Friday, August 26, 2011

A Biotechnological Advancement..

Bio-luminescent bacteria occur nearly everywhere, and probably most spectacularly as the rare "milky sea" phenomenon, particularly in the Indian Ocean where mariners report steaming for hours through a sea glowing with a soft white light as far as the eye can see.

Sea water bacteria of Genera Vibrio and Photomicrobium produce bioluminescence with the help of enzyme Luciferase. In the sea, bioluminescent light is concentrated in the blue window of greatest optical transparency of seawater. Most organisms emit between 440 nm and 479 nm (visible range of light). Some cnidarians have green fluorescent proteins that absorb an initially blue emission and emit it shifted towards the green (~505 nm). By using Recombinant DNA technology we can produce luciferase in vitro. The gene cassette responsible for the expression of Luciferase can be PCR (Polymerase Chain Reaction) amplified in vitro using primers specific for the gene. These primers possess restriction sites which can be cleaved by restriction enzymes and cloned in a suitable expression-based vector system. The vector system is a Plasmid - an extrachromosomal, self replicating DNA entity. The plasmid can be opened using the same restriction enzymes (as for the gene of interest - in this case, Luciferase) and could be ligated with the luciferase cassette. Once cloning is performed the plasmid can then be transformed into the host bacteria, which in this case would be E.coli (most commonly used as a host expression system). Growth of these recombinant bacteria allow expression of the desired enzyme which can be subsequently purified using differential gradient centrifugation and other biochemical techniques.

For those who are unfamiliar with the rDNA technology please refer to following: http://www.blackwellpublishing.com/allison/docs/sample_ch8.pdf


In a continuously aerated system this enzyme can produce light. This chemical reaction consumes oxygen - aerobic system is, thus, necessary. 


Empirical reaction:
FMNH2 + O2 + RCHO -----------> FMN + RCOOH + H2O + "LIGHT" 

Bacterial luciferin is a reduced riboflavin phosphate (FMNH2) which is oxidized (FMN) in association with a long-chain aldehyde, oxygen, and a luciferase enzyme.

Luciferase catalyzed reaction
The reaction is catalyzed by ‘luciferase’. The enzyme itself is never consumed in a reaction, it only catalyzes it. The mere presence of the protein molecule is sufficient enough to trigger the reaction. One enzyme molecule can be engaged only in one reaction at a time, however when carried out in sufficiently higher scale, it will produce light with greater intensity as a cumulative effect.

The enzyme, after the generation of light is free to participate in another such reaction. Thus the enzymes never deplete. However, they loose their functionality - denature - after  a while i.e., shelf life of the protein molecule. Thus, they need to be replaced if used in-vitro. When supply of raw material and aerated conditions are maintained the chemical reaction is triggered. This light, if amplified in a correct manner (using mirrors / bleach), will be an  alternative source of light. 

Kindly refer to our FAQ section on facebook page.


I, on behalf of Bio-Solutions, request all engineers / physicists, interested in making this project a success, to make suggestions here.


Objective - To have a different way of looking at sources of light for household usage
Vision - A brighter India, A better India.
Utility - Rural regions, roads and highways, coastal regions, rail-routes.  
Beneficiaries - Everyone.
Issues - 
  1. Success rate of rDNA cloning is relatively low. 
  2. Applicability in rural regions is a challenge.
  3. Cost effectiveness as compared to solar energy conservation methods - Solar Panels.
  4. Waste in the form of bio-mass disposal.
  5. Handling and awareness.

29 comments:

  1. Great work!!!!
    Fabulous combination of Management,Biology and Chemistry

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  2. Thank you,
    It can answer the question of reach of electricity, specially in the hilly regions of India.

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  3. have u estimated the cost of production..

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  4. yes,

    Raw materials required for a biobulb:

    50 Gms of pre-aerated industry agar medium per bulb made up of translucent plastic material.

    22 microns / 45microns filter to avoid external contamination or leakage.

    e coli culture strain with known doubling time (for the replacement of the media and bio mass)

    it is feasible.
    thanks for a valid concern :)

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  5. hmmm a gr8 idea. I wish it works out. ATB guys.

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  6. another major breakthrough like the CFL.....??

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  7. @Ashish slightly more greener than CFL..
    All the best to BioSolutions

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  8. The idea is fantastic guys. I hope the execution is even better. ATB!

