Heavy metal detection with reporter genes

One month ago I was reporting how GFP has been turned in a mercury biosensor for in vivo application, now it's the turn of another "me too" sensor engineered with bacterial luciferase (luxCDABE). The two technologies are somewhat complementary, in effect the first one resides on a mutant GFP that binding mercury stop the fluorescence, in the second paper the mercury-inducible promoter (P-mer) drive the transcription of bacterial luciferase. Mercury (as far as 100 picoMolar) is able to induce luciferase expression with a maximum of activity around 8 hrs. Such biosensor is engineered in two bacterial strain (P. putida and E. aerogenes) known to be resistant to heavy metals (in fact they were isolated from polluted soil), so this biosensor seems to be adequate for the detection of mercury in soils and associated environments.

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Fu, Y., Chen, W., & Huang, Q. (2008). Construction of two lux-tagged Hg2+-specific biosensors and their luminescence performance Applied Microbiology and Biotechnology, 79 (3), 363-370 DOI: 10.1007/s00253-008-1442-1

b-lactamase reporter for selecting yeast clones


Large scale therapeutic protein production is not so cheap, and it is well known that pharmaceutical industry is under constant pressure to reduce the overall costs. When post-translational modification requirements allow to adopt in production yeast systems instead of mammalian cells, the cost is significantly lowered. In this latter case, the selection of high-producing clones is very important and the possibility to visually discriminate just in 3-4 classes the productivity of thousands of clones in a few days by a single operator is noteworthy, especially if the method is cheaper than classic ELISA or fluorescence/bioluminescence test.

So Hribar and colleagues from the National Institute of Chemistry of Ljubljana, started to deal with alternative “low cost” reporter genes like the beta-lactamase, to find out a rough measure of the protein of interest. The autors worked on Pichia pastoris, a yeast strain just licensed to more than 100 companies also for recombinant heterologous protein production. The short technical report published on the april number of Biotechniques, will not warm up any reporter geek - like me - that loves stupid performances (who really need 9 orders of magnitude linearity???), but the colorimetric method of such 39 kD b-lactamase enzyme would be an asset for smart bio-companies which prefer pragmaticity to geekiness.

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Hribar, G., Smilović, V., Lenassi Zupan, A., & Gaberc-Porekar, V. (2008). β-lactamase reporter system for selecting high-producing yeast clones BioTechniques, 44 (4), 477-484 DOI: 10.2144/000112730

In the beginning was the fat

So you are an imaging scientist. Probably you master an in vivo imaging system (CCD) and spend most of your working time in a lonely dark room collecting some spare photons coming out from a bioluminescent mouse, or frog, or zebrafish, or tobacco plant. Get out! Viviani and colleagues from the Sao Carlos University made some promenades during summer nights to collect adults and larvae of Aspisoma lineatum (Brazilian fireflies). Then they made some in vivo imaging with fireflies, just to discover that bioluminescence was not only a characteristic of adult lanterns. They found that also in larvae, a weak luminescence was associated with the fat body, hypothesizing that the origin of photocytes was from the fat body. Intriguingly, another group (Oba et al.) recently found that firefly luciferase displays fatty acid CoA activity, suggesting that originally luminescence was a side-product of a metabolic activity. So, in the beginning was the fat. This discovery opens other questions: light-emission is useful in adults for reproduction. What is the role of light-productions in larvae?

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Viviani, V., Okawachi, F., Scorsato, V., & Abdalla, F. (2008). CCD imaging of basal bioluminescence in larval fireflies: clues on the anatomic origin and evolution of bioluminescence Photochemical & Photobiological Sciences, 7 (4) DOI: 10.1039/b718016k

OBA, Y., SATO, M., OJIKA, M., & INOUYE, S. (2005). Enzymatic and Genetic Characterization of Firefly Luciferase and Drosophila CG6178 as a Fatty Acyl-CoA Synthetase Bioscience, Biotechnology, and Biochemistry, 69 (4), 819-828 DOI: 10.1271/bbb.69.819

New instruments for reporter assay detection

Directly from Biotechniques Weekly (april 10)...

1) Rapid whole-animal imaging in infrared Experimental Biology
2008, San Diego, CA, Apr. 6—The Pearl Imager, due to be introduced by Li-Cor Biosciences (Lincoln, NE) in May, is a high-speed infrared imaging system designed for whole-animal studies. The small-footprint (41 x 41 x 66 cm) system takes less than 30 seconds to scan on three channels: 700 nm, 800 nm, and white with six logs of dynamic range. Pearl Imager includes a detachable mouse imaging bed equipped for heating and anesthesia. Li-Cor Biosciences www.licor.com

Related reportergene news (infrared imaging):

2) Luminescent microscope
2008 Munich, Germany, Mar. 31—Olympus introduced the LV200 Luminoview Bio-luminescence microscope (BLM). The microscope features a specialized optical design to maximize light collection, enable dual-color luminescence, as well as provide brightfield and fluorescence overlays. The microscope provides bio-luminescence microscopy imaging for small organisms and slice cultures as well as at the single-cell scale. The microscope's optics are designed to provide the straightest path and shortest distance between the object and the camera, to ensure that as much light as possible reaches the CCD chip. The microscope is fitted with a fully controllable environment to enable long-term live cell imaging and is fully integrated with the company's imaging and analysis software. Olympus Life Science Europa GMBH www.microscopy.olympus.eu

Related reportergene news (luminescence microscopy):

BLI and cancer #1

Recently, more than one bacteria strain has been described to germinate and grow only in the hypoxic regions of solid tumours.

It comes to mind the ancient 1890s when Dr WB Coley observed tumour regression after postoperative bacterial infections. The tricky question now is: does bacteria should be a new weapon against cancer? It is conceivable a cancer therapy employing safe-bacteria as magic bullets? To get off the ground, we need first a tool to visualize the process of bacterial-tumour targeting, to quantify bacterial growth in target tissues (better if non invasively), and to monitor bacterial migration in real-time. Bioluminescence imaging? Yes, of course: which other tech is becoming such cornerstone in biomedical investigation?

Nowadays, JJ Min and colleagues from Chonnam National University introduce in Nature Protocols a detailed “how to” for imaging bacterial luciferase (lux) expressing bacteria in small living animals. This genetic engineering and imaging protocol would be a powerful approach for the quantitative visualization of the distribution of bacteria in mouse tumour models.

What about the reporter gene? Compared to more celebrated luciferases, the advantage of the lux luciferase is that it does not require any exogenous source of substrate to produce bioluminescence, and this would be a plus in some hidden niches of tissues where the substrate should be difficult to administer. But some possible drawbacks need to be considered:
  1. the weight: in fact it is more correct to refer to lux luciferase as to the “lux operon”, because such complex is more than one enzyme: the lux operon (like the lux CDABE from Photobacterium leiognathi) encodes, in roughly 10 kb, several proteins required for the bioluminescence, including bacterial luciferase, the substrate producer and the substrate-regenerating enzymes. Managing 10 kb in your vector needs some mastering abilities (and some space).
  2. the light: the light color is bluish-white, a hue strongly absorbed by mammalian tissues because of the haemoglobin. This would really hamper the sensitivity of the system in vivo.
  3. the oxygen paucity: yes, bacterial luciferases require oxygen, an asset in the hypoxic regions of tumours!

Min, J., Nguyen, V.H., Kim, H., Hong, Y., Choy, H.E. (2008). Quantitative bioluminescence imaging of tumor-targeting bacteria in living animals. Nature Protocols, 3(4), 629-636. DOI: 10.1038/nprot.2008.32