Product Information Sheet

Marker Gene Technologies, Inc. University of Oregon Riverfront Research Park

1850 Millrace Drive Eugene, Oregon 97403

1-888-218-4062 www.markergene.com

RubyGlowTM Luminescent Bacterial Detection Kit

Product M1573

19636ELISA

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RubyGlowTM Luminescent Bacterial Detection Kit (Product M1573)

NOTE: This kit provides an easy and quick method to detect and quantify bacterial cells based on ATP present in metabolically active cells. Each kit contains a genetically engineered luciferase which generates very stable luminescent signals. This luciferase emits red upon luciferin conversion and thus can be multiplexed with other assays using blue or green fluorescence. For more information about these techniques, please visit our website at www.markergene.com or contact our technical assistance department at techservice@markergene.com. These materials are intended for research purposes only. Use in drug or manufacturing processes is strictly prohibited. Please contact us for information on use or licensing.

I. OVERVIEW The bacterial enumeration in different bacterial samples is of great importance in many fields of research. Traditional methods usually take several days. For fast results, one of the methods to estimate the number of microbial cells is based on the determination of adenosine triphosphate (ATP) levels, assuming that living cells are generating ATP and the ATP content of microbial cells is relatively constant across all phases of growth. One of the most sensitive techniques for measurement of ATP levels has proven to be the luciferin-luciferase bioluminescent assay. The reaction involved is described as below: Luciferase + ATP + Luciferin + O2 Oxyluciferin + AMP + PPi + CO2 + Light

When ATP is the limiting component in the reaction, the intensity of the emitted light will be proportional to ATP concentration in the sample. Using genetic engineering, we have generated a new luciferase that exhibits a long-wavelength light emission (619 nm), as well as improved thermostability, compared to the native green firefly luciferase often used in these assays. The light output of this proprietary luciferase is stable over several hours and thus eliminates the necessity for substrate injection assay methods. The RubyGlowTM Luminescent Bacterial Detection Kit utilizes this new thermostable luciferase and can detect bacteria from a number of media or biological sources with reasonable detection limits. In addition, a 2 to 4 hour extended incubation at 37 ºC can be used for low level samples. This extended incubation can significantly reduce the detection limit and increase the sensitivity

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of detection while still allowing users to finish the evaluation in one day. In addition, data variation is low and confidence levels have been found to be high compared with several other commercially available “Add and Read” kits. Due to its stable light emission, we have adapted this kit for use in a microtiterplate format enabling the processing of a large number of samples (such as food or clinical samples) simultaneously. This microtiterplate format assay is also amenable to moderate/high throughput applications such as drug and antibiotic screening assays. Our new RubyGlowTM luciferase emits red light (EM 619 nm) upon luciferin conversion, while most of the commercially available luciferases emit green light (562 nm). This unique feature enables this assay kit to be compatible with assays using standard luciferase analyses, or other green (fluorescein-based) or coumarin (blue color) assay methods. The kit components involve no radioisotopes or toxic materials and are environmentally safe. The provided kit components are sufficient for 100 reactions in tube or 96-well microtiterplate format.

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II. MATERIALS

A.) Enzyme: RubyGlowTM luciferase, in solution B.) Buffer Solution: Reaction buffer C.) Substrate: Luciferin, powder D.) Lysis Buffer E.) Phosphate/EDTA Buffer F.) Lysozyme

Storage and Handling. Reagents, standards, and buffers should be handled with care, kept cold when not in use, and stored at -20ºC. In case of contact with skin or eyes, wash thoroughly with soap and cold water. Reagents should be stable for at least 6 months following purchase if stored properly. Equipment and materials not provided but required:

Luminometer or plate reader with luminescence detector

96-well black walled assay plate (Corning #3792 round-bottom or #3915 flat bottom)

96-well clear plate for sample processing in microtiterplate format or microcentrifuge tubes for sample processing in single tube format

Multi-channel pipette

Dry ice

III. PROTOCOL A. Reconstitute to working solutions

1. Thaw RubyGlowTM Luciferase and Reaction Buffer vials. Keep the thawed vials on ice prior to use.

2. Bring the Luciferin Substrate to room temperature. 3. Transfer exactly 5ml of the completely thawed Reaction Buffer to the

Luciferin Substrate ; swirl or vortex gently to dissolve. 4. Transfer the RubyGlowTM Luciferase to the substrate solution

prepared in step 3; swirl to mix. To ensure complete transfer, pipet 1ml luciferin solution to the luciferase vial to rinse and transfer back to the final substrate bottle.

5. The well mixed solution currently in the substrate bottle is the RubyGlowTM Luciferin/Luciferase working solution. This working solution should be kept on ice. Any unused solution can be stored at 4ºC and will remain active for approximately 2 weeks according to our test results. Alternatively, unused portions can be aliquoted and stored at -20ºC for extended periods. Avoid repeated freeze/thaw cycles.

