Expression of the Luminescence Operon in Marine Bioluminescent Bacteria

Abstract

Source: Brodl, E., et al. In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria. J. Vis. Exp. 1 (2018)

This video demonstrates the cultivation of marine bioluminescent bacteria to observe quorum-sensing–mediated activation of the luminescence operon, enabling real-time monitoring of gene expression and light emission for studies in microbial communication and gene regulation.

Protocol

1. Expression of Bacterial Bioluminescent Strains

NOTE: Bacterial bioluminescent strains require specific growth medium/artificial seawater medium for growth and light production.

1. Prepare an artificial seawater medium, composed of two separately prepared medium components.​
   NOTE: The following amounts are for 1 L of liquid medium or 1 L of agar medium.
   1. For the artificial seawater medium, weigh in the following salts: 28.13 g NaCl (sodium chloride), 0.77 g KCl (potassium chloride), 1.60 g CaCl₂ · 2H₂O (calcium chloride dihydrate), 4.80 g MgCl₂ · 6H₂O (magnesium chloride hexahydrate), 0.11 g NaHCO₃ (sodium bicarbonate), and 3.50 g MgSO₄ · 7H₂O (magnesium sulphate heptahydrate).
   2. Add 1 L of distilled water and dissolve all components.
   3. For LB (Luria-Bertani) medium, weigh in the following ingredients: 10 g yeast extract, 10 g peptone, and for agar plates, an additional 20 g agar.
   4. Add 250 mL of tap water and dissolve components.
   5. Autoclave both prepared media separately at 121 °C for 20 min.
   6. For agar plates, combine 250 mL of LB medium with 750 mL of artificial seawater medium directly after autoclaving and prepare plates.
   7. For liquid medium, combine 250 mL of LB medium with 750 mL of artificial seawater medium either directly after autoclaving or when cooled down.
      NOTE: The artificial seawater medium may get turbid through salt precipitation.
2. Streak the bacterial bioluminescent strains on artificial seawater medium agar plates and incubate overnight at 24 - 30 °C.
   NOTE: Long-term storage of bacterial strains is normally achieved through freezing glycerol stocks of the bacterial culture. Strains should always be streaked on agar plates first to ensure uniform starting conditions for all strains, prior to usage for liquid cultures, due to a lag phase in growth after thawing.
3. Prepare an ONC (overnight culture) by inoculating 100 mL of artificial seawater medium with a single colony from the plate. Incubate the ONC at 24 - 30 °C and 120 rpm in an incubator shaker overnight.
4. Inoculate 800 mL of artificial seawater medium with 8 mL of ONC.
5. Incubate the bacterial cells at 24 - 30 °C and 120 rpm in an incubator shaker.
   NOTE: The light intensity profile of bioluminescent bacteria strongly varies with temperature. Depending on the regulatory mechanisms of the light production of the respective bacterial strain, light emission may start after approximately 1 - 6 h.
6. Observe bacterial cell culture until they start shining (approximately 1 - 6 h).
   NOTE: Depending on the purpose of the expression, the cells are grown until the next day and are then harvested, or the cells can be kept shaking as long as they are shining. Harvesting the cells and purification of any proteins can be done according to standard procedures.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Multitron Standard Incubator Shaker	Infors AG