Synthetic mRNA Expression
mRNA encoding plasmids were transformed into BL21 (DE3) or BL21 Star (DE3) (Invitrogen) E. coli strains, typically used for recombinant protein production. 5ml starter cultures were inoculated with a single colony, and grown overnight in Luria-Bertani (LB) broth (Thermo Fisher), containing 50 ug/ml kanamycin (Thermo Fisher) at 37C, 200 rpm. For small scale expression, 100 ul of starter culture was used to inoculate 10 ml of LB medium containing 50 ug/ml kanamycin, and cells were grown at 37C, 200rpm, until the OD600nm reached 0.4-0.6. For large scale expression, 200 ml of LB was inoculated with 5 ml of overnight culture. IPTG was then added to a final concentration of 1 mM. Where RNase E inhibitor was used, it was added from a 100x stock at the point of IPTG addition 500 ul of culture was pelleted at 30 minute intervals post induction, and pellets stored at -80C. Total cellular capacity (integral of viable cell concentration), cell-specific growth rate and cell‐specific productivity were calculated as previously described (Brown et al., 2019).