BISC209: Lab5
LAB 5: Con't. Project: Soil Microbial Communities & Diversity
Preparing your clones for sequencing analysis
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 and allows the clones to grow on this media with kanamycin that normally would disrupt bacterial protein synthesis and kill the cells. We also know that each of the vectors imparting Kan resistance contains a 16S rDNA insert from one of your soil flora. 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.
You and your partner will be given a 96 well sterile plate to fill with a bit of each of the clones you want to send away for sequencing.
Preparing Glycerol Stocks to send out for sequencing
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: Complete or repeat any work from the prior week's 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:
- Use your stock slant or a colony from your newest isolation streak plate to make a replacement stock slant.
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Activity 3: Morphologic tests
At this point, based on your Gram stain data, the morphologic observations of the colony growth, and reference to the PROKAROTES and/or Bergey's Manual, you may have a tentative idea about your organisms taxonomic groupings. Use these morphologic tests to confirm or update your data.
Check for the presence of spores, a capsule, and motility.
Check for the presence or absence of the following enzymes:
Catalase and oxidase
Check for the ability of an organism to catabolize glucose only in the presence of oxygen (oxidative catabolism of glucose) or it ability to ferment glucose (fermentative catabolism) using OF-Glucose medium. - test for the ability of an organism to digest starch using a starch agar plate.
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Testing
Perform the next set of tests to id
Examine role of microbes in soil ecosystem
Student Plan based on The Prokaryotes?? and Bergeys??Links to Labs in the Soil Microbes Project
