Low‐light image analysis of transgenic organisms using bacterial luciferase as a marker

Abstract

Methods for measurement of a novel light-emitting reporter gene system in bacteria, yeast, plant cells, plant tissues and intact plant organs are described. The principle underlying the assay procedures is the bacterial luciferase catalysed oxidation of reduced flavin mononucleotide (FMNH2) in the presence of the ten carbon aldehyde decanal, to yield FMN, decanoic acid, water and a photon of light at 490 nm which can be captured by X-ray film, a photomultiplier tube or, for in vivo measurements, an image-intensifier coupled to a video camera. This light measuring assay system is sensitive, easy to use, inexpensive, does not require radioactivity, and has been used successfully for rapid detection of bacterial transformants, the quantitative measurement of transient and stable gene expression in bacteria and yeast, and in vivo measurement of temporal and spatial gene expression throughout plant and animal development.

Real-Time Monitoring of Single Bacterium Lysis and Leakage Events by Chemiluminescence Microscopy

“…Monitoring CL from Live Single Bacterium. Imaging luminous bacteria colonies on a plate is not difficult for conventional cameras, but visualizing the faint light from a single bacterium on the microscope is challenging. The successful detection of a single luminescent bacterium is based on an ultrasensitive optical detector−ICCD camera.…”

Imaging of light emission from the expression of luciferases in living cells and organisms: a review

“…In a series of studies (93)(94)(95)(96)(97) it was shown that Vibrio harveyi luxA, luxB and a luxAB fusion could be successfully expressed under various bacterial, mammalian and viral promoters and visualized by photoncounting imaging in Gram-positive Bacillus thuringiensis and B. megaterium (cf. Fig.…”

“…Bacterial luciferase expression was also successfully imaged in tomato leaves and fruit (Fig. 5a, b) (97). Firefly luciferase (Pluc) was also expressed in some of these experiments.…”

“…Langridge et al have also imaged bacterial luciferase expression in the tomato, Lycopersicon esculentum (see Fig. 5a, b), and in the potato, Solanum tuberosum (97).…”