub-cultured clones overnight and send these the transformed E. coli off to Beckman Genomics (formerly called Agencourt) 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 the tropical habitat,
Culture-Dependent Analyses
Identification of your Bacterial Isolates by 16s rRNA Gene Sequencing
You have been working, through traditional microbiological culturing techniques, to isolate and characterize a few of the culturable bacteria in your soil community through morphology, physical, and metabolic differentiation.
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 physical, morphologic, and metabolic characteristic of the bacteria you have chosen to study. You will continue learning about how these bacteria are different from one another and how they contribute to their community through research and by 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 separate the bacterial genes by cloning each of them into a vector and transforming a recipient bacterium whose job is to keep the 16s rRNA genes separated and available for analysis. Since you have already gone through the laborious process of isolating bacterial strains to pure culture, all the 16s rRNA genes from each isolate should be identical. All we have to do now is to lyse the cells, amplify each separate 16s rRNA gene by polymerase chain reaction, and send the pcr products off for clean up and sequencing. For our pcr, we will again use Finnzyme Phusion™ polymerase, a proof-reading DNA polymerase. In schematic, the process goes as follows:
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Part A: Prepare Lysates from pure cultures of 4 Bacteria of Interest
1. Each student will sequence DNA from 3-4 unique organisms. Use pure colonies from your best plate that looks like a pure culture. DON'T use the stock slants.
2. Get 3-4 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 (A, B, C, etc.) and the unique set of numbers that we have used to identify each isolate. 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 sterile toothpick or a P10 micropipet tip (the tiny ones, not the P20 tips) 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 DNA polymerase 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 (contains DNA 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 to continue using the unique code numbers for your isolates.
Component TABLE
| Component | amt. in a 25 μl reaction |
Final Conc. |
|---|---|---|
| Purified DNAase free Water |
8 μl | |
| 2x Phusion Master Mix | 12.5 μl | 1x |
| 27F primer | 1.25 μl | 0.5 μMolar |
| 1492R primer | 1.25 μl | 0.5 μMolar |
| template DNA | 2 μl | optimum is 100ng of DNA/reaction |
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, similar 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.
Thermal Cycler Program:
3 step program
| Cycle Step | Temperature | Time | # of Cycles |
|---|---|---|---|
| Initial Denaturation | 98C | 5 min. | 1 |
| Denaturation Annealing Extension |
98C 55C 72C |
10 sec 30 sec 30 sec |
35 |
| Final Extension | 72C 4C |
10 min Hold |
1 |
The pcr will run for 45min or so. Before you go home you will load your pcr products and run a gel to assess your success at amplification of the 16S rDNA gene from each of your bacteria. Your instructor will photograph and label the gel according to the template and post the results to the conference.
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 Start or continue the Antibiotic Production/Sensitivity test.
If you completed the test using the control Micrococcus, Staphylococcus, and E. coli, you could test any non antibiotic producing isolates against the isolates that produced antibiotics in order to examine their sensitivity.
Start or check on your Cellulolytic digestion plates and/or your Starch Digestion (amylase) plates or the Phosphatase plates. If there is sufficient growth the starch digestion plates may be ready for iodine treatment. Check with your instructor.
Record all your results and observations.
- NEW TESTS
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Tests for Motility
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 and in Enzyme tests ).
If you want additional confirmation of motility, you can perform the flagella stain. The directions for the flagella stain is found in the Protocols section of the wiki at: Special Stains: Flagella.
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Special Stains:
Perform appropriate special stains as indicated. All isolates should be tested 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 for flagella stain.
Directions are found in the Protocols section of the wiki.
Stains (Special): Endospore, Capsule, and Flagella, Gram Stain
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.
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
