Lab Report on Gene Transfer Linkage Mapping of Bacteria

Interchange of genetic materials between cells is necessary for the evolution of life. Eukaryotic cells and bacteria realize gene shift in a variety of ways. Normally, when a cell undergoes the meiosis, genetic materials have been interchanged among chromosomes and homologous at the first meiotic division. This procedure births new gene combination in gametes not found in a diploid progenitor cell.

Genetic recombination occurs in several ways. This happens by gene shift, transduction or conjugation. The shifting process occurs when bacteria gather some pieces of DNA from the environment (Harrison and Micheal,264). On the other hand, transduction is a process where bacteria phage takes DNA from the environment (Harrison and Michael, 264). On the other hand, transduction is where the bacteria phage takes DNA from one bacterium cells and injects it into other bacteria where such DNA will be incorporated into chromosomes. Also, conjugation happens when a bacteria transfers DNA to another bacteria. This experiment aims to investigate the process of conjugation stated well how it happens, the procedures as well as other things necessary for it to be successful.

Conjugation process was first carried out by scientist, William Hayes in the year 1952. He showed that bacteria could transfer DNA to another one’ cell and this process is said to be directional as one cell acts as the donor while another acts as the recipient. Conjugation requires the cell to cell contact for it to occur successfully.

The main purpose of this experiment is to determine the locations of various markers on the E.coli chromosomes through performing a gradient of transfer. Primarily several Hfr stains were used to locate the approximate positions of the gene marker after which a gradient of transfer exercise is carried out to place the map positions for the unknown marker.

Methods and Materials

The experiment was done in pairs. Two different strains of E.Coli: CSH121 (Hfr) and CSH125 (F^-). In the first week of the experiment, a mating was set up between the two stains and serial dilutions were plated onto selection Petri dishes lacking leucine but containing streptomycin.

On the second week, we picked isolated colonies from week one Petri dishes then inoculated them onto several different negligible media Petri dishes. One did not have adenine; one was containing tetracycline, one without tryptophan, one without histidine and one without arginine. On the third week, we checked the growth of the dishes to find out the result of the experiment.

Materials for week one experiment

Striker

Biohazard bench-top waste container

Test tube rack

Three sterile capped 17 x 100 test tubes

Two micro-centrifuge tubes with 1ml each of 3-hour culture of CSHI21 and CSH125

Lab marker

P-1000 pipette

P-200 pipette

Box of sterile pipette tips (p1000, p200)

5 Petri dishes (C Petri dishes minus Leu-)

Microcentrifuge tube rack with closed sterile microcentrifuge tubes

Bacterial spreader

Beaker with 95% ethanol

Labeling tape

2-mL of sterile LB broth in 5 ml test tube

1-mL of sterile minimal media in sterile microcentrifuge tube

Water bath set at 37°C with test tube rack for test tubes

Empty test tube rack to carry tubes to the shaker

Materials used in week two

Small beaker with sterile toothpicks (50/person)

Biohazard waste container with autoclave liner

Bench-top waste container with biological autoclave bag

14 Petri dishes: 4-C, 2-H, 2-A, 2-E, 2-G, 2-LB+Tet

Sharpie

Labeling tape

Bins for student's dishes for incubator

Students’ Week 1 Petri dishes

Students’ Week 2 Petri dishes

Method for Week 1 experiment: Mating

Actively growing cultures of both strains were started for the students by the laboratory staffs. Several micro-centrifuge tubes that had this liquid culture of each strain were placed in TA bench for students to pick and return to their benches with it and then light a Bunsen burner.

Using a marker, three pieces of tapes were labeled with initials as;

a. CSH121/CSH125

b. CSH121 (control)

c. CSH125 (control)

Then three pieces of tape were placed on different glass test tubes. Then lid of the tube containing CSH121 microcentrifuge tube was then opened carefully while my lab partner was removing and flaming mouth of the test tube with the label CSH121.

P200 pipette and the sterile pipette were used to transfer 200 μL of CSH121 to the test tube. The mouth of the glass test tube was flamed before placing the lid. This procedure was repeatedly done until 200 μL of CSH121, and 200 μL of CSH125 was added to the test tube marked CSH121/CSH125 and tubes labeled CSH125 and CSH121/125 respectively.

