LAB 5: Getting the 16S rRNA genes from the Soil Genomic DNA in the E. coli clones ready to send away for DNA sequencing
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.
You and your partner will be given a 96 well sterile block. 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 habitat and which sampling site (if there was more than 1 sample/site). We also need to differentiate the culture independent bacteria from the culture dependent bacteria that we will id from future 16s rRNA gene sequencing. Therefore, you should label the wells of the 96 well block using the template provided by your lab instructor according to the following schema:
Orient the plate so that A1 is on the top left and start with that well and move down the row to A12 before starting with the B row.
Use letter and numbers S 101-148 for clones from sample site A in the seasonal display room (Durant camellia room).
Use letters and numbers S 149-196 for clones from sample site B in that room.
Use letter and numbers T 101-148 for clones from sample site A in the Tropical House.
Use letters and numbers T 149-196 for clones from sample site B in that room.
Use letter and numbers H 101-148 for clones from sample site A in the hydrophyte house (bamboo area).
Use letters and numbers H 149-196 for clones from sample site B in that room.
The Wednesday lab should use the same letter scheme except that their numbers should start with 200 rather than 100. Since the Wed. lab has two sampling sites from the Durant Camilla room only, the tropical team and and hydrophyte team should sequentially number a random sample of 96 white, well-spaced colonies from any of their transformation plates representing the bacteria in their single soil sample starting with 200 and ending with 296.
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 0.25-0.50 μL/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 for the habitat is completely full, apply the sterile sealing mat carefully and label a piece of your team color tape to identify your block plate. This label should include your lab section, team color, and initials of partners, data. Use this identifier on the template that contains the well information. Take your block to the 37C room and place it carefully on the platform shaker. Your block will incubate at 37C overnight with rotation.
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), S, T, or H (for habitat code) 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.
Culturable Bacteria Characterization continued
Complete, continue, or repeat work started previously.
Check all work in progress and record results or observations on your latest isolation streak plates and stocks of your soil bacteria isolates.
Use a fresh stock slant or a colony from your newest isolation streak plate to make a replacement stock slant. Careful attention to replacing stocks will ensure that your cultures remain pure and grow satisfactorily.
Interpreting Selective/Differential PEA and EMB plates.
Examine and record the growth and appearance of soil bacteria isolates inoculated to PEA and EMB plates last week. See the description in the Protocol BISC209: Selective,and/or Differential, and/or Enriched media to help you with the evaluation of cell wall type and other information you might glean from growth appearance on these selective and differential media. Are your Gram stain findings supported? If not, explain why and/or repeat your Gram-stain.
Metabolic Activity Tests
Check for the presence or absence of the following enzymes:
Catalase and oxidase. Use the directions in the Protocols section: Enzyme tests
Follow the instructions in Protocols for determining the roles of soil isolates in the soil to begin or perform all of the role tests. Test all of your isolates for OF/Glucose, for Antibiotic production, for starch digestion and cellulolytic activity. Only test isolates that you suspect are Azotobacter or in the Ammonia Nitrofying groups for the presence of Ammonia or Nitrate.
3A. Inoculate tubes of OF-Glucose medium (2 tubes per isolate) to determine whether an organism is oxidative or fermentative or both using the carbohydrate glucose. Use the instructions found in Protocols for determining the roles of soil isolates in the soil
3B. Start the protocol to examine cellulose degradation. This will examine the ability of your isolates to participate in carbon recycling through the digestion of leaf cellulose. This protocol involves collecting leaf discs from your team habitat in the greenhouse. You will use a microfuge tube to "punch" the discs from leaves. It is preferable to use fallen leaves collected from the soil surface, if you need to use fresh leaves still on the plants in the greenhouse, pick one or two leaves from areas of the plant that are inconspicuous. Remember to wear gloves and try hard to avoid contaminating the leaves with skin flora when you collect them. Rinse the leaves briefly in sterile water before making your leaf punches. When you have obtained your leaves, follow the directions found in Protocols for determining the roles of soil isolates in the soilfor getting leaf disks in the medium and inoculating your isolates.