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B. Protocol for measuring ATP from bacteria

Note: Extracting ATP from bacteria can be performed in a single tube or in a microtiterplate format if multiple samples are required to be processed at the same time.

1. Prepare the Bacterial Lysis Mix: thaw Phosphate/EDTA Buffer as well as Lysis Buffer and store at 4ºC until use. To the Lysozyme vial, add 0.4mL Phosphate/EDTA Buffer plus 3.6ml of DI water and bring final mixture to complete solution.

2. Transfer this 4mL lysozyme solution to the Lysis Buffer ; swirl to mix. This lysis mixture is called “Bacterial Lysis Mix” and can be used to lyse and extract ATP from bacteria.

3. Transfer 45L of bacterial culture sample(s) to a tube or microtiterplate

well containing 5L of Phosphate/EDTA Buffer . Mix well by pipetting up and down several times, and then place on dry ice to freeze completely. (Include control wells containing only culture medium to obtain a value for any background luminescence.) Thaw the mixture to room temperature,

followed by addition of 150L of Bacterial Lysis Mix prepared in step 2 above (IIIB). Allow this mixture to stand at room temperature for 10

minutes. The total lysate volume will be 200L.

4. Transfer 50L of this cell lysate to a 96-well black-walled flat or round bottom microtiterplate. (NOTE: Please consult your microtiterplate reader’s instruction manual for the appropriate plate to use). Dispense

50L of the RubyGlowTM Luciferin/Luciferase working solution to each well, mix and record luminescence [using a time for integration of ≥ 1 second] with a luminometer or a plate reader with luminescence recording capability.

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Example protocols: Test the activities of antibiotics:

1. JM109 or another laboratory bacterial strain is cultured in LB medium to its stationary phase at 37ºC overnight.

2. Prepare a 50x working solution of antibiotic reagent and dispense 5L to a microtiterplate or microcentrifuge tube. In some cases, a series of dilutions of the antibiotic reagent is made to evaluate a dose-response effect on the bacteria.

3. Dilute the overnight culture at 1:50 with LB medium and dispense 245L to each well containing antibiotic reagent. Incubate at 37ºC for 5 hours. Remember to include wells which contained no antibiotic to serve as control wells.

4. Transfer 45L of bacteria/antibiotic and mix with 5L of Phosphate/EDTA Buffer . Mix well by pipetting, then place on dry ice to freeze the cells

followed by thawing at room temperature. 150L of the Bacterial Lysis Mix (prepared at step 2 Section IIIB) is added. Allow the mixture to stand at room temperature for 10 minutes.

5. Transfer 50L of the lysate to a 96-well black-walled flat or round bottom

microtiterplate. Dispense 50L RubyGlowTM Luciferin/Luciferase working solution to each well, mix and record luminescence [time for integration≥ 1 second] with a luminometer or a plate reader with luminescence recording capability.

Figure 1: flow chart of the assay

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6. To evaluate the activity of antibiotic reagent, compare the luminescence

readings of known antibiotics to the control wells described in step 3. 7. Plot the light output (RLU) versus antibiotic reagent concentration to

obtain a dose-response evaluation as shown in Example Figure 2a & 2b. Detection limit of bacterial culture:

1. W3110 or other wildtype E. coli strain is cultured in LTB medium to its

exponential phase at 37ºC. This can be achieved by overnight culture at 1:50 in LTB with culturing at 37ºC until OD600 reaches 0.8. Or simply inoculate W3110 colony in LTB medium until OD600 reaches at least 0.2 (NOTE: This can take up to 8 hrs.). Place the culture at 4ºC overnight and re-culture a second day to reach OD600 of 0.8.

2. Place this culture on ice. Make a series of dilutions with LTB medium while keeping all samples on ice.

3. Transfer 45L of the diluted bacterial culture(s) to a microtiterplate

containing 5L of Phosphate/EDTA Buffer/well . Remember to include control wells containing only culture medium to obtain value for background luminescence. Mix well by pipetting up and down, and then place on dry ice to freeze completely. Next, thaw the samples to room

temperature, followed by addition of 150L of Bacterial Lysis Mix prepared in step 2, Section IIIB. Let this mixture stand at room temperature for 10 minutes.

4. Transfer 50L of the lysates to a 96-well black-walled flat or round bottom

microtiter assay plate. Dispense 50L RubyGlowTM Luciferin/Luciferase working solution per well, mix and record luminescence [time for integration: 1 second] with a luminometer or a plate reader with luminescence recording.

5. Plot the light output (RLU) versus OD600 as shown in Example Figure 3. 6. To determine the colony forming unit (CFU) of each dilution (OD600 of

0.001 or below), apply 45L of the culture to a LTB plate and let incubate at 37ºC overnight. Count colonies the next day.

