Biology 212 Genetics
Spring 2007
Lab 7: Conjugation and
Transformation in E. coli
Purpose:
1. To study the processes of gene transfer in bacteria.
2. To use microbiological methods, especially sterile techniques and selective plating, to analyze genetic characteristics of bacteria.
3. To compare the frequency of conjugation and transformation in strains of E. coli.
Reference: Jones and Hartl, Chapter 7
Introduction:
Bacteria are haploid organisms that lack the "true nuclei" of eukaryotic organisms. Since they only have one copy of each gene, typically the genotypes of bacteria can be deduced easily from the phenotypes. Unlike the traits typically studied in eukaryotic organisms, bacteria have few distinct external features and are mainly categorized based on their shape, color (if any), colony properties (clonal growth) and metabolic characteristics. The traits we will examine are based on their ability to synthesize most of their own building blocks (such as amino acids, vitamins, and nucleotides) their flexibility in sugar metabolism (ability to use sugars such as lactose or galactose, in addition to glucose) and tolerance of many antibiotics and drugs (displayed as resistance to ampicillin, tetracycline, or other drugs). Variation among strains of bacteria is typically due to genetic differences that result in the presence or absence of particular metabolic enzymes (proteins).
Bacteria reproduce by asexual means (fission), but are able to transfer genes by several mechanisms: conjugation, transformation, and transduction. Conjugation is the transfer of genes via a conjugation bridge, mediated by a fertility plasmid, the F factor in E. coli. Transformation is the uptake of naked DNA by bacteria; many bacteria are able to take up DNA readily from their environment, although E. coli must first be treated with calcium chloride or other salts. Transduction is the transfer of genes mediated by viruses. This lab will introduce the transfer of genes by conjugation and transformation, in order to demonstrate the relative efficiency of each mechanism.
This lab will introduce you to the genetic variation in two strains of E. coli. Strains are two organisms of the same species that differ slightly in their genetic characteristics. The strains you will be working with are not disease-causing types of E. coli and they possess several additional characteristics that ensure they are unable to grow outside of the "cushy" life in a laboratory dish. The lab will consist of three parts. In the first part, you will examine the behavior of their growth on different selective media to deduce their genotypes. In the second, you will carry out procedures to demonstrate the process of conjugation. In the third, you will perform transformation to transfer genes into the bacteria. Since time is limited, you will be encouraged to divide up the labor, so one member of each pair will carry out conjugation, while one performs transformation. Analysis of the plates to determine genotypes can be done during longer incubation steps or in week 2 of the lab, when we will also go over the other results and the worksheet questions.
Materials:
P20 micropipets
Sterile yellow tips for P20
Sterile pipets (1 or 2 ml, 10 ml)
Sterile large test tubes and test tube racks
Sterile plastic dropper pipets
Sterile 1.5 ml microcentrifuge tubes
Overnight log phase cultures or freshly diluted cultures (1:10 dilution grown for one hour) of the two E. coli strains of bacteria, BB4 and SCSI; incubator set for 37ºC and 150 rpm.
Plates of BB4 and SCSI grown on various selective media
LB medium: aliquots of 50 or 100 ml in 125 ml or 250 ml flasks
LB + ampicillin plates (100 ug/ml); green stripe
LB + tetracycline plates (10 ug/ml)--1 week old or less; red stripe
LB+ampicillin (100 ug/ml) + tetracycline plates (10 ug/ml)--1 week old or less; red and green stripes
Vortex mixers
Microcentrifuges
Water baths or heat blocks at 37ºC
Dishpans with dilute bleach for dirty test tubes, pipets
Beakers for collecting waste plasticware
General methods: Sterile procedure and disposal of bacterial tubes and cultures
When working with microorganisms, "sterile procedures" are used to prevent other organisms from being introduced or growing in place of the study organism. You should use the following sterile techniques throughout.
- When using sterile pipets, don’t touch the ends or body of the pipets with your fingers or lay the pipet down on the table before you use it.
- Use the pipet-aids, green (for 5 or 10 ml pipets) or blue (for 1 or 2 ml pipets) to pump fluid into the pipet. Do not mouth pipet. Do not force glass pipets into pipet aids--can result in injury.
