(r2) ProtocolsWorkingWithVibrioNatriegens < Lab

-- Main.JaredEller - 14 Dec 2017

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 compared 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. (These dosages are from the literature. May need to do further controls.)

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

<strong>Plasmid Transformation:</strong>

Creation of Vibrio natriegens Electrocompetent Cells:

1. Grow a 10mL culture of V.nat cells in BHIv2 overnight.<br />2. Inoculate 100mL BHIv2 with 1mL of overnight culture.<br />3. Place in orbital shaker at 37C at 200 rpm until OD600=0.5.<br />4. Separate culture into two 50mL falcon tubes and place on ice for 15 min.<br />5. Pellet cells at 6500 rpm for 20 min at 4C.<br />6. Resuspend cells in 5 mL(each tube, 10 mL total) of electroporation buffer (680 mM sucrose, 7 mM K2HPO4, pH 7).<br />7. Fill tube to ~35mL of electroporation buffer and invert gently to mix.<br />8. Centrifuge cells at 6500 rpm for 15 min at 4C.<br />9. Decant supernatant with a pipette.<br />10. Wash the cells with electroporation buffer as described above a total of three times.<br />11. After the final wash, resuspend cells in electroporation buffer as to have OD600=16.<br />12. Aliquot cells at 100 uL each tube and store in -80C. 

 Transformation using Electroporation:

1. Add 2 uL of DNA to cells on ice.<br />2. Transfer cells to cold 0.1cm cuvette.<br />3. Electroporate at 700V<br />4. Immediately recover with 500 uL BHIv2<br />5. Recover for 1 hr at 37C in shaker incubator.<br />6. Plate 100 uL of reaction.<br />7. Grow overnight at 37C. 

<strong>Natural Transformation of Vibrio natriegens:</strong>
Barrick Lab > ProtocolList > ProtocolsWorkingWithVibrioNatriegens
Topic revision: r2 - 2018-02-02 - 19:14:24 - Main.JaredEller