The Bio 6B lab explores bacterial plasmids and operons through a set of connected experiments over multiple lab days. The concepts behind these labs are presented in a set of related pages on this site:
- Operons. Basics of gene expression; both a lecture topic and a lab topic.
- . Background for the labs.
- Plasmid Lab Overview. Lays out the experimental approach for the Conjugation and pGLO labs, which share some of the same methods.
- Bacterial Conjugation. Overview and technique.
- pGLO Background. Including the arabinose/GFP operon.
In addition, there are multiple pages for the experimental methods, which you'll find in the menus.
In Bio 6B, you will perform a multi-step examination of bacterial conjugation, which is the transfer of plasmid DNA from one cell to another. This experiment will be spread over six lab days and will include these steps:
- Observe conjugation plates; perform total nucleic acid extraction
- Total nucleic acid gel; start cultures for plasmid purification
- Plasmid DNA purification
- Plasmid restriction digest
- Plasmid gel
The steps of the conjugation lab will overlap with the pGLO lab.
Bacterial conjugation is the transfer of a copy of a plasmid from one bacterial cell to another. In this experiment you'll allow conjugation to occur, then verify that it occured both by checking for the transfer of antibiotic resistance from one cell to another and by directly examining the cells' DNA.
Before you begin this experiment, read the bacterial strains and plasmids page.
How the experiment works
You’ll start with liquid cultures of with two strains, or genetic types, of E. coli:
- E. coli S17 containing the plasmid pARO180. The plasmid has a gene providing resistance to ampicillin, but the S17 strain isn't resistant to streptomycin.
- E. coli HB101 with no plasmid. The chromosome contains a gene providing resistance to streptomycin, but without a plasmid, this strain isn't resistant to ampicillin.
You'll mix liquid cultures of these two strains to make a new liquid culture:
- "Mix." The two strains mixed together in one culture tube. Some of the cells are expected to conjugate, meaning that the plasmid pARO is copied from S17 to HB101. The resulting conjugated HB101 cells will contain both the amp resistance gene and the strep resistance gene, so they will be able to grow on plates containing both amp and strep (they're double resistant). Not all the cells will conjugate, so the "mix" liquid culture will actually contain both original strains plus the new strain created by conjugation. Only the conjugated cells will grow on amp + strep plates.
Each original strain is resistant to one antibiotic, but not to both, so you should be able to determine whether conjugation occurred by looking for colonies that grow on amp & strep together. As with any experiment, it's important to understand the controls:
- Negative controls: S17 doesn't grow on strep (or A+S); HB101 doesn't grow on amp (or A+S). If either strain already grows on both antibiotics, you will have no way of knowing whether conjugation occurred. If one of the starting strains does grow on both antibiotics, it could either be because it's the wrong strain (already double resistant), or the cultures were cross-contaminated, or because the plates don't contain the antibiotics they're supposed to contain.
- Positive controls: S17 grows on amp, HB101 grows on strep. If you don't get conjugated cells growing on A+S, you'll need these controls to help you understand what happened; these cultures will tell you whether the starting strains actually had the expected resistance.
- Positive controls: The mix culture grow on amp alone and on strep alone. If you don't get conjugated cells growing on A+S, it could be because the cells failed to conjugate, or because you failed to mix the two together when you were supposed to (which has happened many times in Bio 6B!). If both these positive controls grow as expected, it proves that you did actually add both strains to your "mix" tube.
Note that each control serves a specific purpose. When I ask 6B students what a control is, they usually say that it's something you compare your experimental results to. This is true, but it's not meaningful; the question is, what do you learn from the comparison? Also, there are controls in this experiment, but there are no control groups. A control group would be used if you repeated the experiment multiple times with different individuals.
The experimental procedure is very simple; you simply need to mix the two bacterial strains together, let them conjugate, and then spread them on plates with antibiotics to see if they grow. The antibiotic plates will act as a selective medium, allowing some bacteria to grow while others can't.
The procedures shown here are only the beginning. After you perform the conjugation part of the experiment, you'll go on to analyze the DNA directly in a series of additional experiments.
Procedure for day 1: conjugation
- Test tube rack for glass culture tubes
- Your liquid cultures of E. coli S-17/pARO180 (the plasmid donor) and HB101 (the plasmid recipient) in glass culture tubes. These are the cultures you started in the previous lab period and left in the shaker incubator.
- One empty sterile glass culture tube
- Pipetters, tips, and beaker for waste
- Plates: 3 LB with ampicillin, 3 LB with streptomycin, 3 LB with both amp and strep.
- Inoculating loop (metal)
- Optional: plate spinner
Perform the conjugation and plate out the cultures
Warning: glass culture tubes don't seal tightly! They are designed to allow air circulation. Keep the tubes upright by placing them in a test tube rack.
When using glass culture tubes, label them by putting a piece of tape on the tube and writing on that. Don't write directly on the tubes or caps, because we re-use them. Don't put your tape label on the cap, because you might mix up the caps.
