Culturing the Rapidly Growing Bacterium Vibrio natriegens

Abstract

Source: Wiegand, D. J., Lee, et al., Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens. J. Vis. Exp. (2019)

This video demonstrates the cultivation and harvesting of Vibrio natriegens, a fast-growing bacterium, under aerated conditions to prepare bacterial pellets for crude extract generation.

Protocol

Preparation of Vibrio natriegens Crude Cell Extract–Bacterial Culture

1. Prepare V. natriegens bacterial growth media LB-V2 (Lysogeny broth with V2 salts) as per Table 1. Sterilize the growth media by autoclaving. Allow the media to reach room temperature (RT). Store excess media at RT.
   CAUTION: Wear proper personal protection equipment (PPE) and consult lab-specific instructions when operating an autoclave.
2. Use a glycerol stock of wild-type V. natriegens to inoculate 3 mL of LB-V2 media. Grow overnight at 30 °C while shaking at 225 rpm.
3. Wash 1 mL of the overnight culture by centrifugation at 10,600 x g on a benchtop centrifuge for 1 min. Aspirate the supernatant without disturbing the resulting pellet and resuspend in 1 mL of fresh LB-V2 media.
4. In an autoclaved 4 L baffled Erlenmeyer flask with sterile cover, add 1 L of fresh LB-V2 growth media. Inoculate using 1 mL of washed overnight culture (1:1,000 dilution ratio). Grow culture at 30 °C while shaking at 225 rpm.
   NOTE: V. natriegens cultures can be scaled up or down while maintaining the 1:1,000 dilution ratio. For example, add 250 μL of washed overnight culture to 250 mL of fresh LB-V2 growth media in a 2 L baffled Erlenmeyer flask with a sterile cover.
5. Monitor the culture’s OD₆₀₀ using a spectrophotometer. When the culture reaches OD₆₀₀ = 1.0 ± 0.2, harvest culture via centrifugation at 3,500 x g for 20 min at 4 °C. Place pellet on ice.
   NOTE: V. natriegens grows rapidly, so close monitoring of the culture is necessary. Growth to OD₆₀₀ = 1.0 should take approximately 1.5−2 h at the 1:1,000 dilution ratio.
6. Aspirate the supernatant and immediately store the resultant bacterial pellet at -80 °C or directly proceed to cell lysis.
   NOTE: This step is a good stopping point; however, it is recommended that the pellet be processed immediately or within 1−2 days for best results.

Table 1: Reagents for the preparation of 1 L of LB-V2 bacterial growth media

Table 1 A	Preparation of LB-V2 Bacterial Growth Media
Component	Quantity (g)	Final Concentration (mM)	Final Volume (L)
LB Broth (Miller)	25		1
NaCl	11.69	200
MgCl2	2.20	23.1
KCl	0.31	4.2
Table 1 B	Preparation of S30A Lysis Buffer
Component	Quantity (g)	Final Concentration (mM)	Final Volume (L)
Tris Solution (pH 8.0) - 1 M (mL)	25	50	0.5
Mg-glutamate	2.72	14
K-glutamate	6.10	60
Dithiothreitol (DTT) - 1 M (mL)	1	2

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2 mL Tubes	Eppendorf	22363352
HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)	Sigma	H3375
LB (Luria-Bertani) Broth (Miller)	Sigma	L3522
Magnesium chloride (MgCl₂)	Sigma	M8266
Potassium chloride (KCl)	Sigma	P9333
Sodium chloride (NaCl)	Sigma	S7653
Tris Solution (pH 8.0) - 1 M	Invitrogen	AM9856
Vibrio natriegens (Wild-type) Lyophilized Stock	ATCC	14048