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Building the horse - Engineering a novel strain for antibiotics-free selection

Our E. Trojan that carries the T4MO gene must remain sensitive to antibiotics while it degrades intracellular indole, otherwise it would mean violation of our original intention since it would introduce another high resistant strain into the population, and decrease the effectiveness of antibiotics.

Nonetheless, molecular cloning needed to be performed for bringing the T4MO gene and the bcr gene into antibiotics-sensitive bacteria. Here we encounter a problem: how to perform cloning without using antibiotics as the selection marker?

Other common and readily available selection methods include auxotrophic and those that utilize toxin-antitoxins interactions, yet they have their own drawbacks in our study of bacterial population dynamics.

Thus we propose the construction of EX, which would be a new bacterial strain that in theory can be selected for plasmid while remains possesses no antibiotic resistance. Selection can be performed under a simple change in physical environment – increasing the incubation temperature. The trick behind the mechanism originates from yeast artificial chromosome. It is the removal of an essential gene nadE and its relocation to a piece of extra-chromosomal DNA that we wish to maintain.