Exercise 18: Transformation

Introduction

Bacterial cells reproduce through the process of binary fission which produces genetically identical clone daughter cells. This process creates very little genetic diversity in the population. To compensate bacteria have developed several horizontal gene transfer mechanisms. Each mechanism is a unique way to introduce DNA from another cell and integrate it with the host cell DNA in a process called recombination. Transformation occurs when competent bacteria acquire “naked” DNA from the environment and integrate it with the host chromosome. Transduction occurs when viruses accidentally package host DNA into the viral capsid instead of viral DNA. The faulty viral particle can now transfer the packaged DNA to another host cell. Conjugation is the transfer of plasmid or chromosomal DNA through a tube-like pilus. This allows direct genetic exchange between two bacterial cells. These three processes allow for cells within a bacterial population (culture) to share genes and create diversity. Genetic diversity provides the basis for competition and natural selection that leads to evolution.

In this lab students will mechanically induce bacterial cells to initiate transformation through heat shock. Small, circularized DNA molecules called plasmids will serve as the source of “naked” DNA. These plasmids contain specific genes that confer characteristics such as metabolic enzymes or antibiotic resistance (Figure 18.1). Each gene coding region is preceded by a promoter sequence that initiates transcription of the gene. The expression of most genes is regulated by controlling access to the promoters. This lab exercise will demonstrate some of the factors that control expression.

Bacterial cells will be placed in a transformation solution containing a high concentration of calcium. Plasmids will be added as the source of the “naked” DNA. The suspension of cells and plasmids will be temporarily heated to make the cells “competent” for acquiring the extracellular DNA. After the transformation process has been initiated the suspension will be plated on several types of media to identify the cells that have acquired the genes on plasmid.

In this lab students will be using a commercially prepared kit that provides the test cultures, specially designed plasmids and all reagents. Students must follow the manufacturer’s instructions to insure a successful experiment.

Course Intended Outcomes

Describe and perform at least 3 biochemical tests and describe their roles in microbial identification. Interpret results obtained and draw conclusions from biochemical tests performed.

Materials Used

Students will work in small groups. Each group will need the following materials for this exercise.

• Inoculation loops
• Microtubes
• Hot water bath
• Ice bath
• Additional materials and cultures will be assigned by your instructor

Procedure, Day 1

Technique:

1. Refer to Exercise 2 for proper use of instruments and aseptic technique.
2. Wear gloves during all molecular procedures to reduce the risk of introducing DNase enzymes or other contaminants to your samples.
3. Follow the kit instructions provided by the manufacturer.

Sampling/Inoculation:

1. Your instructor will assign the cultures to be used for inoculation by your group.
2. Collect the required materials for the lab.
   • Place all microtubes in the tube rack provided.
   • Bacterial culture tubes are always to be shared with your table group.
3. Clearly label plates (on the bottom side) with your group name or initials, organism name(s), and section day/time.
4. Place inoculated plates in the rack labeled “to be incubated” on the back bench. Always place the plates with lids down and bottoms up.

Procedure, Day 2

1. Collect your group’s plates from the back bench area labeled “last week’s lab”.
2. Follow the manufacturer’s instructions for identifying and confirming the presence of transformed bacterial cells/colonies.
3. Retain your plates for use in the next molecular exercise.

Lab Clean-up

Return bacteria cultures to the front bench after inoculations are complete.

Dispose of plastic microtubes and inoculating loops in the biohazard trash.

Disinfect your benchtop and wash your hands before leaving the lab.

Results and Interpretation

1. Complete the table below.

Critical Thinking Questions

Which gene on the plasmid provided the characteristic that was used to determine if transformation occurred?

Were all genes on the plasmids automatically transcribed after transformation? If not, what must be added to the medium to turn on transcription?