The field of molecular biology developed in the 1950s and 1960s from the disciplines of genetics and biochemistry. Studies of bacterial viruses were a principle focus of molecular biology, partly because during the viral life cycle millions of genetically identical individuals, clones of a single virus that have entered an infected cell, are produced. To apply the approaches of molecular biology to the study of plasmids it would be necessary to find a way to isolate the descendants of a single molecule of plasmid DNA. How could this be done?
The First Ingredient: E. Coli
Earlier work by others had shown that treating Escherichia coli (E. coli), the bacterial host for the plasmids we were studying, with a simple chemical, calcium chloride, made their walls permeable so that they could take up DNA. However, prior efforts to grow E.coli cells that had their genetic properties changes or in genetic language, transformed by this DNA, were unsuccessful.
In late 1971 Leslie Shiu, a first year Stanford medical student carrying out research in my lab, found that bacteria treated with calcium chloride took up plasmid DNA -- producing offspring that contained cell propagating plasmids. Genes on the plasmid made these transformed bacteria resistant to antibiotics, allowing them to survive and reproduce when exposed to the drug. Since all the descendants of a transformed bacterial cell contain plasmids identical to the one that originally had entered the cell, it was now possible to make multiple clones of individual molecules of plasmid DNA.
The ability to clone plasmid DNA provided an important ingredient for genetic engineering, but other ingredients were also required. One of the goals of my research was to map and characterize plasmid genes. To do this I wanted to be able to take plasmids apart and put them back together again one segment at a time.
In nature various genes have been joined to the replication machinery of plasmids by natural recombination mechanisms working within cells. Potentially the plasmid replication regions might also be able to propagate DNA segments attached to them outside of living cells. But how to attach the fragments?
  
|
Winding Your Way Through DNA Resource Book Index
