Team:HKUST-Hong Kong/overview.html

1.1. Theory – how to select?

The E. trojan is a synthetic E.coli strain that is engineered to lack an essential gene, nadE, in its genomic DNA. To survive, this strain has to rely on a pre- introduced plasmid (pDummy) bearing the essential gene; thus forcing the bacteria to maintain the plasmid until an alternative source of nadE gene is present. The pDummy, however, has been designed to have a temperature- sensitive origin of replication which would cease to function if the bacterial cells are incubated under higher incubation temperatures (>42ᵒC???).

For sub-cloning purposes, an E. trojan – compatible vector plasmid is designed. This carrier vector, like the pDummy, contains the nadE essential gene. Once a gene of interest is inserted into this vector, the plasmid can be transformed to the E. trojan for amplification. Incubating the transformed bacteria at a temperature high enough to inactivate the heat sensitive replication origin of the pDummy would result in pDummy loss, making it necessary for the cells to retain the insert- bearing pCarrier for survival. Bacterial cells that do not take up the pCarrier and its insert would be deprived of the nadE gene product and die; while those who do would survive and continue dividing.