- Pour off used medium and place used glass test tubes and pipets in dishpans containing bleach. Used glass pipets should be placed in the dishpans or used pipet holders.
- Used plasticware (pipets, droppers, microfuge tubes, tips) should be discarded in the biohazard waste beakers on the benches.
- Although this strain of E. coli cannot grow outside the laboratory, all materials in contact with bacteria will be treated with bleach or autoclaved as a precaution.
Part 1: Determine genotypes of strains, BB4 and SCSI
Procedure: Can be done during an incubation step during the conjugation or transformation procedure.
1. Observe the growth of the strains BB4 and SCSI on various selective media, complete the table (question 1), and determine the genotypes (question 2) on the worksheet.
2. Design a conjugation mapping experiment to answer question 3 on the worksheet.
Part 2: Conjugation
Procedure: Note that lab partners should do either part 2 or part 3 and observe their partner's results in week 2.
1A. Label a 1.5 ml microcentrifuge tube, "mix". Mix 0.5 ml strain BB4 with 0.5 ml strain SCSI in the tube. Sterile dropper pipets can be used to pipet the bacterial cultures.
Incubate in a water bath or heat block adjusted to 37ºC for 15 min. During this incubation, the F plasmid is transferred by conjugation from the donor strain (BB4) to the recipient strain (SCSI).
1B. Label a second 1.5 ml microcentrifuge tube, "SCSI". As a no conjugation control, add 1.0 ml of SCSI cells to the tube. Incubate in a water bath or heat block at 37ºC for 15 min.
2. Vortex the tubes from step 1 for 2 min.
3. Centrifuge for 2 min. at 12,000 rpm in a microcentrifuge; NOTE: tubes must be balanced! If needed, balance against a blank tube with 1 ml distilled water.
4. Remove and discard the supernatants (the liquid in the tubes), using sterile dropper pipets.
5. Use a sterile 1 ml pipet or dropper pipet to add 1 ml sterile LB to the cell pellets (material at bottom of tube). Briefly resuspend the pellets in the LB by vortexing.
6. Prepare a 1:10 dilution of the tube from step 1A (conjugation mixture) as follows. Mix 0.9 ml of LB with 0.1 ml of cell mixture (tube from 1A) using glass pipets in a large sterile glass test tube labeled "mix 1:10", vortex.
7. Obtain 2 plates LB+ampicillin+tetracycline and label one "mix" and one "mix 1:10". Plate 0.1 ml of original mixture (from step 1A) on the appropriate plate and plate 0.1 ml of the 1:10 dilution on the second plate. See instructor for demo on how to spread the solution evenly on the plates using a sterile glass spreader.
8. Prepare serial dilutions of the controls from 1B as follows. Obtain 2 large sterile glass test tubes. Label the tubes "SCSI 1:100" and "SCSI 1:10,000". Transfer 9.9 ml sterile LB medium to each tube using a 10 ml glass pipet.
9. Add 0.1 ml of SCSI from step 1B to the tube marked "SCSI 1:100" and vortex. Transfer 0.1 ml of the solution from the SCSI 1:100 DILUTION TUBE to the tube marked "SCSI 1:10,000" and vortex.
10. Obtain 2 plates, LB+ampicillin, and label one "SCSI 1:100" and one "SCSI 10,000". Transfer 0.1 ml of the diluted solutions from step 9 on the appropriate dilution plates. Spread using a sterile glass spreader.
11. Incubate the plates from steps 7 and 10 at 37º C overnight. Your instructor will remove them from the incubator tomorrow and store them in the refrigerator until next week's lab.
Part 3: Transformation
Procedure: Note that lab partners should each do either part 2 or part 3 and observe their partner's results in week 2.
1. Label two microcentrifuge tubes, one "pBR" and one "no DNA". To the first tube "pBR" add 2 ul (80 ng) of pBR322 DNA (@40 ng/ul) using a P20 micropipet. To the second control tube "no DNA", add 2 ul of TE buffer.
