Advanced Bacterial Conjugation

Alright, this can be kind of confusing, but you should do okay....First of all, what is conjugation? In the shortest number of words possible, it's bacterial sex. Another, more scientific way of saying that is two bacteria exchange genetic information, usually in the form of neat little rings of DNA called plasmids. And what are plasmids, you ask? Well, they are small DNA rings that usually have 4 or 5 genes (but can have up to 25). Each of these genes codes for some sort of protein, just like chromosomal DNA can. However, these rings are completely independent of the bacterial cell's DNA. It can replicate on its own. They can also be exchanged from one bacteria to another through conjugation.

Plasmids are NOT needed for bacterial survival. In normal situations, most bacteria will never need a plasmid. But, plasmids can make a bacteria's life easier. For example, the reason why you HAVE to take all of the antibiotic you are prescribed is that plasmids exist that can make a bacterium resistant to that antibiotic. Once resistant, that bacteria can divide, and divide, and divide, until you have a whole colony of antibiotic resistant microbes! Yipes! If all of the antibiotic is taken, the risk of the bacteria developing resistance is reduced.

I guess you can think of plasmids as a sort of evolution for bacteria. Those that have them are a step ahead of those that don't.

So, now you know what a plasmid is. And now you get to use that knowledge! There is a plasmid, call the F-plasmid (F for fertility) that will allow a bacterial cell to form a pili. This pili is a hollow tube that will allow bacteria to pass plasmids from the "male" cell to the "female" cell. This is how antibiotic resistance is often propagated. The resistant plasmid can be passed--even from specie to specie!--to another cell, and that cell will then be resistant.

Now, you will need to different strains of E. coli. One should be resistant to streptomycin (labeled Strain I), and another that is resistant to ampicillin and naladixic acid (labeled Strain II). These should be grown in pure cultures overnight in LB broth. The LB broth should have NO antibiotics in it!! This is important!

The different antibiotic resistances are either on a chromosome or a plasmid. For ampicillin (amp) resistance, the gene is present on the plasmid. For streptomyocin (str) resistance, the gene is present on the chromosome. The gene for resistance against naladixic acid is also on the chromosome.

First Lab Day: This is the procedure in which you will test each strain of bacteria to see if they are resistant to the antibiotic they are supposed to be resistant to, and to make sure that they are not already resistant to the other antibiotic.

Procedure for Day One:
1. You should have 5 different agar plates. They should be: LB agar (no antibiotics), LB agar and streptomyocin, LB agar and ampicillin, LB agar and both amp and str, and LB agar and naladixic acid. On the bottom of each plate, a line should be drawn down the middle, and one side labeled "I," and the other side labeled, "II." This is what it should look like.
2. Using aseptic technique, carefully place a SMALL amount of Strain I on each plate, on the side designated "I." This should be done for EACH plate, and ONLY on the side marked "I."
3. Using a NEW loop (or use the same one after it has been properly cleaned. See the above link for aseptic technique), repeat step 2 with Strain II. This should be done for EACH plate, ONLY on the side marked "II."
4. Once the small amount of liquid is dry, invert the plates (so that the agar is on the TOP), label the plates according to names or groups (if they are going to be incubated together), and place in the incubator at 37 degrees Celsius overnight. If you do not have an incubator, place them in a dark place that will be undisturbed, and let them sit for 2 days or so.
5. Your students should now predict what type of growth will occur on each plate. For example, growth should be expected for BOTH strains on the plain LB agar. There are no antibiotics in the agar, so both strains should grow well. Have them make a table, showing their predicted growth for each plate for each strain, as well as a chart for showing actual growth for each plate for each strain.

Second Lab Day: This is the day where growth of the plates will be observed, and where the procedure for conjugating the strains will be done.

Procedure for Day Two:
1. Look at the plates and record the results of the growth on the chart the students have created. If there are abherent growth patterns, perhaps the plates should be redone, or the original colony might have been contaminated. In this case, more culture should be regrown and the procedure redone.
2. A mating plate should now be prepared. This plate should have both streptomycin and ampicillin. The lines should be drawn on the bottom of the plate, and each section labeled appropriately, like this. Using sterile loops, a drop of each strain should be put in the separate area. Then, another drop of each strain should be placed near each other on the "mating portion" of the plate (Region III). This is what the plate should look like.
3. When the drops are on the plate, combine the two strains ONLY on Region III. The liquid should them be let dry, the plate inverted (agar side up), and the plates labeled and incubated as above. This is what the plate should now look like.
4. Have the students record their expected growth in a chart.

Third Lab Day:

This is the day the students will observe the mating plate, and will take individual colonies off of the plate and test for the antibiotic resistances.

Procedure for Day Three:
1. Look at the mating plate and record observations. There should be some growth on Region III, but there should be NONE in Region I & II. This is because the strains should be susceptable to one of the antibiotics in the agar.
2. Prepare four plates as shown here. One plate should contain only streptomycin, another only ampicillin, another with both str and amp, and one with naladixic acid.
3. Here's the tricky part: Using aseptic technique and a sterile loop, take ONE colony (that will be one "dot" of bacteria), and touch it to EACH of the plates in the first square. This will be Colony #1, and should ONLY be placed in the box marked #1!! Using another STERILE loop, take another colony (try to space out the colonies...do not take them all from the same place, and try to only get ONE colony. Don't try to take samples from large globs of bacteria, for these are probably more than one colony, and can skew the results), and touch it to each of the squares marked #2. This will be Colony #2, and should ONLY be placed in the box marked #2!!! Repeat this for each box, each time using a sterile loop and a different colony.
4. Incubate for 24 hours in an incubator, and record the expected growth for each plate and strain.


Afterwards: Record observed growth and answer the following questions regarding the procedure and results.

1. Why did strain I not grow on the LB/amp plate?
2. Why did strain II not grow on the LB/str plate?
3. Why did neither strain grow on the media containing both antibiotics?
4. What type of growth was expected on the mating plate?
5. If growth is present on the plate containing ampicillin and streptomycin, does this support the prediction of recombination of antibiotic resistant genes into a new strain? Explain.
6. Based on the results obtained in this study, can one determine whether the str gene from Strain I was transferred to strain II or if the amp gene on the plasmid was transferred from strain II into strain I? Explain.

And here are my results.