These tubes were then placed in a 37°C water bath for about one hour. After this, 2mL of LB broth were added to each tube using same sterile transfer technique. The tubes were then placed in 37° C shaker water bath for 45 minutes.

Three Petri dishes containing streptomycin and no leucine (Leu-) were labeled during incubation.

Two more Petri dishes were labeled CSH121 and CSH125 for control purposes.

Also during the incubation stage, four microcentrifuge tubes were labeled 1:10, 1:100, 1:1,000 and 1:10,000. 90 μL of minimal media was then added to each tube.

For the control experiment, minimal media were added to the tubes labeled CSH121 1:10 and 1:100 and CSH125 1:10 and 1:100.

After incubation, sterile transfer technique was used to remove ten μL of the CSH121/CSH125 mix and then it was added to the microcentrifuge tubes labeled 1:10.

Ten μL of the bacterial suspension was then transferred from 1:10 tube to the 1:100 tubes. This is a process called serial dilution. Bacteria spreader was used to spread the bacteria over the surface of the media.

Bacteria spreader was dipped into a 95% ethanol and then flamed before plating each of 1:1,000, 1:10,000 dilutions and the controls.

Petri dishes were incubated for in 37°C for two days after which it was placed in 4°C refrigerator waiting for the next lab period.

Week 2 experiment: gradient of transmission

Petri dishes were retrieved from TA benches, and then the amount of growth on each dish was estimated and recorded.

A total of 100 colonies from the CSH121/125 dish were picked. There were three pages with grids for picking colonies. Dishes were to be placed on appropriate grids. The toothpick was used to pick a single colony from CSH121/CSH125 dish.

After picking the number of colonies required, these dishes were stack on top of each other and then placed in a 37°C incubator for two days.

Week three was only for data analysis

Result

Part 2: Gradient of Transmission (Week 2)

Growth on Petri dishes:

CSH121/CSH125 1:100……3...

CSH121/CSH125 1:1,000…4……

CSH121/CSH125 1:10,000……4…..

CSH121…………………2………

CSH125………………2…………

Part 3: Data Analysis (Week 3)

Number of colonies on dish#

1. Leu- __50____

2. His- __50____

3. Arg- ___50_____

4. Ade- ___50___

5. Trp- __50_____

6. Tet __50____

7. Leu- __50____

Discussion

The number of colonies for all the dishes was equal according to the grid to which they were placed. It is because the transfer of genes occurs on each colony the same. Needless to say, one bacterium requires one donor for the gene (Domingues et al. 23). This means that the colonies were paired and thus the successful transfer of the gene or what is called conjugation.

A control experiment was used to set up a baseline that would be used to compare results of the main experiment. It rules out the environmental variables that might affect the outcome of the test. It was important to be able to obtain single colonies in this experiment so as to ensure that one is only working with a single genetic makeup (Fisher, 24). Each separate colony is derived from individual bacteria, and all colony expansion is on a selective antibiotic plate and should contain one’s plasmid.

The approximate location (in minutes) for Arg, Ade, Trp, and Tet are 2, 8, 15 and 20 respectively.

Given another Hfr strain with CSH119 that has all the same markers as CSH121 an experiment can be designed to locate approximate location and orientation of the origin in CSH119 as follows;

Hfr strains with these markers are mated with F&ndash strain possessing the markers. The time of entry for each marker will be recorded (Harrison and Michael, 262). Based on the result obtained, a map of Hfr chromosomes will then be drawn to determine the position and orientation of the origin of CSH119.

Work Cited

Domingues, Sara, et al. "Natural transformation facilitates transfer of transposons, integrons and gene cassettes between bacterial species." PLoS pathogens 8.8 (2012): e1002837.

Fisher, Robert A. Optical phase conjugation. Academic Press, 2012.

Harrison, Ellie, and Michael A. Brockhurst. "Plasmid-mediated horizontal gene transfer is a coevolutionary process." Trends in microbiology 20.6 (2012): 262-267.