Step Four: Selecting Bacteria with Recombinant Plasmids

Now the importance of the antibiotic resistance and LacZ genes in the gene cloning process can be appreciated. The strain of E. coli bacteria used in gene cloning will be susceptible to all antibiotics. The only way these bacteria will be able to grow on media that contain the Amphicillin antibiotic is for them to acquire resistance when they are transformed with a plasmid. If an antibiotic-sensitive bacteria is transformed with the nonrecombinant plasmid, the bacteria could grow on Amp and produce a blue color. Cells that can grow will divide and divide and form colonies. If the bacteria lack a resistance gene, the antibiotic will either kill the cell or prevent it from dividing. Either way, no colonies will form on the media plates. So how does a gene cloner identify bacteria that have a recombinant plasmid? This is where the LacZ gene is important. When cloning genes with a plasmid that has the Amp resistance and LacZ genes, the insertion of new DNA occurs in the LacZ gene. Because new DNA will combine with the plasmid in the LacZ gene, recombinant plasmids will have an inactive LacZ gene. Bacteria colonies that have recombinant plasmids will be white, bacteria with the original, nonrecombinant plasmid will be blue. Therefore, the gene cloner will select white colonies from their plates and assemble a gene library that hopefully has the gene they are interested in.

Fig.13: Bacteria that are sensitive to antibiotics (A) can be transformed to grow on media with antibiotics and form a colony (B). (Image by D. Lee)

Fig.14: Colony color is used to determine if bacteria in the colony have a recombinant plasmid (white) or a nonrecombinant plasmid (blue). Bacteria with a recombinant plasmid are white because the gene of interest has interrupted the color gene that the plasmid contains. (Image by D. Lee)

To summarize, the gene cloning process with Amp/LacZ plasmids would proceed as follows:

1. Cut the plasmid and recombine with the cut DNA that you want to clone.

2. Transform antibiotic susceptible bacteria with the mixture of recombinant and nonrecombinant plasmids.

3. Plate the bacteria on Amp/X-gal media, grow overnight. The next day the gene cloner will observe a mixture of blue and white colonies on the plates. (What bacteria will contain a cloned gene, the blue or the white?)

4. Select the white bacteria colonies.