Stanley Cohen and Herbert Boyer transform bacteria with a recombinant plasmid, and Doug Hanahan studies induced transformation.

Hello, I’m Stanley Cohen… Hello, I’m Stanley Cohen…and I’m Herbert Boyer. In 1972, we were at a biology conference in Hawaii. At the time, I was studying bacterial resistance to antibiotics, and Herbert was studying restriction enzymes. We realized we could work together to recombine genes from different bacteria into one DNA molecule. We used genes from two drug-resistant strains of E. coli bacteria — one gene provides resistance to the antibiotic tetracycline, and the other provides resistance to kanamycin. Each gene is carried on a plasmid in E. coli. Plasmids are small rings of DNA that exist independently of the main bacterial chromosome. They can be replicated and passed on to progeny. I named these plasmids p for plasmid and SC for Stanley Cohen. The plasmid pSC101 carries a gene for tetracycline resistance, and pSC102 carries a gene for kanamycin resistance. We grew the bacterial strains that carried these plasmids, and then we isolated the plasmid DNA. We added the restriction enzyme EcoRI to the plasmid DNA. EcoRI cuts each DNA strand off-center of the recognition site, producing short, single-stranded sequences called "sticky" ends. We mixed the cut plasmids and added DNA ligase. Fragments with EcoRI ends are complementary. This allows fragments to recombine with any other. Hydrogen bonds align two sticky ends, until the ligase repairs the sugar-phosphate bonds to create a stable recombinant molecule. Our objective was to combine the kan r gene and the tet r gene on one plasmid. However, other sorts of molecules were ligated together from the parts. Before we could isolate the recombinant plasmid we wanted, we needed a way to get our ligated plasmids into E. coli. Classic experiments by Oswald Avery and his group showed that Pneumococcus bacteria are "transformed" to virulence when they take up DNA from virulent strains. However, natural transformation is a rare event, so we used a chemical method developed in 1970 by Mandel and Higa at the University of Hawaii. This involved mixing the bacteria and DNA in a suspension of cold calcium chloride at freezing temperature. Then, we rapidly raised and lowered the temperature to create a "heat shock." This technique induces the bacteria to take in plasmid DNA. We spread the transformed bacteria onto a culture plate containing tetracyline and kanamycin. Only transformed bacteria containing both kinds of resistance genes could grow in the presence of both antibiotics. This result was consistent with the bacteria being transformed with a recombined plasmid containing both the tet r and the kan r gene. However, it was also possible that some bacteria had been doubly transformed by religated versions of the original plasmids. Restriction analysis showed that some of the colonies had, indeed, been transformed by a recombinant plasmid. We were able to tell which was which when we cut the plasmids and ran them out on an agarose gel. (Roll over each band to see the difference.) We had made the first recombinant plasmid. Several months later we showed that these same methods could be used to recombine genes from eukaryotic and prokaryotic organisms. We inserted a frog gene into an E. coli plasmid. The resulting bacteria produced frog RNA. Hi, I’m Doug Hanahan. As a graduate student at Harvard, I made the first thorough study of induced transformation of E. coli. Here are my ideas about what happens when bacterial cells are transformed using the Mandel and Higa method. During rapid growth, the cell membrane of E. coli has hundreds of pores, called adhesion zones. The cell membrane itself is made up of lipid molecules that have negatively-charged phosphates. Even though the adhesion zones are physically large enough to admit plasmid DNA, the negatively-charged phosphates on the DNA helix are repelled by those on the lipids. Theoretically, Ca 2+ ions from added calcium chloride can complex with the negative charges, creating an electrostatically neutral situation. Also, lowering the temperature congeals the lipid membrane — stabilizing the negatively-charged phosphates and making them easier to shield. Heat shock creates a temperature imbalance on either side of the bacterial membrane, which may set up a current. With the "ionic shield" in place, the DNA is then swept through the adhesion zone. Techniques like transformation and recombinant DNA have created the field of biotechnology. It is now possible to engineer bacteria to make important human proteins like insulin. However, in order to get bacteria to make insulin or any other eukaryotic protein, a number of factors need to be considered. As you learned in Concept 24, genes in eukaryotic animals have introns — sections of noncoding DNA. Bacteria do not have introns in their genes, and so they do not have the biochemical machinery to remove introns. There is also another consideration. Some eukaryotic proteins are processed after translation. For example, insulin is first translated as preproinsulin, which is 108 amino acids long. The first 24 amino acids are the signal sequence that leads preproinsulin out of the cell. As the protein leaves the cell, the signal sequence is cleaved off, leaving proinsulin, which is stored in the pancreas for further processing. Proinsulin folds into a looped structure and disulfide bridges are made between cysteine amino groups spanning the protein. A 33 amino acid stretch is cleaved off leaving the mature insulin protein. Bacteria cannot process preproinsulin into insulin. So, to get bacteria to make usable insulin, a few tricks were used. First, instead of copying the insulin mRNA, DNA was made based on the protein sequence of the two insulin chains — A and B. Then DNA polymerase was used to make the second strand. These are the double-stranded DNA fragments that are inserted into plasmids. Each DNA fragment is inserted into the -galactosidase gene on a plasmid. The plasmids also have the tetracycline resistance gene. The plasmids are then transformed into bacteria. Tetracycline is added to kill off any untransformed bacteria. The transformed bacteria are grown, then the -galactosidase and insulin fusion protein is harvested and purified. The -galactosidase part of the protein is cleaved off and discarded. Finally, the two protein chains are mixed together. Under the right conditions, the disulfide bonds form and usable human insulin has been made from bacteria.

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