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
cellāspecific productivity were calculated as previously described
(Brown et al., 2019).