Overview:

Today’s protocol will allow us to quantitatively assess the levels of transfected luciferase in the cells; recall that these levels are likely to be influenced by the chosen ‘drugs’ (Fas Ligand, TNF〈, etc) and (perhaps) the presence of your transfected GFP-fusion proteins. We will indirectly assess the levels of luciferase by quantitation of its enzymatic activity (light emission); this value provides an indication of the level of reporter gene transcription resulting from the external stimuli that were applied on Wed and Thurs (as compared to baseline, non-stimulated conditions.)

In the ‘reporter plasmids’ you were given last week, the gene for Firefly luciferase, a light-generating enzyme, is immediately downstream from one of three common regulatory elements (transcription factor binding sites). Cells employ these transcription factor binding sites in the regulation of many genes that carry out important cellular functions, in a coordinated manner. In the present lab exercise, luciferase activity in detergent lysates of the transfected cells will be measured as an indicator of the quantity of luciferase present in the cells, given that there is a direct relationship between the two over a range of several orders of magnitude (~8).

In contrast, the “Renilla” luciferase plasmids have Renilla luciferase (a slightly different enzyme) downstream of a strong promoter1 that is not under the control of our drugs. This plasmid is designed for high, constant expression of this distinct luciferase, to be measured separately, and is therefore used as a control for cell number/transfection efficiency.

Cell lysates have been prepared from your transfected cells, using a ‘gentle’ (non-denaturing) detergent2 to penetrate the cell membranes and allow proteins to ‘escape’ into solution, still in their native (and active) states. A portion of the lysates (i.e., soluble protein samples) will be applied to a luciferase assay, in which the enzymatic activity of luciferase is quantitated in a luminometer upon addition of its substrate (luciferin). We will save the remainder of the lysate for western blotting with specific antibodies to the GFP ‘tag’.

1 CMV, or cytomegalovirus promoter; similar to the promoter in your GFP plasmids

2 “Non-denaturing” detergents break open the cells and solubilize membrane proteins; but they leave intact the non-covalent interactions (H bonds, van der Waals bonds, ionic bonds) that maintain the proteins in their native, active conformations.   Enzymatic activity (of luciferase) can therefore be assessed under these conditions.

Overview of luciferase and bioluminescence (adapted from Promega brochure):

Luciferase is a generic, or common, name for a group of structurally unrelated enzymes that catalyze reactions producing light in living organisms (referred to as “bioluminescence”). These exothermic chemical reactions yield photons as a consequence of energy transitions from excited-state molecular orbitals to lower energy orbitals. Analogously, “Luciferin” is a generic term for the various substrates of these enzymes, which are a class of biological pigments. Well-known examples of organisms that produce visible bioluminescence are the firefly, glow-worm, and marine invertebrates such as the jellyfish.

The divergent chemical reactions producing bioluminescence all have in common the formation and destruction of a dioxetane3 structure. Specifically, the firefly luciferase that is coded for by our plasmid gene (a 61 kDa protein) catalyzes the oxidation of its luciferin substrate in a reaction that requires ATP, Mg+2, and O2. This is a 2-step oxidation reaction that occurs through the formation of a luciferyl-AMP intermediate; in the second step, this activated intermediate reacts with oxygen to create a transient dioxetane that breaks down to the oxidized products, oxyluciferin and CO2, with the emission of light in the green/yellow region (550-570 nm):

In living organisms, nature brings enzyme (luciferase), substrate (luciferin ‘pigment’), and co-factors (ATP, etc.) together in a microenvironment conducive for light formation. In the test tube, we will provide the substrate (luciferin) and co-factors necessary for the luciferase in our lysates to generate light that will be detected and quantitated in the luminometer.

Reagents, supplies:

• Lysis buffer “LB”, already added to your sample wells (and subsequently frozen); machine will be blanked with some remaining LB.
• Luminometer tubes (12 x 75 mm) (12 per team)
• Luciferase substrate mix for ‘Firefly’ luciferase (luciferin plus ATP; named

“LAR II”, for Luciferase Assay Reagent II); aliquotted for teams

• Luciferase substrate mix for ‘Renilla’ luciferase (“Stop and Glo”); (not aliquotted; to be used at the luminometer.

Lab Instrumentation:

• Luminometer machine (back of 406)

Luminometer

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Protocol: Detection of luciferase activity in transfected cells

1. Preparing the samples (lysates):

Thaw your frozen plate of transfected cells on the benchtop for a few minutes, before beginning with sample preparation. Plates and assay reagents should be at room temperature.

1. Label 12 microfuge (eppendorf) tubes #1-12, and write your team name.

2. Working with one well at a time, using a p200 micropipette (set at ~100 ul), pipette the thawed lysate up and down over the well a few times to break up clumps and ‘rinse’ the well as much as possible.

