BISC209/S11: Lab6

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Wellesley College-BISC 209 Microbiology -Spring 2011


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", inserted the different 16s rDNA fragments from your pcr product into a cloning vector, and transformed special genetically modified E. coli with a plasmid in order to separate and amplify the 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.


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. Because the vector plasmid contains a kanamycin resistance gene, kanamycin resistance is a selectable marker. Kanamycin is an antimicrobial compound that disrupts bacterial protein synthesis and kills the cells. The genetically engineered strain of E. coli that we transformed is sensitive to kanamycin; therefore, E. coli that did not take up a cloning vector plasmid and express its genes should not form colonies on media with kanamycin.

There are two reasons that we are able to determine that the vector plasmids in the transformed E. coli that formed colonies on your plates contain a 16S rRNA gene insert from the genomic DNA isolated from your soil sample. 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 transcription of the ccdB gene and turned off expression of the protein that inhibits DNA gyrase, allowing the cell to live, replicate and form colonies that should appear white, NOT blue. The second reason that we know any white colonies are transformed with the vector plasmid that contains a soil bacterial 16s rRNa gene and that any blue colonies are likely to contain an "empty" vector (lacking a soil bacterial 16srRNA gene) is because there is a lacZ gene positioned next to the ccdB gene and transcription of BOTH is disrupted by ligation of a 16s rRNA gene insert. That transcription disruption turns off expression of the lacZ gene product, beta-galactosidase. Beta-galactosidase is an enzyme that catalyzes the breakdown of several substrates, including lactose and X-gal. X-gal is a colorless substrate that is cleaved into a blue colored product by beta-galactosidase. The Luria-Bertoni agar medium that the transformants grew on 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 because we can see that there was minimal disruption of expression of the lacZ gene. 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 since separation and amplification of individual soil bacterial 16srRNA genes was the goal of our cloning and transformation). We want to pick colonies containing 16s rRNA genes from DIFFERENT soil bacteria, but we have no way of detecting which white colony contains a 16s rRNA gene that is different from the 16s rRNA gene in the cells forming a different white colony. Since we can't sequence the gene in every colony on your plates, we will id a random sample.

We will fill one 96 well sterile block with 96 clones from each soil sample. Follow the directions below, carefully, to inoculate each well with a different, well isolated, non-blue colony.


1. We need to keep track of which DNA sequences come from which sampling site. Therefore, you should make sure that each sampling site has a separate, well-labeled 96 well plate. Put a piece of your color tape on both the cover and the bottom or side of a plate and make sure you label it with your sample code (A-F), your lab section (Tues or Wed, the date, and Wellesley College.

2. In the hoods in the lab, you will find 3 prep areas for transferring colonies to the 96 well block plates.

You will use your P1000 to inoculate one well at a time (beginning with well A1) with 1 ml (1000μL) of LB broth containing 50 μg/ml kanamycin (NO X-gal). (Note that you will eventually fill every well with broth, but you will alternate adding 1ml of broth with adding bacterial cells from a colony to that well before you fill the next well with broth.)

3. Find and select a well-isolated, white colony from your transformation plates.
If the colony is large, use the flat end of a sterile toothpick to pick up that 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, starting at the beginning A1. Leave the toothpick in the well after inoculating it as added insurance that you can distinguish which wells have been inoculated with both LB and a colony and which have not yet been filled!!!! If the colony is tiny you can use the sharp end of a sterile toothpick to pick up growth only from that colony. Make sure you have a visible amount of growth when you inoculate a well of your plate moving from A1, to A2, etc. Leave the toothpicks in the wells you have inoculated.

4. Once all the wells in your plate are filled with both medium, a colony (as indicated by the presence of a complete set of toothpicks), carefully pull out each toothpick by wiping it on the edge of the well (to scrape off the organism). Do this 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 all 96 wells are inoculated, apply a sterile sealing mat carefully. Place your full block on a platform shaker at 37C and tape it down before turning on the shaker. Your block will incubate with constant shaking at 37C overnight.


3. Seal the plate with an aluminum foil special seal and label the plate clearly: Wellesley College, BISC209, Tues or Wed (for lab day), (sampling site code A-F) and the date.
4. Freeze at -80C and send away for sequencing on dry ice. Our sequences will be done by chain termination sequencing (fluorescence marker) which is also called Sanger sequencing at Beckman Coulter Genomics, 36 Cherry Hill Drive, Danvers, MA 01923 USA. (We are not getting high throughput pyrosequencing.)
The sequences should come back in a week or two.

