Deleting tonB4 and tonB6 in B. thetaiotaomicron

Complex carbohydrates are composed of diverse monosaccharide subunits and various glycosidic linkages; a plethora of substrate-specific enzymes are needed to digest them for primary metabolism. A unique feature of the Bacteroidetes genome is the presence of polysaccharide utilization loci (PULs), a group of localized and co-regulated genes whose products orchestrate the detection, transportation, and digestion of complex carbohydrates. B. thetaiotaomicron (B. theta) is a gram-negative bacteria that resides in the human gut microbiome; its starch utilization system (Sus) has been studied extensively as a model organism for polysaccharide saccharification. ExbB, ExbD, and TonB make up the Ton Complex, a set of proteins which work in tandem with Sus proteins to transport starch oligosaccharides into the periplasm. This project is a deletion of two homologs of TonB, of which 11 homologs have been identified. In-frame deletions of the full gene for each of the 11 TonB homologs suggests that TonB4 is the main TonB protein involved in transport; TonB6 may act as a backup in the absence of TonB4. Previous attempts to design a ΔTonB4/ΔTonB6 strain through homologous recombination have been unsuccessful. In this project, we insert an aTC-inducible copy of TonB6 into a ΔTonB6 strain then delete TonB4 through homologous recombination to answer: is it essential for TonB4 or TonB6 to be expressed?