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. Take a look at your plate for well spaced, white, colonies. Using sterile toothpics select and pick up a whole single colony with the flat end of a sterile toothpick and place it in a singe well of a 96 well (each pair will prepare a 96 well block) plates will be prefilled with Kan etc and leave each toothpick in the well. The colonies will grow overnight. Once the whole block is filled with toothpicks, carefully pull out the toothpick wiping it on the edge of the well (scaping off the organism) Discard the toothpicks properly. Incubate each well overnight.

Glycerol stock day 1. the next day aliquot 50 ul of 50% glycerol in costar round bottom plates. Use the multichannel pipet and transfer (with mixing) 50 ul of the culture into the costar plates.

your labeling clear.

Stick on dry ice and send to . They will send back sequencing.

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. Careful attention to replacing stocks will ensure that your cultures remain pure and grow satisfactorily.
• Complete, continue, or repeat work started previously
• Check all tests in progress and record results or observations on your latest isolation streak plates and stocks of your soil bacteria isolates.
• Examine and record the growth and appearance of soil bacteria isolates inoculated to PEA and ENDO 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, repeat your Gram-stain.

Activity 2:
Morphologic tests
At this point, you should have significant knowledge about the cell wall type, the characteristic arrangement of the bacteria (individual, Strep., Staph, etc.), and some colony growth information. You should also find out whether or not each of your organisms of interest has a capsule, flagella, makes endospores, or might be acid-fast. The pattern of results, just from those morphologic characteristics 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 and to decide if you should perform any extra stains on one or more of your organisms.

If you decide to do more stains, use the appropriate Protocols for Stains and Motility testing in the Prtocols section: Special Stains: Endospore, Acid fast, Capsule, and Flagella and 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 an experiment to examine the ability of your isolates to participate in carbon recycling through the digestion of leaf cellulose. Follow the Test for leaf carbon recycling (cellulose degradation) found in the Protocols for determining the roles of soil isolates in the soil

Activity 6
Find and begin the protocol for testing a suspected antibiotic producer, Test for Antibiotic Production, among the tests described in Protocols for determining the roles of soil isolates in the soil

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