- Label your empty culture tube "mix." Pipet 0.5 ml of E. coli S-17/pARO180 and 0.5 ml of E. coli HB101 into this tube, mixing the two cultures so they can conjugate.
- Let all 3 culture tubes (S-17, HB101, and mix) sit in the rack for 30 minutes, allowing conjugation to occur. The cells will take care of the conjugation on their own, but you need to give them enough time to complete the process.
- Label your plates. You will need 9 plates, as listed above and shown in the results table below. For the 3 amp plates, label them S17, HB101, and mix. Also add your name and the date. Label the plate containing the agar, not the lid (you might mix up the lids). Repeat this process for the strep plates and the amp+strep plates.
- Turn on your Bacti-cinerator when you have about 5 minutes left on your conjugation. It needs to warm up until the inside of the tube is glowing orange.
- Swirl the S-17 culture gently, and pipet 10 μl of the liquid onto the S17/amp plate. Be careful not to spatter, and don't reuse the pipet tip.
- Sterilize your inoculating loop with the bacti-cinerator and use it to spread the 10 μl of liquid culture as evenly as possible, all over the plate. You can use a plate spinner, but it's not necessary. Sterilize the loop again immediately after spreading.
- Repeat the pipetting and spreading steps for all your plates. You should end up with each of your cultures (S17, HB101, and mixed) being cultured on three kinds of plates: amp, strep, and amp + strep.
- Put the plates in the plate incubator to grow until the next lab period.
- Once you've made your plates, you don't need the liquid cultures any more. Peel off the tape labels and put the tubes in a tube rack in the used glassware tub on the cart. Keep these tubes upright; don't lay them down anywhere, ever. Don't pour out the liquid culture medium; the tubes will be autoclaved to kill the cells.
That's it for today; you will see your plate results in the next lab.
Procedure for day 2: plate results & total nucleic acid extraction
Observe and record your plate results. Do your results prove that conjugation occurred? Which control plates are necessary to prove that conjugation occurred? What are the other controls for?
Here are the expected results (as shown in the diagram at the top of the page):
"+" means that colonies grow; "-" means that they don't. In principle, growth vs. no growth is simple, but plate results can sometimes be difficult to interpret. The antibiotics don't necessarily kill the cells, they only slow their growth. If you put a lot of non-resistant cells on a plate, you might see some living cells after incubation. For this experiment, you should look for the formation of new colonies: isolated dots of bacteria that form when a single cell grows into a large blob.
Satellite colonies are another potential problem with this procedure, specifically in the mixed culture. That tube could potentially contain three different strains: S17/pARO180, HB101, and conjugated cells (HB101 with pARO180). All the cells containing the pARO180 plasmid have the ampicillin resistance gene, so they are producing and secreting β-lactamase, the enzyme that breaks down ampicillin. If you put some of that culture liquid on the A+S plate, the excreted β-lactamase might break down the amp in the plate, allowing the non-amp-resistant HB101 cells to grow. To be sure that conjugation occurred, you need to see isolated colonies on the A+S plate, spread far away from the place where you initially transferred the culture liquid to the plate.
If you're unsure whether conjugation occurred, restreak some colonies from the A+S Mix plate onto a new A+S plate. If isolated colonies grow, then they're resistant, meaning that conjugation occurred. As positive controls, restreak some of the same colonies onto amp alone and strep alone. If conjugation failed, you'll probably find that these colonies grow on strep and not on amp.
We don't have the satellite colony problem with strep, because strep-resistant cells tolerate strep, but they don't destroy it.
After studying and recording your plate results, extract total nucleic acid from your colonies, and save some plates as described in the instructions.
Terms and concepts
- Ampicillin & streptomycin
- Antibiotics and antibiotic resistance
- Controls in this experiment: positive and negative
- Liquid and plate media
- Satellite colonies
- Selective media
- Compare and contrast ampicillin and streptomycin in this experiment. How do these antibiotics act? What are the mechanisms of resistance? Where are the genes located (chromosome or plasmid)? Why do we use antibiotics in this experiment?
- In this experiment, which plates are controls? Why do we need controls?
- How do you know whether conjugation occurred? How can you tell the difference between conjugated cells and the original plasmid donor?
- If you want to culture bacteria that contain the plasmid pARO, which plates could you use?
Conjugation in Escherichia coli: A laboratory exercise Phornphisutthimas et al., 2007. Biochemistry and Molecular Biology Education. I based the Bio 6B lab procedure on this article, but found it necessary to change some of the procedures in order to get results comparable to those presented in this article.
Transfer of Conjugative Plasmids and Mobilizable Plasmids in Gram-Negative Bacteria. Kaiser & Suchman, 2013. ASM. Conjugative plasmids and mobilizable plasmids are slightly different things; pARO180 is a mobilizable plasmid. This page includes an old-fashioned graphic that shows the difference.
Mobility of Plasmids. Smillie et al., 2010. Microbiology and Molecular Biology Reviews. Detailed background on the characteristics of plasmids that make them mobilizable.