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The bacterial pellet was then resuspended in 800 L of GMML liquid medium (M9 salts, 1% glycerol, 1 mM MgSO 4 and 0.1 mM CaCl 2 and 0.3 mM Leucine) containing Amp, Spe, IPTG (0.5 mM) and a specific ncAA (1 mM), This suspension was then transferred to 96-deep-well plates (2.2 mL) and incubated at 37 C, 220 rpm for 11 h, with monitoring of fluorescence intensity (excitation 485 nm and emission 515 nm) as well as OD 600 by Microplate Readers

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[1] Males are affected more often than females

Energy estimation with Cartesian_ddg protocol The Cartesian_ddg protocol was utilised to estimate the energetic effects of pAcF mutations on protein stability 52,57,58