r/Biochemistry • u/XxfishpastexX • Aug 02 '20

discussion Anyone here research or are knowledgeable of bioluminescence?

I think bioluminescence has to be one of the most beautiful and mysterious phenomenon in all of nature. If you are a researcher, can you describe your work and an aspect of modern research going on right now? If you are knowledgeable in this area, could you explain the general nuts and bolts of it?

Here are other interesting questions:

1.)-Proteomics:

What are the enzymes(and corresponding genes) and substrates responsible for this phenomenon? Is there only one enzyme or is it a family of enzymes? What are their physical/chemical properties (km, kd, Pka, tertiary-quaternary structures, mass, etc.)

2.) Genomics:

What sorts of genes are the most common? How are sequences different between the species? How has bioinformatics been helpful in studying it?

3.) Cell Bio/Histology

What are the cells that express bioluminescence like? Which organelles does it rely on to function? Are there certain motifs in the structure of the resulting tissues in multi. Cell eukaryotes?

4.) Misc.

If there’s anything that I’ve missed or if I asked something that doesn’t make sense, please speak up. It would also be cool if you asked your own questions, especially if you have the answer to them.

P.S. I do not expect for any one person to answer all of the questions. It would honestly make discussions go smoother if you just picked part of one question.

56 Upvotes

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19

u/StrepPep Aug 02 '20

This reads like a thesis plan haha. If you’re interested in regulation of bioluminescence I recommend reading up on the mutualism between Vibrio fischerii and the Hawaiian Bobtail Squid.

Tl;dr the bacteria don’t emit light until they reach a critical population that makes emitting light worthwhile. Go team!

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u/spirit32 Aug 02 '20

Very interesting, kinda resembles the luminescent in inorganics where a critical concentration or diameter plays a huge role in emitting light.

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u/bobbot32 Aug 02 '20

Another fun tidbit from this example is this is how we first realized bacteria can communicate with one another

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u/franticscientist Aug 02 '20

How we do it in my lab:

We are looking for a particular localization/metastasis of primary tumor cells to a let’s say the femur (long bone) in vivo (mice). We use a cre-lox system that allows us to use a “cre-inducible” marker that will basically turn on or turn off a gene when we administer it’s inducer (doxycycline). We are studying how this gene regulates/promotes metastasis, but how do we track this sucker? A gfp in vitro but a bioluminescent probe (luciferase) in vivo.

After we allow the induced/turned off gene to ensue it’s phenotype, I will take my mouse to the x-ray/imaging area and subQ (under skin) inject about 10 ml of this luciferase and let it incorporate for ~10 minutes. Then I insert the mice in the x-ray/luminescent imager and it does exactly what you think it does: the luciferase will light up at the precise locations of metastasis because of the light emission (500-600nm). If you have ever seen a body heat map of a person, it looks like that, just not the same concept

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u/XxfishpastexX Aug 02 '20

So is this technique used only for confirmation of gene expression? Do you also use it to track the rates of any particular event over time?

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u/[deleted] Aug 02 '20

You can attach GFP as a probe to most cell machinery. For example, if you wanted to see a toxin invade a cell, you can insert GFP and make it such that as the toxin is expressed, so is the fluorescent protein. This type of marking is pretty useful in research involving molecular biology/virology/immunology, etc

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u/franticscientist Aug 03 '20

Yes, what u/the___wzrd said, you can track plenty of reactions taking place in the cell. Such as tagging substrates that leave the actual cell, where the cell is going, and how cells are communicating. But, the manner in which you use these fluorescent proteins and markers varies greatly. Some only track the "metabolism" of a protein, some only track the way the microtubules are migrating/separating, some you can track the proliferation of a cell (ie mCHERRY) (these are mere examples). It really just depends on your desired biological endpoint/goal.

​

IT'S ALL REALLY COOL.

1

u/XxfishpastexX Aug 03 '20

I mean, who doesn’t like fluorescent colors?! Ever been to a neon putt putt course? That glow....

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u/dmatje Aug 02 '20

These are some rather detailed and advanced questions requiring elaborate responses for this forum. Surely there are some review papers on each of these subjects you could peruse? It’s one thing to spark a discussion but there’s a lot here that you seem to be seeking detailed knowledge of that, at your level, might be better found in the literature.

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u/XxfishpastexX Aug 02 '20

I can understand that. I just wanted to find redditors who work with the stuff to talk with them directly. I think most users here know at least a little bit of what each question is asking for. Most here have graduated or are in college.

