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, Expression levels were measured by flow-cytometry and expressed in REU. Data correspond to the mean of 3 independent experiments performed in triplicates and error-bars correspond to standard deviation over these 3 experiments. Grey bars correspond to exponential phase and black bars to stationary phase. (A) Expression level of the reference construct in absolute unit in exponential and stationary phase. (B) (C) (D): Expression levels of basic promoters and RBSs! in exponential and stationary phase expressed in REU (B), Characterisation of promoter and RBS libraries was performed on exponential and stationary phases

, Linear correlation were performed for each construction sets and a coefficient of determination between 0.83 and 0.999 were found. ! 1. Thaw gently competent cells on ice (aliquot of 100µL), one tube per Gibson assembly reaction

, Add 10µL of the Gibson Assembly reaction (keep cells on ice)

, Incubate 30min on ice

, Heat-shock cells at 42°C during 45s (in water bath)

, Put back on ice after heat-shock (2 to 5min)

, Add 900 µL pre-warmed SOC (37!)(rich medium)

, Incubate cells at 37°C with agitation during at least 30min

, Centrifuge cells at 4000rpm during 1min, remove 800µL of supernatant and plate the rest

, Incubate at 37°C overnight Note For multiple transformation (more than 10), competent cells aliquoted in PCR strip can be used. A similar protocol is used. To adapt to large volumes, cells are incubated in SOC in 96 well