REPORTERS IN MOLECULAR BIOLOGY

DefinitionsIn molecular biology, a reporter gene (often simply reporter) is a gene that researchers attach to a regulatory sequence of another gene of interest in bacteria, cell culture, animals or plants. Certain genes are chosen as reporters because the characteristics they confer on organisms expressing them are easily identified and measured, or because they are selectable markers. Reporter genes are often used as an indication of whether a certain gene has been taken up by or expressed in the cell or organism population.

Common reporter genes

Use of reporter gene:

• β-galactosidase is derived from the Lac-Z gene of E. coli lac operaon.

• β-gal is a hydrolase enzyme which catalyzes the hydrolysis of Β-galactosides into monosaccharides.

• In E.coli, its natural function is to use Lactose as a carbon source in the absence of glucose

β-GALACTOSIDASE as a reporter system

STRUCTURE

•1024 amino acids

•464 Kda

•Homo-tetramer

•β-galactoside into monosaccharide

BIOCHEMICAL MODE OF ACTION

• It hydrolyzes a colorless substrate X-gal to produce a Blue colored compound

• With ONPG as substrate, it produces o-nitrophenol which is yellow in color

β-Galactosidase in Reporter Assay

• LacZ can tolerate N-Terminal Fusions without any change in its activity.

• Therefore it can be translationally fused with the gene of interest to be used as a reporter gene.

BLUE-WHITE SCREENING

• Vector contains Lac-Z gene

• After transformation, cells are grown in a media containing X-gal

• The transformed cells produces blue color due to hydrolysis of X-gal

• Un-transformed cells remain white

Fluorescent proteins 

 

are members of a structurally homologous class of proteins that share the unique property of being self-sufficient to form a visible wavelength chromophore from a sequence of 3 amino acids within their own polypeptide sequence.

From Discosoma  -

DsRed 

RED FLUORESCENT PROTEIN

bright fluorescent Signals in 8-10 hrs after transfection

From sea anemone Entacmaea quadricolor - TurboRFP

(excitation/emission maxima are 553and 574 nm)

CYAN FLUORESCENT PROTEIN

A close-up of the adult red-cyan morph of the stony coral Acropora millepora. The red fluorescence is attributable to amilRFP and the cyan fluorescence is attributable to amilCFP 

ORANGE FLUORESCENT PROTEIN

Use of green fluorescent protein (GFP) as a reporter gene.

AIDS Research

CAIRO: In the latest string of research to combat AIDS, American researchers have inserted a fluorescent protein known as GFP (Green Fluorescent Protein) in cats to monitor the activity of altered genes which scientists say are more resilient to the feline from AIDS.

Vibrio fischeriGene: luxSubs: N-decyl aldehyde

Renilla reniformisGene: RlucSubs: Coelenterazine

FIREFLYGene:Fluc GeneSubs: Luciferin

IN-VIVO CELL TRAFFICKING(Jonathan Hardy et al)

• Collagen Induced Athritis

• T-cells as vehicles to deliver Immuno-regulatory proteins

• CD4+ T-cells with GFP-Luc translational fusion gene

• Luciferin injected

• Imaging

Bacterial Bioluminescence• Can be used to test for a specific bacterial

species or possible food born pathogen such as Salmonella typhimurium.

• The lux gene, responsible for bacterial bioluminescence, has been isolated and cloned.

• The cloned lux gene can be injected into a host-specific phage, which does not have the ability to express the gene.

• If the phage infects a host bacterium, light emission results.

GLO- FISHES

Tobacco Plants