pET21b-TH4G

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	To express biopolymers in BL21(DE3), BL21(DE3)pLysS or BL21(DE3) LOBSTR host, a single colony was picked and cultured in 5 mL Miller’s LB Broth (LB) starter culture containing 100μg/mL carbenicillin (Sigma-Aldrich Co. LLC., US). The starter culture tube was incubated overnight at 37°C under constant shaking at 350 rpm. The next morning, the whole starter culture volume was added to a flask containing 500 mL autoclaved Terrific Broth (TB) media (25.4g of TB powder, 2 mL of glycerol in 500 mL of Milli-Q water). The flask was shaken at 37°C/350 rpm and protein expression was induced at OD600 of 0.4–0.6 by 1mM Isopropyl β-D-1-thiogalactopyranoside (IPTG).

Protocol tips

To express biopolymers in BL21(DE3), BL21(DE3)pLysS or BL21(DE3) LOBSTR host, a single colony was picked and cultured in 5 mL Miller’s LB Broth (LB) starter culture containing 100μg/mL carbenicillin (Sigma-Aldrich Co. LLC., US). The starter culture tube was incubated overnight at 37°C under constant shaking at 350 rpm. The next morning, the whole starter culture volume was added to a flask containing 500 mL autoclaved Terrific Broth (TB) media (25.4g of TB powder, 2 mL of glycerol in 500 mL of Milli-Q water). The flask was shaken at 37°C/350 rpm and protein expression was induced at OD600 of 0.4–0.6 by 1mM Isopropyl β-D-1-thiogalactopyranoside (IPTG).

Publication protocol

"The plasmids encoding TH2G, TH4G, TH6G and TH8G constructs were first transformed into BL21(DE3) (Novagen, San Diego, US), BL21(DE3) pLysS (Novagen, San Diego, US) and BL21(DE3) LOBSTR (Kerafast Inc., MA, US) E. coli expression hosts.

To express biopolymers in BL21(DE3), BL21(DE3)pLysS or BL21(DE3) LOBSTR host, a single colony was picked and cultured in 5 mL Miller’s LB Broth (LB) starter culture containing 100μg/mL carbenicillin (Sigma-Aldrich Co. LLC., US). The starter culture tube was incubated overnight at 37°C under constant shaking at 350 rpm. The next morning, the whole starter culture volume was added to a flask containing 500 mL autoclaved Terrific Broth (TB) media (25.4g of TB powder, 2 mL of glycerol in 500 mL of Milli-Q water). The flask was shaken at 37°C/350 rpm and protein expression was induced at OD600 of 0.4–0.6 by 1mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). While the expressed biopolymers in BL21(DE3) and BL21(DE3) pLysS hosts were collected four hours post induction, those expressed in BL21(DE3) LOBSTR were collected 2.5 hours post induction. The E. coli pellets were collected by centrifugation and stored at −80 °C Freezer. The above mentioned protocols are an adaptation of a previously published protocol for high yield expression of recombinant peptides in E. coli [14]."

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