Chemically competent BL21(DE3) E. coli (New England Biolabs, Ipswich, MA, USA) were transformed with the POE-W ELP expression plasmids and plated on 2XYT agar + 100 µg/mL carbenicillin solid medium. Fresh transformants were picked with a sterile loop and used to inoculate 15 mL starter cultures of 2XYT or SuperBroth + 100 µg/mL carbenicillin. Starter cultures were grown at 37 °C shaking at 200 rpm until visibly cloudy, typically 2-4 h. The full volume of culture was then used to inoculate 1 L of freshly autoclaved 2XYT or SuperBroth + carbenicillin. Liter cultures were returned to shaking at 37 °C for 24 h following inoculation. The leaky T7 promoter resulted in high levels of recombinant protein production without induction.