7. To examine how extended incubation at 37ºC will affect the high dilution cultures, we removed all the diluted cultures off ice and cultured them at

37ºC for 2 and 4hours. 45L of the incubated diluted bacterial cultures were processed and assayed similarly. Light output (RLU) versus OD600 was plotted and compared to non-incubated cultures.

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IV. DATA FROM EXAMPLES

Figure 2a. Evaluating the growth inhibition of bacteria by antibiotic reagents: Cultures from JM109 or BL21(DE3)pLysS (chloramphenicol resistance) strains harboring an expression vector (confer ampicillin resistance) were diluted and mixed with a number of antibiotics. This mixture was incubated at 37ºC for 5 hours. Then cells were lysed, ATP extracted and measured with RubyGlowTM luciferase. Luminescence was recorded. RLU was plotted against the different antibiotics. AMP:ampicillin; Tet:tetracyclin; Strep:streptomycin; CAM:chloramphenicol. Ampicillin and chloramphenicol inhibited the growth of JM109, but didn’t affect the growth of resistant strain.

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Figure 2b. Dose-response growth inhibition of bacteria by Hygromycin: Cultures from JM109 were diluted and mixed with a series of Hygromycin dilutions. This mixture was incubated at 37ºC for 5 hours, lysed, ATP extracted and measured with RubyGlowTM luciferase. Luminescence was recorded. RLU was plotted against Hygromycin concentration.

Figure 3. Detection limit of bacteria by RubyGlowTM kit: Dilutions from exponentially grown W3110 were lysed, ATP extracted and luminescence recorded. To examine whether extended incubation at 37ºC will help the detection of high dilution culture, we tried 2 and 4 hour incubation periods and found that the detection limit was dropped at least two magnitudes.

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Notes: F=store at or below -20o

C; C=store cold (4o

C); L=lyophilized; T=avoid repeated freeze/thaw; R=read protocol/instructions carefully prior to use.

M1573 KIT CONTENTS

DESCRIPTION QUANTITY PART NO. STORAGE

REAGENTS

Luciferin Substrate 1 x 1.4mg M1573-001 F, L

RubyGlowTM Luciferase 1 x 0.5mg M1573-002 F, T

Lysozyme 1 x 20mg M1573-003 F, L

Buffers

Reaction Buffer 1 x 6mL M1573-004 F

Lysis Buffer 1 x 12mL M1573-005 F

Phosphate/EDTA Buffer 1 x 2mL M1573-006 F

DOCUMENTATION

MSDS Sheets 1

Product Information Sheet 1

Figure 4. Linearity of luminescence vs growth of bacteria: Dilutions from exponentially grown W3110 were lysed, ATP extracted and luminescence recorded. RLUs were plotted against the bacterial growth which was indicated by OD600. Linearity was observed up to OD600 of 0.8. The insert shows the linearity below OD600 of 0.1.

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REFERENCES

1.) Sharpe AN, Woodrow MN, Jackson AK (1970) “Adenosine triphosphate (ATP) levels in foods contaminated by bacteria.” J. Appl. Bacteriol. 33: 758-767.

2.) Stannard CJ, Wood JM (1983) “The rapid estimation of microbial contamination of raw meat by measurement of adenosine triphosphate (ATP).” J. Appl. Bacteriol. 55:429-438.

3.) Stanley PE (1986) “Extraction of adenosine triphosphate from microbial and somatic cells.” Methods Enzymol. 133:14-22.

4.) Karl DM (1980) “Cellular nucleotide measurement and applications in microbial ecology.” Microbiol. Rev. 44:739-96.

5.) McElroy WD, DeLuca MA (1983) “Firefly and bacterial luminescence: Basic science and applications.” J. Applied Biochem. 5:197-209.

6.) Ulrich PG, Wannlund JC (1984) “Bioluminescence in the microbiology laboratory.” American Clinical Products Rev.

7.) Tuncan EU, Martin SE (1987) “Lysostaphin lysis procedure for detection of Staphylococcus aureus by the firefly bioluminescent ATP method.” Applied and Environmental Microbiology 53: 88-91.

8.) Lundin A, Thore A. (1975) “Analytical information obtainable by evaluation of the time course of firefly bioluminescence in the assay of ATP.” Anal. Biochem. 66:47-63.

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Contact and Support

For questions or comments on this or any product from Marker Gene Technologies, Inc., you may contact us by phone or via our website. We welcome customer feedback and we make every effort to improve our products based on input from our clients.

To ask a question or make a comment or suggestion, you can call us at 1-888-218-4062 or fax to 541-342-1960.

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Custom Synthesis Info We want to thank you for your purchase and hope that you will

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Marker Gene Technologies, Inc.

University of Oregon Riverfront Research Park 1850 Millrace Drive

Eugene, Oregon 97403 1-888-218-4062

www.markergene.com

http://www.markergene.com/