3C. Begin or continue the protocol for Antibiotic Production by testing your isolates for antibiotic production. Follow the instructions in Protocols for determining the roles of soil isolates in the soil. Next week you will use these plates to assess susceptibility in several different types of control bacteria (Staph sp., E. coli, Micrococcus). If you like, you can set up an additional plate of each of your isolates to test them next week for susceptibility to the antibiotic producers in your soil community to look for interesting interactions.
3D. Begin the protocol to test for Ammonification by soil microbes.Follow the instructions found in Protocols for determining the roles of soil isolates in the soil.
3E. Examine starch digestion by your isolates by inoculating starch plates. Follow the instructions found in Protocols for determining the roles of soil isolates in the soil.
Morphologic tests: Special stains and Motility tests.
Do the stains last. If you don't have time, there will be time next week to start or to continue this activity.
At this point, you should have significant knowledge about the cell wall type, the characteristic microscopic appearance of the bacteria (individual, chains, pleomorphic, dimorphic, etc.), and colony growth information. You could also find out whether or not each of your organisms of interest has a capsule, flagella, or makes endospores by performing some special stains or tests.
To do any of the special stains, follow the Protocols for Stains and Motility testing in the Prtocols section: Special Stains: Endospore, Capsule, and Flagella and Motility Tests.
Only do these stains if you have reason to believe they will be positive. Suspected Bacillus, Streptomyces, Actinomycetes or other isolates that showed a clear area inside the cell when Gram stained, may contain endospores and should be stained for spores.
Colonies that show moist, slimy, or spreading colonies may have a sigificant capsule and should be tested with the capsule negative/positive stain.
Flagella are the bacterial motitility organelle, but the flagella stain designed to allow you to see them is tricky and may not work. Set up a SIM's test from the MOTILITY protocol sections and read the results of that before deciding to do a confirmatory wet mount or flagella stain. If you get a positive motility test from a SIMS test (or from a wet mount), but you get a negative flagella stain, you should doubt the stain result and consider your isolate motile.
The pattern of results from all of your characterization tests might allow you a preliminary identification for some of your organisms of interest. Use the Link to the electronic edition of | The Prokaryotesthrough Springer ebooks and the
Link to the electronic edition of | Bergey's Manuals to try to determine the taxonomic grouping of each bacteria strain.
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 two Materials and Methods Sections for your final paper:
Isolation of soil bacteria to pure culture
Identification of bacteria by 16S rRNA gene sequencing from soil genomic DNA
Both of the M&M sections that you will write for this assignment should have multiple subsections.
Although it is unlikely that you know the genus and species name of the bacteria you have cultured and isolated by this point in our project; you probably do have some idea to which of our desired bacterial groups each is likely to belong. Therefore, when you write this M&M section on "Isolation of Soil Bacteria to Pure Culture", you should, at an appropriate point, divide it into subsections, each subsection having a title such as "Isolation of Myxobacteria", etc. Either describe the enrichment protocols you followed to culture and isolate a specific bacterial group, including the ingredients for all media used (found in the wiki) OR give a citation to a published reference (NOT the wiki!)that includes all necessary information and any modifications you used. You do not have to write up all 11 groups, but only the 4 isolates that are your part of your group's work.
The M&M section that you wrote previously, "Amplification of 16S rDNA in a Soil Sample", can be revised and included as the first part of the more complete Materials and Methods section you will write this week as "Identification of bacteria by 16S rRNA gene sequencing from soil genomic DNA". Please use the feedback you received on your previous M&M to improve this Methods submission.
Continue monitoring and following the appropriate protocols to enrich and isolate the culturable bacteria.
Actively begin/continue to research how the morphologic, metabolic, biochemical, and other test results on your bacterial isolates fits with your expectations for the group you enriched for based on the guidance of The Prokaryotes and Bergey's Manual. Link to the new interface for electronic access to the e-book form of The Prokaryotes and Bergey's Manual at | http://0-beta.springerlink.com.luna.wellesley.edu.
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