Overview
- Luciferin substrate must be added.
- D-Luciferin is too large of a chemical to cross the plasma membrane of E. Coli so cell lysis is required to extract luciferase.
- After cell lysis, the reagent solution can be added to the lysis buffer. Light should be emitted within 5 to 10 seconds of adding the reagent solution.
- The luciferase/luciferin reaction at 22.5 ºC theoretically offers the greatest light intensity.
- Solutions of D-Luciferin should be aliquotted and stored in darkness at -80 ºC
- We were only able to see the color in a very dark room.
Materials
D-Luciferin free acid
ATP
MgSO4 · 7H2O
1M HEPES Buffer
Lysozyme
10 mM Tris-HCl
Lysis Buffer
For E. coli cell lysis, use a freshly prepared lysozyme solution (10 mg/ml) in 10 mM Tris-HCl, pH 8.0.
Reagent Solution
Prepare using ATP free water. Combine 1 mM luciferin or luciferin salt, 3 mM ATP and 15 mM MgSO4 in 30mM HEPES buffer, pH 7.8. Store substrate solution at -20ºC in polypropylene or glass.
Preparing 1mM D-Luciferin
Directions for a 5 mg sample: Dissolve 0.034 gr dithiothreitol in 22 mL of QH2O. Add 0.2 mL of this DTT solution to the 5 mg d-luciferin. Add 4μl of 10M NaOH to dissolve the luciferin. Dilute this into the remaining 21.8 mL of DTT solution and store as aliquots at - 80 ºC in darkness until use.
Procedure
Bacterial lysis:
- After 12-18 hours of inoculation of bacteria expressing luciferase plasmid, pipette 2 mL of cell culture into a clean 2 mL tube. Centrifuge at 10,000 rpm for 1 minute. Pour out liquid into a collection beaker. Continue this process until all of the cell culture (in the inoculation tube) is gone.
- Resuspend the pellets in 350 ml of STET buffer (10 mM Tris-HCl with 1 mM EDTA)
- Add 25μl - 30μl of lysozyme buffer to the resuspended pellet.
- Mix by vortexing for 3 seconds.
- Incubate for 2 hours at room temperature.
- If the reagent is not added immediately, store the lysed bacteria in the -20 ºC freezer until use.
Addition of Reagent Solution:
- Following the above instructions, prepare a 1mM sample of D-Luciferin.
- Following the above recipe, prepare the reagent solution.
- In a dark room, add about 250-350μl of reagent solution to each sample of lysis product.
- Light should be emitted within two-three seconds.
Example Calculations
Lysis Buffer (Desired Total Volume: 15mL)
| Chemical Name |
Tris-HCl |
EDTA |
NaCl |
| Molecular Weight |
N/A |
292.23 g/mol |
58.44 g/mol |
| Molarity Desired |
10 mM |
1mM |
0.1M |
| Calculation |
Dilute 1M Tris-HCl: |
|
|
| Final Amount |
150μl (+14.85 mL ddH2O) |
0.00438 g |
0.08766 g |
Lysozyme Solution (Desired Total Volume: 15mL)
| Lysozyme Solubility |
10 mg lysozyme in 1 mL of 10 mM Tris-HCl |
| Desired Amount of Lysozyme Solution to Make |
15 mL |
| Amount of Lysozyme Needed |
10 mg x 15 = 150 mg |
| Amount of 10 mM Tris-HCl Needed |
15 mL |
Reagent Solution (Desired total volume: 22 mL)
| Chemical Name |
ATP disodium salt trihydrate |
MgSO4•7H2O |
HEPES Buffer |
D-Luciferin free acid |
| Molecular Weight |
605.24 g/mol |
246.5 g/mol |
238.3 g/mol |
280.33 g/mol |
| Molarity Desired |
3 mM |
15 mM |
30 mM |
1 mM |
| Calculation |
|
|
|
Use the 1 mM stock solution created earlier |
| Final Amount |
0.039945 g |
0.081345 g |
0.1573 g |
22 mL |
Controls
No arabinose added during inoculation
Use bacteria without luciferase plasmid and go through steps to induce color
