Vibrio natriegens has the fastest doubling time of any known organism and has the potential to shorten experimental timelines and increase bioproduction levels with realtively easy to use protocols. We received several V. nat. cultures from the Dalia Lab at Indiana University. These cells have been stored in glycerol stocks and categorized as follows:

SAD1302: V. natriegens ATCC14048

SAD1304: V. natriegens ATCC14048 spontaneous Rif100 resistant colony

SAD1306: V natriegens ATCC14048 spontaneous Rif100 resistant colony w/ pMMB-tfoX(Vc) (Amp100ug/mL or Carbenicillin 100ug/mL)

SAD1495: V natriegens ATCC14048 spontaneous Rif100 resistant colony w/ pMMB-tfoX(Vn) (Amp100ug/mL or Carbenicillin 100ug/mL)

SAD613: Sm10 lambda pir (E. coli mating strain) w/ pMMB-tfoX (Vc)

CAH602: S17 (E. coli mating strain) w/ pMMB-tfoX (Vn)

Most of these methods were adapted from Dalia Lab or Gibson Lab methods:

http://pubs.acs.org/doi/abs/10.1021/acssynbio.7b00116

http://www.nature.com/articles/nmeth.3970

Vibrio natriegens is meant to largely function using E. coli laboratory methods but there are a few factors that mark important differences between the two.

Media Conditions:

V. nat. cells can grow in LB or BHI for liquid cultures, but grow faster in liquid media that have been supplemented with v2 salts (v2 salt: 204 mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl2). I have had the most success growing V.nat strains in BHIv2, especially during reproliferation. V. nat will grow on LB agar plates with no special changes comapred to E. coli.

Temperature Conditions:

V.nat cells grow rapidly at 37C and grow well at 30C also. Plates or liquid cultures containing V.nat should not be stored at 4C, as this will prevent reproliferation from these sources. Plates can be stored at room temperature for 2-3 days and the colonies should exhibit contact growth inhibition to keep the colonies separate. For long term storage V.nat can be stored in glycerol stocks (~15%-25%) at -80C.

Antibiotic Resistance:

V.nat has a natural resistance to Kanamycin but is more susceptible to other antibiotics. When selecting on kan plates, V.nat with KanR will simply grow faster than V.nat without KanR. Larger colonies=KanR. Other antibiotics select against V.nat but must have a dosage change compared to E.coli.

Liquid media Antibiotics : Solid Media Antibiotics:

Amp/Crb = 2-25 ug/mL : 2-50 ug/mL

Kan = 200 ug/mL : 100 ug/mL

Tetracycline = 2.5 ug/mL : 2.5 ug/mL

Chloramphenicol = 12.5-25 ug/mL : 12.5-25 ug/mL