Saturday, 26 October 2019

Bacterial Transformation

Bacterial Transformation

Learning Objectives

Each student should be able to
1. Describe the process of transformation
2. Perform a DNA extraction procedure
3. Demonstrate the presence of competent bacterial cells in a bacterial culture

Materials per Group of Students

24-hour nutrient broth culture of Bacillus subtilis strain SB 100 (ATCC 29056) 24-hour nutrient broth culture of Bacillus subtilis wild type (ATCC 6051) lysozyme solution (2 mg/ml) 1 glucose–minimal salts agar plate containing tyrosine and histidine (see appendix J) 1 glucose–minimal salts agar plate containing tyrosine and tryptophan 2 glucose–minimal salts agar plates containing tyrosine, tryptophan, and histidine 1 glucose–minimal salts agar plate containing histidine sterile test tubes sterile phosphate buffer (0.1 M, pH 6.2) chloroform sterile centrifuge tubes sterile toothpicks centrifuge ice-water bath sterile distilled water 2 sterile 250-ml flasks 50-ml Difco Antibiotic Assay Medium 3 in a 250-ml flask 15 ml of MM1 medium (see appendix J) 15 ml of MM2 medium 5-ml pipettes with pipettor 1-ml pipettes with pipettor thermometer glass spreader 70% ethanol Bunsen burner

Why Is the Above Bacterium Used in This Exercise?

In this exercise, the student will learn how to form recombinant bacterial cells by the transformation. To accomplish this objective, the authors have chosen a wild-type strain of Bacillus subtilis as the donor strain and the SB 100 B. Subtilis strain as the recipient. As a result of multiple mutations, SB 100 B. Subtilis is an auxotroph that requires the amino acids tyrosine, tryptophan, and histidine for growth. It has lost the ability to synthesize these amino acids. The wild-type strain of B. Subtilis can synthesize these amino acids and grow on glucose–minimal salts medium lacking them. By mixing DNA extracted from the wild type with viable cells of the SB 100 auxotrophic strain, transformants will be produced that now have the ability to grow in the absence of tryptophan or tyrosine.

Principles

Transformation is the uptake by a recipient bacterium of a naked DNA molecule or a fragment from a culture medium and the incorporation of this molecule (or fragment) into the recipient chromosome in a heritable form. In the natural transformation of the DNA comes from a donor bacterium. The process is random, and any portion of the genome may be transferred between bacteria. When are bacteria lyse, they release considerable amounts of DNA into the surrounding environment? If a fragment contacts a competent bacterium, one able to take up DNA and be transformed, it can be bound by the cell and taken inside. Transformation may be an important route of genetic exchange in nature.

In this introductory transformation exercise, a wild-type strain of Bacillus subtilis will be used as the donor strain and B. Subtilis strain SB 100 as the recipient strain. This recipient strain is an auxotroph (a mutated bacterium that lacks the ability to synthesize an essential nutrient and must obtain it from its surroundings) that requires the amino acids tyrosine, tryptophan, and histidine to grow because it has lost the ability to synthesize these amino acids. The wildtype strain, however, can synthesize these amino acids and grow on a glucose–minimal salts medium lacking these amino acids. By mixing DNA extracted from the wild strain with viable bacteria of the auxotrophic strain, you will 
(1) look for transformants that have gained the ability to grow in the absence of tryptophan, and  
(2) look for those which have acquired the ability to synthesize tryptophan and/or histidine and no longer require that the particular amino acid be supplied to the medium.

Procedure

First Period

Preparation of Donor DNA

1. Aseptically transfer 5 ml of the wild-type Bacillus subtilis to a centrifuge tube and centrifuge at 5,000 g for 10 minutes.
2. After centrifugation, using a new pipette, discard the supernatant and resuspend the bacteria in 5 ml of sterile phosphate buffer.
3. Add 0.05 ml of the lysozyme solution and incubate (with gentle agitation) for 30 minutes at room temperature.
4. Cool the centrifuge tube to 5°C in an ice-water bath.
5. Add 5 drops of chloroform to lyse the bacteria whose walls have been weakened by lysozyme digestion. Agitate the tube to mix the chloroform and medium. This is the source of transforming DNA for the second period of this procedure. This preparation can be stored at 5°C for several days.

Bacterial Transformation
Figure.1 Grid Pattern for a 90–mm Petri Plate.

Bacterial Transformation

Bacterial Transformation

Preparation of Competent Bacteria

1. Aseptically inoculate a culture of B. Subtilis SB 100 into a 250-ml flask containing 50 ml of Difco Antibiotic Assay Medium 3.
2. Incubate for 24 hours at 37°C. The culture should be gently agitated.

Second Period

Preparation of Competent Bacteria (continued)

1. Harvest the bacteria by centrifugation at 5,000 g for 10 minutes and resuspend them in 15 ml of MM1 medium.
2. Add 12.5 ml of the resuspended culture to a sterile 250-ml culture flask and incubate with shaking for 5 hours.
3. Harvest these bacteria by centrifugation and dilute them fivefold by resuspending in 15 ml of MM2 (transformation medium). These are the competent bacteria that will be used in the next part of this experiment.

Transformation of Competent Bacteria

1. Aseptically transfer 0.9 ml of the competent bacteria (B. Subtilis SB 100) into a sterile test tube. Carefully, without adding any chloroform, add 0.1 ml of the DNA solution (the donor DNA) prepared from the wild-type B. Subtilis. The white DNA layer is at the chloroform-water interface.
2. Incubate the test tube at 37°C for 30 minutes with gentle shaking.
3. Pipette 0.1 ml of the transformation mixture onto glucose–minimal salts agar plate containing tryptophan, tyrosine, and histidine. Spread the bacteria with a flame-sterilized glass spreader. Label the plate with your name, date, and amino acid mixture.
4. Dilute the transformation mixture 1/10 using sterile distilled water. Pipette 0.1 ml of this dilution onto a glucose–minimal salts agar plate containing a mixture of tryptophan, tyrosine, and histidine. Spread out the bacteria as before. Label with your name, date, and amino acid mixture.
5. Incubate the plates at 37°C for 48 to 72 hours.


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