Editing Protein Expression Vibrio natriegens (section)

== Preparation of electrocompetent Vibrio natriegens ==

Grow Vibrio natriegens overnight.

Prepare a&nbsp;subculture &nbsp;by inoculating an overnight culture, washed once in fresh LB3, at 1:100 dilution into fresh media. For example, spin down 500μL of the overnight culture &nbsp;(centrifugation for 1 minute at 20,000rcf), resuspendein 500μL fresh LB3 media and inoculated in 50mL of LB3 media.

Incubate the culture &nbsp;at 37℃ at 225rpm for 1 hour reaching OD ~0.4.

Spin down cells (centrifugation at 3500rpm for 5min at 4℃)

Wash&nbsp;by resuspension in 1ml of cold 1M sorbitol followed by centrifugation at 20,000rcf for 1 minute at 4℃.

Repeat wash for a total of three times.

Resuspend the &nbsp;cell pellet in&nbsp;250μL of 1M sorbitol.

Use 50μL of concentrated cells for each&nbsp;transformation.

For long term storage, aliquot the concentrated cells &nbsp;in 50μL shots in chilled tubes, snap frozen in dry ice and ethanol, and stored in 80℃ for future use.