BISC209: Lab5

LAB 5: Con't. Project: Soil Microbial Communities & Diversity

Preparing your clones to send away for sequencing analysis of your 16S rDNA When you examine your transformation plates after an overnight incubation, you should see hundreds of identical looking well isolated colonies, all containg the vector which expresses the kanamycin resistance gene. Kanamycin resistance is a selectable marker emparted by the cloning vector that allows kanamycin sensitive E. coli to grow on this media. E. coli that did not take up a cloning vector plasmid and express it's genes do not form colonies on media with kanamycin. Kanamycin is an antimicrobial compound that disrupts bacterial protein synthesis and kill the cells.

We also know that each of the vectors imparting Kan resistance contains a 16S rDNA insert from the genomic DNA isolated from your soil sample. We know that insert is in the vector because it is responsible for disrupting expression of the ccdB (control of cell death) gene which, if not disrupted, expresses the ccdB protein that poisons bacterial DNA gyrase, causing degradation of the host chromosome and cell death. We hope there are hundreds of 16s rDNA gene fragments from DIFFERENT soil bacteria in many transformed clones.

You and your partner will be given a 96 well sterile plate to inoculate broth with kanamycin with a bit of each of the clones you want to send away for sequencing. After overnight culture, we will prepare glycerol stocks of each clone to send out for sequencing of the 16s rDNA insert on the vector.

Preparing Glycerol Stocks

1. Streak the original colonies out on LB plates containing 50 μg/ml kanamycin-labeling each with your team color initial, eg. Tues red= TR-1, R-2, R-3, etc up to 96. Incubate at 37C overnight.
2. Isolate a single colony and inoculate into 1-2 ml of LB containing 50 μg/ml kanamycin. Keep your labeling clear.
3. Grow with shaking to log phase (OD600 = ~0.5)
4. Mix 0.85 ml of culture with 0.15 ml of sterile glycerol and transfer it to a well in the 96 well plate. Keep the numbering straight!!! Make a template in your lab notebook.
5. Give the plate to your instructor to seal and send away on dry ice.
The sequences should come back in a week or two.

Culturable Bacteria Identification continued

Activity 1

• Use your newest stock slant or a colony from your newest isolation streak plate to make a replacement stock slant.
• Complete or repeat any work from the prior weeks' lab to ensure your cultures are pure and growing satisfactorily.
• Check and record any important observations on your newest isolation streak plates and stocks.
• Examine and record the results of your PEA and ENDO plates. Is your Gram stain data supported?

Activity 2: Morphologic tests
At this point, based on your Gram stain data, the morphologic observations of colony growth, and reference to The Prokaryotes and/or Bergey's Manual, you may have a tentative idea about your organisms taxonomic grouping. Use the appropriate protocols Special Stains: Endospore, Acid fast, Capsule, and Flagella to continue to learn more.
This week check for the presence of spores and a capsule.
Set up the tubes needed to examine motility Motility Tests.

Activity 3
Check for the presence or absence of the following enzymes:
Catalase and oxidase. Enzyme tests

Activity 4
Use tubes of OF-Glucose medium to check for the ability of an organism to catabolize glucose only in the presence of oxygen (oxidative catabolism of glucose) or to ferment it with or without oxygen (fermentative catabolism).
Use Starch agar plates to test for the ability of an organism to digest starch.

Activity 5
Today you will set up and begin the experiment to examine the ability of your isolates to participate in carbon recycling through the digestion of leaf cellulose and begin the steps to test your isolates for antibiotic production. Follow the appropriate Protocols for determining the roles of soil isolates in the soil

Procedure for cellulose digestion:
Prepare the plates needed to examine leaf cellulose digestion and inoculate your organisms. Be sure you use melted pours of the appropriate medium for each of your samples and leaves collected from the research site in the greenhouse. These plates will be examined multiple times over the next several weeks. To prevent the medium from drying out seal the edges of the plates with parafilm and place these plates (upside down) in a resealable plastic bag as suggested in the protocol. You may also add sterile water after 2 weeks if you see any evidence that the agar is drying up.

Procedure for antibiotic production:
Inoculate the appropriate plates as described under week 1 in the protocol for the test of Antibiotic production.

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