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BTN - 305
Harnessing Bioluminescence
Fireflies for food securityAbhishek Guddu
Sonali SrijanIndian Institute of Technology, Roorkee
Contents
1.Details of the paper
2.Discussing Bioluminescence
3.Luciferase- luciferin
4.Fauna that exhibits bioluminescence
5.Applications
6.Opportunities
About the paper : Bioluminescence-Sensing Assay for Microbial Growth Recognition
Authors: Heba Ramadan Eed, Nora S. Abdel-Kader, Mahmoud Helmy El Tahan, Tianhong Dai, and Rehab Amin
Regional Center for Food and Feed (RCFF), Agricultural Research Center (ARC), Giza, Egypt; Chemistry Department, Faculty of Science, Cairo University, Giza, Egypt; Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, USA; National Institute of Laser Enhanced Sciences, Cairo University, Giza, Egypt
Source: www.sciencedirect.com/
What is Bioluminescence? ➢ Production and emission of light by a living organism
➢ A form of chemiluminescence
➢ Principal chemical reaction involves the light-emitting pigment luciferin and the enzyme luciferase, assisted by other proteins such as aequorin in some species
➢ In some species, the type of luciferin requires cofactors such as calcium or magnesium ions, and sometimes also the energy-carrying molecule adenosine triphosphate (ATP).
➢ Luciferin reacts with oxygen to create light
➢ Aequorin is a calcium-activated photoprotein that generates blue light by itself upon contact with Calcium ions.
Shimomura O. (August 1995). "A short story of aequorin". The Biological bulletin. Source: Wikipedia
Going with the glow : Fauna that exhibit Bioluminescence
➢ occurs widely among animals, especially in the open sea, including fish, jellyfish, crustaceans, and cephalopod molluscs
➢ Fungi and bacteria
➢ invertebrates including insects: Fireflies and Glow worms
➢ Dinoflagellates present in the surface layers of the sea
1. An angry female angler fish from “Finding Nemo”
Source: www.popsci.com
2. FirefliesSource: www.pinterest.com
3. Dinoflagellates: Bioluminescent Mosquito Bay in Puerto RicoSource: thebackpackers.net
4. The yellow bioluminescent ring on this female octopus may attract mates.
Source: http://ocean.si.edu/bioluminescence
5. Bioluminescent Mushroom Source: all-that-is-interesting.com
Luciferins and Luciferases
Luciferin is a complex molecule that is similar to Chlorophyll. The bioluminescent light that is emitted when the luciferin is oxidized is a byproduct of the reaction.
Also, this light is called, "cold light" because there is almost no heat that accompanies the emission of light.
It is thought that this reaction was NOT initially evolved to produce light, but rather it was a mechanisms of binding up excess oxygen. Excess oxygen, particularly when it is in radical form (not paired with another oxygen atom and having an one excess electron) cause great damage to cells, so it is vital that all cells have mechanisms of getting rid of oxygen radicals. However, the luciferin-oxidation reaction was probably "switched" to serve the purpose of emitting light as its primary function millions of years ago.
Applications
More than 10 million cases per year (India)Sources: Apollo Hospitals and others.
source:https://www.google.com/image/jpeg64
WHO Global data (2010)
582 million: The number of cases of 22 different food-borne diseases experienced in 2010
351,000: The number of associated deaths
52,000: The number of deaths caused by the bacteria Salmonella
37,000: The number of deaths caused by the bacteria E. coli
35,000: The number of deaths caused by norovirus (a virus that’s the leading cause of disease outbreaks from contaminated food in the United States)
source:http://www.searo.who.int/entity/world_health_day/2015/whd-what-you-should-know/en/
The food industry has long
used fireflies’
light reaction to
detect good food gone bad
Traditional Method
● Expensive laboratory equipments
● Time taking(longer incubation period around 24 hours)
● Tedious and labour intensive
Source:www.essential-microbiology/bioluminescence-19857376
Food testing using ATP (Bioluminescence) Technology
Test kits containing firefly luciferase and luciferin are used to detect the presence of ATP, a compound that’s found in all living cells; this includes any live microbes like Salmonella or E. coli that might be lurking in our food.
Luciferin +ATP +Luciferase Visible Light (Intensity detected by luminometer)
Source:www.essential-microbiology/bioluminescence-19857376
Intensity vs ATP concentrationThe more ATP, the brighter the light, so the
intensity of luminescence even reveals how many bacteria are present.
Since 1960s, this bioluminescent test has been able to detect even tiny amounts of microbial contamination, using very sensitive instruments to measure light production.
Quick: It requires mere minutes instead of the days needed to detect contaminated food by growing bacterial cultures.
This handy bioluminescent ATP assay, which now employs synthetic luciferase, is still widely used to ensure food safety by detecting microbial contamination in milk, soft drinks, meat and other commodities.