Culture-Dependent Isolate Characterizations: Examples of Competition and Co-operation in Selected Soil Community Bacteria



Activity: Preparing a bacterial smear slide



4.Now you are ready to make smears of your isolates. Using your best aseptic technique, flame sterilize your loop. Apply a very small loopful of deionized water (bottle on your bench) to the slide and then add 1/8th of a 1/8th of a pure colony from a fresh culture on solid medium of your isolates other than Hyphomicrobia. Create the smear as you did for the controls moving from left to right and spacing so that 3 isolates can fit on one slide. Repeat for the other isolates (except Hyphomicrobia) on the slide. For your Hyphomicrobia, use your fresh broth culture of your isolate. Remove the culture tube top (NOT placing it on the bench) and flame the lip of the culture tube. Dip your loop into the broth culture and place a VERY small drop of broth culture of that isolate on the appropriate place on the labeled slide. (The labeling from top to bottom should refer to isolates placed left to right.) Mix the drop with your loop to disperse the broth over an area that will not touch the next culture but is large enough to allow your slide to dry quickly.



An example of a multiple smear labeled slide:

The Gram Stain

Background on Using Stains in Bacteriology










Activity: Preparing a Gram Stain

Use the smear slides you have prepared of your isolates and the controls and follow the Gram Stain Protocol found below. This protocol can also be found in BISC209/S11: Stains in the protocol section of this wiki.

Gram Stain Procedure:










Use of the Compound Light Microscope

Activity: View your stained bacteria.

Gram Reaction Confirmation by use of Selective/Differential Media


Image: quadrant_plate.jpg

Cultured Bacterial Isolate Characterization by Metabolic and Physical Tests

Testing for Antibiotic Production

Start the Testing for Antibiotic Production.

Many microbes secrete antimicrobial compounds to help them compete with other microorganisms for habitat niche. Common antibiotic producers are the Actinomycetes (including Streptomycetes species)and many of the Bacillus species; although they are just a few among many, many antibiotic producing bacteria.

(This testing will take 3 weeks.)
Week 1:
Since the soil is the main source of microbes that supply the world's antibiotics, it's possible that you might discover the next great antimicrobial drug and get very rich by selling the patent for your discovery to a drug company. (Remember that the discovery of penicillin was completely accidental.) In order to find out if any of your isolates are potential antibiotic producers, we will grow your isolates for 1 week on NA before we add the control bacteria or other isolates that may be sensitive to the antibiotic produced and secreted into the medium by the first isolate. You should test all of your isolates for antibiotic production, IF they will grow on NA (not the Hyphomycrobia). Be espectially sure to test any isolates that grew originally on glycerol yeast extract medium, because those bacteria are likely to be spore formers. Endospore formers are often antibiotic producers.

1) To begin the antibiotic testing protocol, you will use the fresh broth culture of each of your isolates that you prepared prior to lab.

2) Pipet one ml of the broth culture into a sterile glass tube and adjust the concentration of the cells by matching by eye the "cloudiness" (turbidity) of the cells in the glass tube to a standard provided by your instructor. We will use a McFarland 0.5 standard(Remember that cell concentration can be measured as optical density at 600nm in a spectrophotometer); the 0.5 refers to the approximate concentration of organisms in solutions which is 1.5X108 cfu/mL for the 0.5 standard. Other common standards are shown in the table below.


3) Add bacteria from a colony of the same isolate grown on solid medium to concentrate the cells in your tube if your culture seems too dilute or add more sterile Nutrient broth to dilute the culture aliquot if it is already cloudier than the standard. The ending volume is irrelevant. Vortex to mix.

4) Using a sterile swab, dip the swab in the concentration adjusted culture tube and make an inoculation (as shown below) down the middle of a plate of nutrient agar.

5) Make a plate exactly like the first for each isolate to be tested. Label them carefully and incubate the plates for ~1 week at RT.














12. Wash your hands.


One to 3 days(depending on how fast your organisms grow) prior to Lab 7, please prepare a fresh both culture of each isolate into sterile nutrient broth or PYC whichever is appropriate.

On the same day you make the broths, subculture by isolation streaking EACH of your isolates onto nutrient agar or onto PyC medium (if your isolate won't grow on NA). In Lab 7 these cultures will be used by your team for the interaction assay, quorum sensing assay and tests for nitrogen cycle role.

Discuss the theory behind the following methods that we used to identify our bacteria by molecular tools: Genomic DNA isolation;
Polymerase chain amplification of the 16s rRNA genes;
Use of the Zero Blunt® TOPO® PCR Cloning Kit to create a library of unique plasmid vector with our 16S rRNA gene inserts; Transformation of One Shot® TOP10 Competent E. coli cells with our vector plasmid that allowed us to select, separate, and amplify our 16S rRNA genes; DNA sequencing by the fluorescent-labeled ddNPTs chain-termination (Sanger) method.(Do not describe the radioactive labeling method.

Links to Labs

Lab 1
Lab 2
Lab 3
Lab 4
Lab 5
Lab 6
Lab 7
Lab 8
Lab 9
Lab 10
Lab 12

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