Table 2.

Cultivation conditions of the nanoliter reactors.

NLR type	Vessel type	Washing solution	Medium	Incubation conditions	Comment
GABA-producers	100 ml sterile plastic container	Washing buffer: 10 mM CaCl2, 10 mM MES pH6, 0.05% cysteine	MRS or SHIME	Anaerobic, static, 37ºC, up to 96 h	–
Transcription factor-based biosensor	Sterile 50 ml shake flask	10 mM CaCl2	2xYT with with 5 mM CaCl2	Aerobic, 37 °C, 250 rpm, up to 45 h	Several flasks were prepared with different final concentrations of GABA (between 0 and 62 mM)
Growth factor-based biosensor	Sterile 50 ml shake flask	Washing buffer	M9 (alanine-free)	Aerobic, 37 °C, 250 rpm, up to 45 h	Several flasks were prepared having different final concentrations of GABA (between 0 and 32 mM). A positive control was included with 2 mM alanine instead of GABA
Gut-like conditions	15 ml Falcon tubes	Washing buffer	SHIME	Micro- and an-aerobic, 37 °C, up to 45 h	Different cultivation conditions were tested to ensure proper growth of the strain and good mixing of the turbid medium