BIOL 1406

PreLab 9a.4

How can I induce E. coli cells to absorb plasmids?

Because a plasmid is only a small circle of DNA, it does not contain the enzymes, nucleotides, and other molecules needed to copy itself. In order to replicate, a plasmid must get into a living host cell. To get a plasmid into a host cell, molecular biologists exploit the natural tendency of many bacteria to absorb DNA fragments from the surrounding environment. This process is called bacterial transformation.

At first glance, E. coli might seem like a poor choice for use in genetic engineering experiments because it does not naturally or easily undergo transformation. However, E. coli has many other advantages: its genetics and life cycle are better understood than most other organisms, it is extremely easy to grow, and there are many different strains available for the molecular biologist to choose from. When it was discovered that cold temperature and a high concentration of calcium ions could induce E. coli to absorb plasmids, scientists were able to devise a simple procedure for transforming E. coli cells. Through the use of cold and a high calcium ion concentration, E. coli cells are said to be rendered “competent,” that is, competent to take in plasmids by transformation.

E. coli cells viewed with a scanning electron microscope

 

Your Turn
During lab, E. coli cells will be transformed using pUC18 plasmids.  The basic steps for transforming E. coli cells are listed below. Read the Lab Procedures for Exercise 9A. Then, in the spaces provided, write down which steps will be performed by the instructor and which will be performed by the students.
 First, the E. coli cells are suspended in a CaCl2 solution and placed on ice.  Check your answer.
 After about 20-30 minutes, plasmids and the E.coli//CaCl2 suspension are mixed together.  Check your answer.
The plasmid/E.coli mixture is incubated on ice for another 30 minutes.  Check your answer.
 The plasmid/E.coli mixture is given a heat shock at 37-45 °C for a few minutes, which improves the efficiency of the transformation.  Check your answer.
The plasmid/E.coli mixture is mixed with nutrient broth and placed in a warm water bath for 30 minutes to an hour. During this time, the E.coli cells will proceed with their normal metabolism. This gives the cells a chance to produce some of the proteins encoded by the plasmid DNA, which will allow us to determine which E.coli cells did in fact absorb a plasmid.  Check your answer.

 


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