Source:https://www.google.co.in/search?q=atp+standard+curve+luminescence&espv
Luc gene as “reporter” for the activity of other genes
In this application, researchers splice the luc gene together with a specific gene they want to study, and then insert this spliced DNA into living cells.
Whenever the spliced DNA gets transcribed, the cells will manufacture luciferase. When luciferin is added, these cells will respond by lighting up. This technique has been used, for instance, to find out exactly when and where specific plant genes get turned on.
To learn about particular genes regulating plant growth, biologists have spliced the luc gene into different bits of plant DNA. When plants are sprayed or fed with luciferin-containing water, the leaves will glow whenever luc gene gets turned on. This allows researchers to identify specific genes regulating plant growth at different times and locations.
Such reporter genes have also provided powerful tools for studying diseases, for developing new antibiotic drugs and for gaining new insights into many human metabolic disorders.
source:https://pubs.acs.org/cen/science/84/8414biolum.html
Bioluminescent ImagingFireflies have also helped scientists develop real-time, noninvasive imaging to see what’s happening inside living organisms.
When luc genes are used to label particular cell or tissue types, very sensitive cameras can be used to detect their light inside the live animal.
Xenogen Corporation based in Alameda, California, has developed mouse assays that use the luciferin-luciferase reaction to trace tumor growth in live mice. Mice bearing tumors are tagged with luc gene which express luciferase and are further injected with luciferin. Researchers then use a sensitive camera system to view, without killing the mice, the tumor and any effects of the different cancer agents .
Tumor cells can be grown in culture medium and then treated with different drugs. Using luminescence-based tests to measure cell viability, those drugs most effective at killing tumor cells can be quickly identified.This way,potential new chemotherapies for treating cancers can be tested.
source:https://pubs.acs.org/cen/science/84/8414biolum.html
source:https://www.google.co.in/search?espv=2&rlz=1C1PDZP_enIN707IN707&biw=1920
Comparison between conventional methods and BLI
Conventional methods(MRI,PET etc.)
600k USD - 1.2 million USD
30minutes - 90 minutes
15 - 20 minutes
30 - 90 minutes
Required
Yes
BLI
<500k USD
20 minutes
1s - 2minutes
20 minutes
Straight forward
No
Equipment cost
Preparation Time
Scanning Time
Data Analysis Time Experts Exposure to radiations Source:https://www.google.com/imaging/techniques/9763456
Technology Comparison
source:http://www.slideshare.net/Funk98/bioluminescence-19857376
Bioluminescent AssaysBioluminescence is also frequently used in assays for measuring cell proliferation, apoptosis, drug
metabolism, and kinase activity. The simplicity and sensitivity of bioluminescence assays make them easy to automate, especially for drug screening. Pharmaceutical researchers can screen the activity of more than 100,000 compounds a day using bioluminescence chemistry.
To help discover new treatments for antibiotic-resistant tuberculosis, scientists have infected mice with luciferase-labeled tuberculosis bacteria. They then treat the mice with various anti-tuberculosis drugs and use bioluminescence imaging to monitor the bacteria inside. source:https://pubs.acs.org/cen/science/84/8414biolum.html
source:https://www.google.co.in/search?q=bioluminescent%20assay source:https://www.google.co.in/search?q=bioluminescent%20assay
Other usage Marine-based systems also have practical applications.
Because the photoprotein aequorin requires Ca2+, it is often used to measure calcium changes inside cells.
● One of the most well-known developments to come out of bioluminescence research is the discovery of the green fluorescent protein (GFP). Ocean Research & Conservation Association's Widder says "it's probably the most important discovery in the last two decades" because of GFP's impact on genetic engineering and cell biology. While GFP is not a bioluminescent protein, it serves as an accessory emitter by receiving energy from a luciferin-luciferase reaction and re-emitting it as green light. Osamu Shimomura, a retired chemist from Marine Biological Laboratory, in Woods Hole, Massachusetts, discovered the protein while he was researching the bioluminescent jellyfish Aequorea victoria.
source:https://pubs.acs.org/cen/science/84/8414biolum.html
GFP mice have been engineered as models to study disease.Source: imgur.com
Interesting Opportunities
Military
Biodegradable landing zone markers
Bioluminescent “Friend vs foe” markers
Plants and trees
Smart crop
When crops need water or nutrients they could go red,yellow or green “alert” to give farmers early warning for the same and even for disease or invasion by the pests.
Trees as Street lights:Luminescent trees can replace street lights.Trees would require air,water and soil nutrients only to maintain their duty of lighting.They would come “on” at night and go “off” during the day time.
source:https://www.google.co.in/search?espv=2&rlz=1C1PDZP_enIN707IN707
source:http://www.slideshare.net/Funk98/bioluminescence-19857376
Thank you
Thank you