Luciferase Assay

Genetic reporters are commonly used in the field of biological science to study gene expression and other cellular events coupled to gene expression. Genetic reporters are used commonly in cell biology to study gene expression and other cellular events coupled to gene expression, such as receptor activity, intracellular signal transduction, mRNA processing, protein folding and protein-protein interactions. Firefly luciferase is widely used for this purpose in several reasons

1.      reporter activity is immediately measurable upon translation since the protein does not require any post-translational modification.

2.      the assay is highly sensitive because its light production has the highest quantum efficiency known for any chemiluminescent reaction and no background luminescence is found in the host cells or the assay chemistry.

3.      the assay is rapid, requiring only a few seconds per second.

Light is produced by converting the chemical energy of luciferin oxidation through an electron transition, forming the product molecule oxyluciferin. Firefly luciferase, a monomeric 61kDa protein, catalyzes luciferin oxidation using ATP-Mg2+ as a cosubstrate.

 

The luciferase activity is measured by the use of the luminometer which can quantitatively measure the intensity of light being emitted via activation of luciferase. Reporter vectors of this system retain cDNA of luciferase and vector backbone which has been designed to enhance reporter gene expression. Certain modifications are made to the reporter vector, such as i) the C-terminal tripeptide is substituted to eliminate peroxisome targeting of the expressed reporter enzyme; ii) codon usage is improved for increased expression in plant and animal cells; iii) two potential sites of N-glycosylation are modified; and iv) several DNA sequence changes are incorporated to disrupt extended palindromes, remove internal restriction sites and eliminate consensus sequences that may be recognized by genetic regulatory binding proteins.

 

X-ray crystallography

This is to understand the three-dimensional structure of the protein of interest. X-ray crystallography is a technique in crystallography in which the pattern produced by the diffraction of X-rays through the closely spaced lattice of atoms in a crystal is recorded and then analyzed to reveal the nature of that lattice. This generally leads to an understanding of the material and molecular structure of a substance. The spacings in the crystal lattice can be determined using Bragg's law (When X-rays hit an atom, they make the electronic cloud move as does any electromagnetic wave. The movement of these charges re-radiates waves with the same frequency. These re-emitted X-rays interfere, giving constructive or destructive interferences; this is the diffraction phenomenon.). The electrons that surround the atoms, rather than the atomic nuclei themselves, are the entities which physically interact with the incoming X-ray photons. This technique is widely used in chemistry and biochemistry to determine the structures of an immense variety of molecules. X-ray diffraction is commonly carried out using single crystals of a material.

Crystallisation of proteins

In order to solve a protein crystal structure, you must first crystallise the protein. This is because a single molecule in solution has insufficent scattering power by itself. A crystal can be considered to be an (effectively) infinite repeating array of the molecule of interest. The constructive interference between diffracted X-rays that are in-phase reinforce each other, so that the diffraction pattern becomes detectable. The geometric conditions where diffraction occurs can be visualized using

(a) the wavelength of the incident beams of light,

(b) the angle of diffraction for a given reflection,

(c) the unit cell and reciprocal unit cell of the crystal, and

(d) the distance between the crystal and the film.

 

Nuclear Run-On Assay

Nuclear run-on assays are transcription assays designed to look at the genes being transcribed in a cell nucleus at a specific time. The principle involved is that, when you lyse a cell and retrieve the intact nuclei, the isolated nuclei contain transcription complexes stalled on the DNA template due to acute loss of ribonucleotide substrates. Transcription has thus been halted at the point in time where the nuclei were removed, but can be started up again in vitro with the addition of new ribonucleotides.
By using radiolabled nucleotides, the experimenter can allow transcription to finish, and then observe via autoradiography which mRNA transcripts were produced from the stalled RNA polymerase reactions and thus which genes were being transcribed at the time the cells were lysed. By comparing the amount of gene-specific radiolabled RNA synthesized in one nuclei preparation with another you can get an idea of the transcriptional initiation events in the cells of interest. Generally, purified mRNA is hybridized to cDNA on a membrane and analyzed via autoradiography. For example, you could treat one group of cells with a signaling molecule such as a growth factor and then perform nuclear run on for these cells and another group of untreated cells, then compare the radiolabled mRNA produced, looking specifically at genes expected to be upregulated. In this protocol, one dish of confluent cells in culture would receive the growth factor, while another identical dish would not. Both dishes would then be harvested, the cells would be lysed and the nuclei purified. It is important to isolate nuclei free of cytoplasmic material and cell membranes and not to damage the nuclei in the process, as both can lead to poor incorporation of radiolabled nucleotides. The difficulty in this varies largely with cell type, but is generally the biggest potential obstacle in the assay. After isolation, the nuclei are incubated with 32P-UTPs and transcription is allowed to finish. RNA is then purified from the nuclei and hybridized to cDNA on a nitrocellulose membrane. cDNA should be from the genes of interest along with positive and negative control genes (expected high or low expression from both groups of cells), and autoradiography should give a good sense of the level of transcription relative to the untreated control.

This procedure can be difficult to perform with certain cell types due to difficulty isolating nuclei. Furthermore, it may be possible that new RNA synthesis may be initiated in the isolated nuclei during the run-on reaction, leading to radio-labeled mRNA that does not represent "stalled transcription". Use of this assay to assess gene expression levels has largely been supplanted by the use of microarray analysis, however this assay is still useful in assessing whether changes in mRNA levels are a function of transcription or of subsequent RNA degradation or transport.

 

Bacterial Transformation

Since DNA is a very hydrophilic molecule, it won't normally pass through a bacterial cell's membrane. In order to make bacteria take in the plasmid, they must first be made "competent" to take up DNA. This is done by creating small holes in the bacterial cells by suspending them in a solution with a high concentration of calcium. DNA can then be forced into the cells by incubating the cells and the DNA together on ice, placing them briefly at 42oC (heat shock), and then putting them back on ice. This causes the bacteria to take in the DNA. The cells are then plated out on antibiotic containing media.

Generating Competent Cell Lines

1. Grow 5 ml overnight of the strain in 2 X YT in a standard container. Screw cap tight and lie flat on the shaker for good aeration.

2. Dilute overnight culture 1/100 - 1/200 to 25, 50 or 100 ml 2 X YT broth in flasks 5 - 10 X culture volume (i.e. 25 ml in 250 ml flask etc.).

3. Grow to early log phase (OD600 = 0.2 - 0.4) (90 - 180 min depending on the strain)

4. Collect cells by centrifugation (4000-5000 rpm for 5 min or 5000 x g for 5 min) at 4 C

5. Keep the cells ice cold in all further steps.

6. Resuspend the cells in 1/2 culture volume of 0.1 M ice-cold CaCl2. Hold on ice for minimum of 30 min, prefably 1 - 2 h.

7. Collect cells as before and gently resuspend them in 1/10 culture volume 0.1 M CaCl2.

8. Competent cells can be stored almost indefinitely by adding ice-cold sterile glycerol to a final concentration of 10% (v/v). Mix and leave on ice for 30 min, then store at -70 C

Transformation

1. To transform, mix 0.1 ml aliquots of cells with DNA (1 - 10 ng). Leave on ice for 10 - 30 min.

2. Heat shock at 42 C for 2 min (90 - 120 s is OK)

3. Add 0.9 ml 2 X YT or LB and allow expression for 30 - 60 min before plating.