Editing Secretion Expression E coli (section)

=== Fermentor Culture Condition ===

[https://amb-express.springeropen.com/articles/10.1186/s13568-017-0394-1 https://amb-express.springeropen.com/articles/10.1186/s13568-017-0394-1] Small volume fed-batch cultures (3 mL working volume) were performed in a Micro 24 bioreactor system (Applikon Biotechnology, Forster City, CA, USA). Feed solutions and other supplements were sterilized by autoclaving or filtration through a 0.22-μm pore size filter, while the rich growth culture medium [20.3 g/L yeast extract (BioSpringer, Milwaukee, WI, USA), 10.1 g/L sodium sulfate anhydrous (JT baker, Center Valley, PA, USA) and 7 g/L K2HPO4 (JT baker, Center Valley, PA, USA)] was separately autoclaved and added later under aseptic conditions. The culture was initiated by inoculation of 2.8% culture volume into the rich growth medium containing 50 μg/mL of kanamycin (Sigma, St. Louis, MO, USA), 7.6 g/L of trace metal cocktail solution (55 g/L sodium citrate dehydrate (Sigma, St. Louis, MO, USA), 27 g/L FeCl3·6H2O (Sigma, St. Louis, MO, USA), 0.5 g/L CoCl2·6H2O (Sigma, St. Louis, MO, USA), 0.5 g/L Na2MoO4·2H2O (Sigma, St. Louis, MO), 0.95 g/L CuSO4·5H2O (Sigma, St. Louis, MO, USA), 1.6 g/L MnCl2·4H2O (Sigma, St. Louis, MO, USA), 1.3 g/L ZnCl2 (Sigma, St. Louis, MO, USA) and 2 g/L CaCl2 (Sigma, St. Louis, MO, USA) and 0.8% culture volume of the glycerol and Epsom salt solution [315 g/L of glycerol (Sigma, St. Louis, MO, USA) and 31.4 g/L of MgSO4 (Sigma, St. Louis, MO, USA)]. During the fed-batch cultivation, the impeller speed was initially set to 800 rpm and later controlled to keep the dissolved oxygen level (DO) at 60% saturation. In fed-batch mode, 55% (v/v) glycerol for the carbon source and 33% (w/v) yeast extract were used as feed solutions. Recombinant ATH35L gene expression was induced by addition of 0.5 mM isopropyl-β-D1-thiogalactopyranoside (IPTG) (Biovectra, Charlottetown, PE, USA) when the cell reached at an optical cell density (OD600) of 10. Cell culture was continued at 30 °C for an additional 14 h after induction. Cultured cells were collected at 14 h post-induction, and cell culture medium was collected from the harvest samples by centrifugation at 13,300g for 5 min for analysis and quantification of extracellular ATH35L. Periplasmic and cytoplasmic fractions were prepared from cell lysate from the harvest samples using PeriPreps™Periplasting kit (Epicentre, Madison, WI, USA).

Fermentor fed-batch culture processes Large volume fed-batch cultures were performed in a DasGip fermentor (SaniSure Inc, Moorepark, CA, USA) with 1 L working volume. Feed solutions, culture medium and other supplements were prepared as previously described for the small volume fed-batch culture processes. The culture was initiated by inoculation of 2.8% of culture volume into the prepared culture medium containing 50 μg/mL of kanamycin (Sigma, St. Louis, MO, USA), 7.6 g/L of trace metal cocktail solution, 15.8 g/L of glycerol (Sigma, St. Louis, MO, USA) and 1% (v/v) P2000 antifoam (Alfa Aesar, Reston, VA, USA) solution. Air space velocity was 1 vvm and the temperature was maintained at 30 °C. Ammonium hydroxide (23.5% v/v) (Sigma, St. Louis, MO, USA) and glacial acetic acid (50% v/v) solutions (Sigma, St. Louis, MO, USA) were used to maintain cultures at pH 7. During batch experiments, the impeller speed was initially set to 1200 rpm and later controlled to keep the DO at 60% saturation. In fed-batch mode, 55% (v/v) of glycerol for the carbon source and 33% (w/v) of yeast extract solutions were used as feed solutions. During feeding, the impeller speed was maintained constant at 1200 rpm, while the DO saturation was automatically kept at 60%. Recombinant ATH35L gene expression was induced by addition of 0.5 mM IPTG (Biovectra, Charlottetown, PE, USA) at an optical cell density of 80 (OD600). After induction, cell culture was continued in fed-batch mode at 30 °C for an additional 12 h. Cultured cells were collected at different time points post-induction to determine the profiles of secreted ATH35L protein, osmolality and the concentration of glycerol and acetate in culture medium. [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5427057/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5427057/]&nbsp;[https://www.frontiersin.org/articles/10.3389/fmicb.2019.01511/full https://www.frontiersin.org/articles/10.3389/fmicb.2019.01511/full]

'''Strain''' &nbsp;BL21 (DE3),&nbsp; BL21 Star (DE3)

'''Medium'''

Seed medium (20 g/L yeast extract) Rich growth medium (20.3 g/L yeast extract, 10.1 g/L sodium sulfate anhydrous, 7 g/L K2HPO4

'''Shaker Incubator Medium'''

&nbsp;Terrific Broth (TB) medium: glycerol 5g, tryptone 12g, yeast extract 24g,&nbsp; K2HPO4 16.4g, and KH2PO4 2.3g. H2O--> 1L

'''Induction'''

grow OD600 to 0.8 , add 0.4 M IPTG, continue to grow 10 hours, add 0.4% Triton X-100, total induction time is 24 hours.
[[Category:Protocols]]