Supplementary Figure 1: Selenocysteine biosynthesis is toxic and incorporation is poorly maintained.
From: Custom selenoprotein production enabled by laboratory evolution of recoded bacterial strains
Dilution plating of RTΔA cells transformed with pRSF-UX-SelA (wild-type or truncated) and p15A-NMC-A (wild-type or C69U-C238U) demonstrates two related phenomena; toxicity of selenocysteine biosynthesis and loss of selenocysteine incorporation. In addition to expressing the NMC-A β-lactamase, p15A-NMC-A confers tetracycline resistance. (a-b) The presence of functional SelA (panel a) drastically decreases cell growth and viability following transformation compared to cells which lack SelA (panel b). Carbenicillin resistance is directly correlated with tetracycline resistance. (c-d) Cells transformed with NMC-A C69U-C238U and functional SelA can grow in the presence of carbenicillin (panel c), while those with a truncated version of SelA do not (panel d). However, carbenicillin resistant cells are 10-fold less abundant than tetracycline resistant cells (panel c) indicating not all transformants are capable of incorporating selenocysteine (at levels sufficient to support β-lactamase activity). Plates are representative from two biologically independent series.