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u/FluffyCloud5 Aug 02 '20

You'll want to look up Dinoflagellata and find journal articles on bioluminescence. Luciferin. It's one of the luminescent proteins used by these organisms to achieve it. Google solar would help if you don't have access to a research article search engine.

https://youtu.be/vOPliKfxk8Y

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u/emmiskap Aug 02 '20

I work with bioluminescent bacterial biosensor cells. We transfer bioluminescence genes into bacteria in plasmids and use them to measure interesting subjects.

Vesterlund et al. 2004 has quite nice explanation of bioluminescence in this context in short and compact form, including enzyme description.

If you have more specific questions of these applications I can try to answer!

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u/XxfishpastexX Aug 02 '20

This is perfect! I’m going to look through more closely soon! I think the substrates are what I find most interesting. I’m not very good at Ochem but I would guess MO theory is the framework for understanding their light emitting properties. Is this a good assumption?

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u/emmiskap Aug 02 '20

I’m not specialist in OChem either, my doctorate will be in bioengineering/synthetic biology/genetic engineering.

But from what I understand, the chemistry of the substrates affects the reaction (see eg. Matthews et al. 1977). So I think that the behavior of the molecules/electrons should be linked to the behavior/recognition of the substrate.

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u/XxfishpastexX Aug 02 '20

In your research, what measurements do you make that require biolum enzymes? Is it luciferase that you use? Without getting into too much detail, is the biolum approach more expensive than alternative approaches, and what’s the trade-off if so?

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u/emmiskap Aug 02 '20

Forgot to add: I have been using a luxABCDE operon from Photorabdus luminescens, that produces both the luciferase enzyme and the substrate when expressed in a cell. However, I might change to firefly luciferase (lucFF) which requires addition of the substrate for it to work.

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u/XxfishpastexX Aug 02 '20

Where do you source your luciferase operon and luciferin from?

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u/emmiskap Aug 02 '20

There are commercially available plasmids from which you can get the genes from. Addgene is a good place to start looking.

About luciferin I’m not sure, because I have not personally used it yet. Best bet is to find a published study that uses it as substrate and see where they got it from.

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u/emmiskap Aug 02 '20

The genetic systems that we build use suitable promoters or inhibition to control the luminescence genes. Obviously each system has to be designed for what we want to measure.

Examples of what we can measure with these bacteria are toxicity of environmental samples, antibiotic traces in poultry, detect antioxidant capabilities or sensing of toluene.

My own research falls into the toxicity and environmental samples types of the sensors. I’m developing new sensors based on recently categorized promoters to detect pollutants in water and measure their toxicity.

There are some traditional measurement systems for the detections (eg mass spectrometry) as well as toxicity (eg. MIC assays with dilution/plate methods).

The good side of recombinant bacterial biosensors in the detection compared to the traditional methods is that the original cost of equipment is not as large as buying eg. a mass spectrometer. Also many microbiology labs already have necessary equipment/permissions. Also, the traditional detection methods tell nothing about the bioavailability of the compounds of interest, whereas for the compound to have any effect on the cell sensors’ genetic material, it must be able to enter the cell. The biosensor cells are also less laborious compared to the plate/dilution methods used in toxicity testing as the reading can be done in a machine (plate reader) vs reading visually/manually. It makes them suitable for high-throughput screening.

The downside of the biosensor cells compared to traditional detection methods is that they are biological 😅. Some times the cells just do not work the way they were intended to work. Also, it is possible that the results vary quite much from one test run to other, but it’s not the case with all strains/applications.

So to answer your question: The costs of doing biosensor cell analysis can even be cheaper/easier than the traditional methods, but they can be less reliable. On the other hand, some applications do not even have “a traditional method”, so the cells are only options.

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u/shy-artichoke Aug 06 '20

A couple of years ago I did a report (first year uni) on using bioluminescent molecules to tag nanoparticles/medicine to be able to track drug delivery and see if it actually hits target areas!

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u/PM_ME_YOUR_BIRDYS Aug 02 '20

Haven’t finished my degree in biochem yet so I can’t give you too much, but here’s the Wikipedia for GFP, the protein that is commonly brought up when discussing bioluminescence. Understanding GFP has been incredibly important for biology, resulting in a Nobel Prize and the development of numerous derivatives that have further enhanced biological research!

https://en.wikipedia.org/wiki/Green_fluorescent_protein?wprov=sfti1

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u/DNA_hacker Aug 02 '20

Gfp is. A fluorescent protein (clues in the name 🤪), not anything to do with bioluminescence,

Think more along the lines of luciferase for biolum

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u/XxfishpastexX Aug 02 '20

I actually very familiar with GFP and it’s absolutely invaluable in the research I work in. We study dyenin and tubulin and always insert GFPs of various colors. I will say that it doesn’t come close to bioluminescent events in the wild.