2. To each tube add 0.1 ml of competent BB4 cells using a glass pipet (these are stock cultures in calcium chloride in 1.5 ml microcentrifuge tubes located in ice buckets--do not use the liquid cultures used for part 2). Incubate at 4ºC for 30 min. Plasmid DNA binds to the bacteria during this step.
3. Transfer the tubes to a 37ºC water bath for a 5 min "heat shock". DNA is taken into the cells in this step.
4. Add 0.5 ml sterile LB to each tube with a sterile dropper pipet. Incubate 30 min. more at 37ºC. During this incubation, the bacteria will express the ampR gene.
5. Prepare a dilution of competent BB4 cells (to be used in determining the transformation frequency). Obtain 2 sterile glass test tubes and label one "BB4 1:100" and the other "BB4 1:10,000". Transfer 9.9 ml of sterile LB with a sterile glass 10 ml pipet to each tube. Transfer 0.1 ml of competent BB4 cells to the "BB4 1:100" dilution tube using a 1 ml sterile glass pipet. Vortex tube, then transfer 0.1 ml of the DILUTION FROM "BB4 1:100" to the "BB4 1:10,000" dilution tube. Vortex the BB4 1:10,000 dilution.
6. Obtain 2 LB+tetracycline plates. Label one BB4 1:100 and one BB4 1:10,000. Transfer 0.1 ml of the appropriate dilutions from step 5 to each of the plates. Spread on the surface using a sterile glass spreader (see instructor for demo).
7. Obtain 2 LB + ampicillin plates. Label one "pBR", and one "no DNA". Transfer 0.1 ml of the appropriate mixtures from step 4 to each of the "pBR" and "no DNA" plates. Spread on the surface using a sterile glass spreader.
8. Label plates with your initials and incubate in a 37ºC incubator overnight. Your instructor will remove the plates after 24 hours and transfer to a refrigerator until next week's lab.
Name______
Section______
BIOL 212 Conjugation and Transformation Lab Worksheet (20 pts.) due next week
1. Complete the following table after observing the growth of the E. coli strains on selective media. Use + to indicate colony growth and - to indicate no growth.
Strain / Minimal(no proline, no thiamine) / Minimal+ proline / Minimal+thiamine / LB+ampicillin / LB+tetracycline
BB4
SCSI
2. Using combinations of the following symbols, give the full genotypes of the strains
pro+proline prototrophpro-proline requiring auxotroph
thi+thiamine (vitamin B1) prototrophthi-thiamine requiring auxotroph
ampRampicillin resistantampSampicillin sensitive
tetRtetracycline resistanttetStetracycline sensitive
a. BB4 genotype
b. SCSI genotype
3. Suppose you wanted to create a strain that was pro+thi+ampRtetR, outline a conjugation experiment using E. coli strains BB4 and SCSI and indicate the media you would use to select for this genotype.
4. Conjugation frequency
a. Determine the number of colonies observed on the mix plate or the mix 1:10 dilution plate, whichever has a reasonable number of colonies to count. If you count the dilution plate multiply the count by the dilution factor.
Mix plate count (x dilution factor if dilution plate used) = # ampRtetR exconjugates
b. Determine the number of colonies observed on one of the SCSI dilution plates; select whichever has a reasonable number of colonies to count. Multiply your count by the appropriate dilution factor.
SCSI dilution plate count x dilution factor = #ampR recipient cells
c. Estimate the conjugation frequency by determining the proportion of ampRtetR exconjugates/ampR recipient cells.
5. Transformation frequency
a. Count the number of ampR colonies on the "pBR" plate. This is the number of ampR(tetR) transformants.
b. Calculate a transformation frequency based on the number of recipient cells
i) Count the number of tetR colonies from a dilution of the BB4 competent cells
BB4 dilution plate count x dilution factor = #tetR recipient cells
ii) Determine the transformation frequency by dividing the number of ampR(tetR) transformants/tetR recipient cells
c. Calculate a transformation frequency based on input DNA (very useful for recombinant DNA work)
i) Calculate the amount of DNA used
2 ul x 40ng/ul =80 ng
ii) Determine the transformation frequency expressed as # ampR(tetR) transformants/ug DNA
(Note: 1 ug = 1000 ng; be sure to keep your units)
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