Dear Drew Hansan!

Here is my answer. Please check it and make comments and/or questions.

Sincerely yours!

Dr. Bratko Filipič

Email: [email protected]

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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

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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

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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.

Generally there are two sets of elements you need to consider in order to conjugate a plasmid. First you need all the genes that express the conjugation machinery, and secondly the plasmid needs the recognition site for transfer that the conjugation gene products can act upon. Many plasmids are self conjugable because they carry everything, but these are quite large due to the number of genes needed and generally not well suited for cloning experiments. These days most people use a plasmid carrying the mobilization site and then a helper plasmid (either integrated in the chromosome or existing as a plasmid) to supply all the needed proteins. So your donor strain would generally be a strain carrying such a helper plasmid. The recipient strain would be one with a unique selection so that you can select for it and against the donor strain (along with the marker on the plasmid you wish to conjugate). Using a plasmid to carry a transposon does not change this. The specifics as to which strain will depend upon the plasmid you are trying to conjugate.