3. Transfer (pipette) all of the lysate of one well to the correspondingly labeled microcentrifuge tube and vortex briefly. Continue with each well until each lysate has been transferred to its numbered tube (at room temperature).

4. Spin lysates in the microcentrifuge for 3 min at maximum speed. Avoid disturbing the tiny pellet of particulate material at the bottom of the tube.

5. Make a photocopy (or take a picture) of your lid before throwing out the plate.

The Luciferase Assay Set-up:

1. Make sure that your aliquot of luciferin substrate (“Luciferase Assay Reagent”, or LAR II) has warmed to room temperature before use; vortex briefly a couple of times (~medium setting), to ensure a homogeneous mixture.

2. Label 12 luminometer tubes #1-12 (write near tops of tubes). Add 100 µl of LAR to the bottom of each of the tubes. Pick the tubes up to pipette into them, and add the LAR to the very bottom of the tubes while avoiding the sides as much as possible. Numbers 1-12 correspond to the numbering of your samples. DO NOT add your cell lysates to the tubes until you come to the back of the room where the machine is, and are ready to read them.

The reaction proceeds rapidly, and the burst of light that results from substrate conversion can only be detected for a short time.

3. In a convenient rack(s), bring your lysates and prepared luminometer tubes to the luminometer at the back of 406. (We will have p20 and p100 pipettes and tips ready for you to use there.)

Reading luciferase assay results:

The luminometer will have already been turned on by the time you are ready to do your readings; TAs will be there to assist at this stage.

NOTE: you will need to obtain TWO readings for each of your samples: one for Firefly (inducible) luciferase, and one for Renilla (control) luciferase. You may find it helpful to number these values on the print-out as (ex) ‘sample #1 and 1R; sample #2 and 2R; #3 and 3R, etc. (with “R” referring to Renilla).

Ultimately you will express your data as a ratio of 1/1R, 2/2R, 3/3R, and compare those ratios between samples. Admittedly it takes some time to wrap your head around this, but the Renilla value, which doesn’t change with drug treatment, should give a measure of cell number/transfection efficiency and should therefore provide a valuable normalization control.

1. Prior to each new team’s readings, this sequence of steps will be done on the machine: On the main menu, press buttons in the following order: “Measure: Protocols: 6: Enter: Yes: Enter”.

2. For your first sample (#1) add 20 µl of lysate #1 (pipette from the top of the sample; avoid the pellet) to the correspondingly labeled luminometer tube (which has 100 µl LAR). Mix gently by flicking the tube (do not vortex).

3. Place the tube in the machine and press “Start”.

4. While the machine is reading this sample, prepare the next sample (#2) accordingly: add 20 µl of lysate #2 to its labeled luminometer tube, mix gently (do not vortex), then place in machine when it “asks” you to:

Note:

• After you load a tube, it will rotate into the machine to be read.
• The machine holds two samples at a time; it will ask you to load the next sample while it’s still reading the first sample.
• Do not remove any tubes until instructed to do so.
• Pay attention to the order in which you load your samples.

5. Read all 12 samples in this manner for Firefly luciferase, saving the tubes after removal from the machine (in numerical order).

6. Now obtain the ‘Renilla’ readings for each tube: add 100 ul of prepared “Stop & Glo” reagent4; vortex briefly to mix, place back into luminometer. Read all 12 tubes in this manner.

After the entire class’s samples have been read, we will obtain readings (both ‘firefly’ and ‘Renilla’ luciferase) from ‘blank’ lysis buffer, as background. Please subtract these ‘background’ readings from your values (eg, subtract the ‘firefly’ background from your ‘firefly’ readings, and subtract the ‘Renilla’ background from your ‘Renilla’ readings.)

7. Add your readings into the appropriate cells of the class google doc. https://docs.google.com/spreadsheets/d/1zleVWY5Emb3CLu6MQgo94Se SOaFhUs8yu8mS296SwdA/edit#gid=0

***Please save the remainder of your lysates for next week’s western blot protocol. We will provide a separate freezer box for them.

Questions

1. Did your gene of interest (GOI) have an effect on the reporter when compared to the vector control?
2. Did your drug(s) have an effect on the reporter when compared to no drugs?
3. Did your GOI and drugs antagonize, synergize or have an additive effect on the reporter?
4. Do you have good duplicates/can you trust your results?
5. What variable(s) would you like to change for your next set of experiments?Why?

4 This reagent accomplishes 2 things: (1) it ‘quenches’ (suppresses) the firefly luciferase activity; (2) it provides substrate for the Renilla luciferase enzyme, which is a different enzyme.