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  9. I have few genuine questions. What ever small knowledge I have about bio luminescence I believe that its about a phenomenon which allows some organisms to make their presence felt to other organisms. Not a single organism uses it as a source of light to search for food or hunt.
    Thant brings few genuine questions to my mind.

    1>
    "... amplified in a correct manner"
    Light is energy, it cant be amplified without providing external energy?

    2>
    How much light will be generated from 50gms of Agar?
    For comparison a 100watt bulb or a 15watt LED generates ~1500 Lumens which will sufficiently illuminate a rural house
    where as even if I put few kilo-grams of fire-flies together it will still be less than 2-3 lumens. How do you plan to fill up this enormous gap?

    3> Same question is Bhavin's -> Cost estimation?

    4> Wouldn't improvising on Solar energy be a practical answer :)

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  10. Hi Nikhil,
    Both terrestrial and aquatic fauna use this property to attract a prey (food) and as a defense mechanism, also for attracting a partner for mating.

    Answers to your queries:
    1) Yes, you are correct, energy remains conserved, however with amplification we can deflect the light particles in 360 degrees achieving a good spread, the objective is to minimize the loss of energy by reflection.

    (total energy= reflected + refracted + absorbed)

    mirrors, bleach etc create that effect.

    Please watch this to understand what I am saying-
    https://www.facebook.com/photo.php?v=1661193868156

    2) for that you need to go through our youtube channel, where we have shared a couple of informative videos. coincidentally a research on similar lines was conducted by scientists at Cambridge university.

    3) Cost and budgeting is what we are working on for now. we are trying to find alternatives for the industrial agar and media.

    the initial process of cloning is expensive, no doubts, but with the effective positive screening percentage of around 90% we should be able to minimise the cost.

    4) yes, solar energy is the cheapest source of energy, if conserved properly we can solve many issues. we are not challenging it.

    we are just proposing another way of looking at things.
    :)

    thanks for your valuable response.
    do share our work with all your friends and colleagues.

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  11. One of the wonderful work, with due consideration to Complete research..Impressed..

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  12. Hello, Definitely an Interesting Concept, if executed well, can prove beneficial in terms of socioeconomic aspects.. Good going.. All The Best to your team!!!

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  13. It is a great Idea...all the very best! :)

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  14. NIce concept guys... But can these bacterias survive in the harsh, tropical conditions of rural India? I mean these organisms come from 100s of kms below the sea level rite....

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  15. Hi Yogesh,
    Thanks for taking interest.

    You are absolutely right. The marine bacteria - eg., Vibrio, won't be able to sustain, as they require a different media (high salt concentration) and different conditions (pressure)to propagate life. The bacteria which we are going to use are, thus, not the marine bacteria themselves.

    Recombinant DNA technology (genetic engineering) allows us to modify a single celled organism, such as E coli, to our benefit. The gene responsible for the production of enzyme Luciferase will be CUT OUT from the genome of the marine bacteria. And will be INSERTED into the genome of an E coli bacterial cell. Now this recombinant strain of E coli will express the gene.

    Also by tagging or in-tandem cloning of these genes with the genes that express extra-cellular protein molecules eg receptors, channels etc, we would be able to have the enzyme molecules on the outer surface of the E coli cell.

    The outer surface of E coli cell now acts as the site of enzyme catalysed reaction, instead of the cytosol in case of a marine bio-luminescent bacteria. When an aerated medium is provided along with the substrate (luciferin) the chemical reaction would take place and light will be produced inside the solution.

    Regards,

    Team BioSolutions

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  16. very good idea all the very best

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  17. i have a question..
    the emmission of this light is in the blue or green range...how good is it for visibility, since it does not encompass the whole visible rnage from 400-700nm, right?

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  18. Is this idea scalable -- can you culture bacteria to back mass production?

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  19. Good luck for the innovative idea...................my good will is for all who is going to be the part of it......i m also ready if i m needed for it .

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  20. Thank you for your support.
    We surely need it.

    @Bhavin: Hi, apologies for the delay, as we were a little busy with festival.

    Yes you are absolutely right about the natural emission patterns. As always, there is a BioSolution.

    We introduce site directed mutagenesis to produce specific mutant luciferase genes that emit yellow light.
    Citation: http://2010.igem.org/Team:Cambridge/Bioluminescence/Colour

    Now, a mixture of Blue and Yellow light producing bacteria can be used to have the desired white light.

    In case you want to break this light further, using chromatographic filters is an option available.

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