In Vivo Assay to Correlate Bacterial Bioluminescence with Cell Density

Abstract

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

This video demonstrates the measurement and correlation of bioluminescence with cell density in transformed bacteria. A single bacterial colony is used to initiate a liquid culture. The culture is placed in a well at the desired cell concentration. An antibiotic and inducer are added to express the gene required for bacterial bioluminescence. Readings for absorbance and bioluminescence are taken at regular intervals using a plate reader.

Protocol

1. Design, Preparation, and Expression of the lux Operon in Escherichia coli

Note: See Table of Materials for information on commercial kits used in this section.

1. For transferring the lux operon into E. coli choose a standard pET vector with appropriate restriction sites and antibiotic resistance gene of interest (e.g., pET28a; NcoI, XhoI, kanamycin).
2. Design the fragments and overlapping primers for Gibson assembly based on the DNA sequence of Photobacterium mandapamensis 27561 (GenBank: DQ988878.2).
3. Set up a standard polymerase chain reaction (PCR) reaction with the designed primers and the isolated genomic DNA (deoxyribonucleic acid) of Photobacterium mandapamensis 27561 as a template.
   NOTE: Isolation of genomic DNA of the respective bacterial strain enhances PCR efficacy.
4. Purify the PCR product via spin-column purification.
5. Perform restriction digestion of the isolated pET28a vector with NcoI and XhoI at 37 °C for 45 min.
6. Purify the linearized vector and the PCR fragments via agarose gel electrophoresis and subsequent spin-column purification.
7. Determine the DNA concentration of each fragment and the linearized vector, and calculate the optimal quantities for the assembly according to the protocol.
   NOTE: Efficacy of assembly depends on fragment size and number and has to be adjusted according to the manufacturer's protocol.
8. After combining all fragments and the buffer in a PCR tube, incubate the assembly mixture in a PCR machine at 50 °C for 1 h.
9. Transform the assembled vector product according to standard transformation protocols for E. coli bacterial plasmid transformation into an appropriate E. coli system for high-yield plasmid replication (e.g., E. coli TOP10 or XL-1).
10. Pick colonies from the transformation plate and streak on new plates for DNA isolation.
11. Isolate plasmid DNA according to standard protocols.
12. To verify the correct assembly of the plasmid, including all fragments, first perform a colony PCR according to standard protocols using primers specific for every assembled fragment.
13. Additionally to the colony PCR and subsequent agarose gel electrophoresis, prepare all isolated assembly vectors for DNA sequencing to verify the correct assembly and the correct DNA sequences.
14. Transform the verified plasmid according to standard transformation protocols for E. coli bacterial plasmid transformation into an appropriate E. coli system for high-yield protein production (e.g., E. coli BL21).
    NOTE: Continue directly with expression protocol below. For longer storage, the preparation of a glycerol stock is recommended.

2. Expression of Modified E. coli Strains

1. Prepare an overnight culture (ONC) for expression by inoculation of an appropriate volume of Luria-Bertani (LB) medium (e.g., 100 mL) with the previously prepared glycerol stock of the E. coli BL21 cells transformed with the assembled plasmid or directly from a transformation plate. Add 100 µL of kanamycin (50 mg/mL; antibiotic resistance gene of pET28a) and incubate the ONC at 37 °C and 120 rpm in an incubator shaker overnight.
2. Inoculate the main expression culture (e.g., 800 mL of LB medium) with 8 mL of the ONC and add 800 µL of kanamycin (50 mg/mL).
3. Incubate the main culture at 37 °C and 120 rpm in an incubator shaker until the cell density reaches an OD₆₀₀ of 0.6 - 0.8 (approximately 2.5 h).
4. Reduce the incubation temperature to 28 °C.
5. Induce protein expression by adding propyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM.
   NOTE: Empirical tests showed that the reduction of the temperature to 28 °C gave the highest light intensity.
6. Observe cells until they start shining (approximately 1 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 (maximum 48 h). Harvesting the cells and purification of any proteins can be done according to standard procedures.

3. Expression of Bacterial Bioluminescent Strains

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

1. Prepare 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 sulfate heptahydrate).
   2. Add 1 L of distilled water and dissolve all components.
   3. For LB 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 the 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 time 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 assure 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 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 cultures 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.

4. In Vivo Activity Assay for Bacterial Bioluminescent Strains and Modified E. coli Strains

NOTE: Long time storage of strains is normally achieved through freezing glycerol stocks of the bacterial culture. Strains should always be streaked on agar plates first to assure uniform starting conditions for all strains, prior to usage for liquid cultures, due to a lag phase in growth after thawing.

1. Streak the desired bioluminescent bacterial strain or modified E. coli strain on an agar plate and incubate at 28 °C overnight.
   NOTE: The incubation temperature can vary from strain to strain and has to be evaluated empirically. To be able to compare bioluminescent bacterial strains and modified E. coli strains, growth conditions have to be identical.
2. Inoculate 3 mL of medium with the respective strain with a single colony from an agar plate and incubate the cells at 28 °C and 180 rpm in an incubator shaker for approximately 1 - 2 h.
3. Measure the cell density of a 1:10 dilution of the liquid culture at 650 nm. Calculate the ratio and volume for 1 mL culture with an OD650 of 0.05.
   NOTE: The subsequent plate reader assay will determine the cell density at 650 nm to avoid interference by the light emission of the strains.
4. Pipette the calculated volume of culture and medium into a 24-well black-walled plate with a glass bottom. For the modified E. coli strain, add 1 µL of kanamycin (antibiotic resistance of pET28a vector) and 1 µL of IPTG (induction of gene expression) to the samples. Place a lid on the plate to avoid evaporation during the measurements.
   NOTE: To assure that the pET28a vector containing the whole lux operon does not get lost by the E. coli culture, kanamycin must be added to each E. coli sample in the plate wells and to assure that light production of the E. coli cells can be measured, the gene expression must be induced by IPTG. To avoid crosstalk and measurement interference, black-walled well plates with glass bottoms and transparent lids showed the best results. Nevertheless, crosstalk can be observed, and well positions have to be chosen carefully.
5. Start the measurement in a plate reader.
   NOTE: The plate reader protocol is based on a script specially developed for this assay that combines two measurements, absorbance and bioluminescence. Data points are collected every 10 min with permanent shaking between the measurements and a constant temperature of 28 °C.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NEBuilder High-Fidelity DNA Assembly Cloning Kit	New England Biolabs Inc.	E2621S	for 10 rxndx.doi.org/10.17504/protocols.io.cwaxad
Phusion Polymerase	Thermo Scientific	F530S
Q5 High Fidelity DNA Polymerase	New England Biolabs Inc.	M0491S
GeneJet Genomic DNA Purififcation Kit	Thermo Scientific	K0721	for 50 rxn
Restriction enzymes and buffer (NcoI, XhoI)	New England Biolabs Inc.	R3193S/R0146S
Wizard SV Gel and PCR Clean-Up kit	Promega	A9282	for 250 rxn
Monarch DNA Gel Extraction Kit	New England Biolabs Inc.	#T1020S	for 50 rxn
GeneJet Plasmid Miniprep Kit	Thermo Scientific	K0503	for 250 rxn
GoTaq G2 DNA Polymerase	Promega	M7841
24-well black sensoplate with glass bottom	Greiner Bio One	662892
FLUOStar Omega plate reader	BMG Labtech
OPTIMA/Mars Analysis Software	BMG Labtech		Version 2.20
Microsoft Excel 2010	Microsoft
Multitron Standard Incubator Shaker	Infors AG