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March 2016 Archive

Posted by Chris on March 28th, 2016  ⟩  0 comments

In the Search for the Essentials of a Minimal Life-form - Learn about the obstacles Venter, Smith and Hutchison faced when they set out to examine what those essential components of life are.

There has always been a small portion of humanity that sought to divest of the clutter and accoutrement of daily life…to reduce their dependence on the vulgar wants and desires of civilized people and live a frugal and minimal life. Monasteries and hermits have been promoting that life strategy for hundreds, if not thousands, of years. In the mid-1800s, Henry David Thoreau certainly argued for such a need in his great work “Walden.” And in our own, more modern world, we can see a similar enticement, or at least vicarious amusement, in the nearly two decades of reality show after reality show in which contestants simulate that very notion of throwing off the vestiges of “stuff” and existing in a bare minimal state for a while.

So what are the barest essentials that you need to survive? I imagine that the answer to that question may have changed considerably over the last 40-50 years. We have grown to depend and “exist” on so many technical wonders and achievements; it’s difficult for many of us now to even conceive of our existence without the devices or tools that seem to be permanently attached to us day in and day out.

Of course, the answer depends greatly on what “life” we’re talking about. Any grade school child will rattle off the requisite “food, water and shelter” line that has been taught for generations. But are those the only essential things needed to live?

hen we think about these questions, we take for granted the building blocks of our bodies; our muscles and bones that let us forage for that ‘food and drink’. We forget about the cells that make up those organs, each one running tirelessly for the duration of our lives; replicating, protecting and building us into who we are. We forget about the DNA that is the building block of all those cells; constantly replicating, editing and self-correcting in order to ensure that we remain who we started out to be.

Then there is the fact that all things fail eventually. We know that viruses invade our DNA and change them, altering the genome into something different, for good or for bad. We know that cells can mutate into cancers or can die off unexpectedly due to illness or disease. Either or both of those things can cause our muscles and bones can become damaged. And without some kind of cellular or genetic treatment, the “essentials” of our lives become very different from what our children are taught in school.

More than twenty years ago, a group of scientists led by Craig Venter, Hamilton Smith and Clyde Hutchison, set out to examine what those essential components of life are and to attempt to create such a minimal life form. And not just for any sort of Frankenstein, monstrous amusement, but in order to solve a problem. The problem is that when we attempt to use cells to help us achieve solutions to problems, in the medical field and so on, those cells come with genetic baggage. We don’t know what that baggage does or whether it will adversely affect our goals. So the baggage has to go. Only, we also don’t know what part of a cell’s genome is baggage and what part is essential. At long last, Venter’s group finally did it, and the science news world literally erupted over their success as reported in the journal Science.

In 1996, Mushegian and Koonin estimated that the “minimum set of necessary genes” for a bacterial cell was close to 260 genes by cross-comparing the miniscule genome of the bacteria, Mycoplasma genitalium (which has only 525 genes) to Haemophilus influenza (which has 1703 genes). Working with that estimate, Venter’s group began to experiment with Mycoplasma mycoides, a bacterium that, while larger than M. genitalium, also replicates faster which makes it easier to research. The idea was to build a bacterial cell that had only the barest essential genes required in order to survive, reproduce and thrive.Certainly, they did not think that it would be as difficult as it turned out to be.

They quickly ran into two key problems: First, there are a LOT of genes which we still do not fully comprehend or even know whether they are essential. How many? Well, it ends up that after all was said and done, we still don’t know what 1/3 of the essential genes that Venter’s final M. mycoides (named syn 3.0) needed.That’s 149 essential genes that we have no idea at all about! Second, life has a habit of enjoying a duplication of effort in order to ensure survival. So if one vital gene gets mutated or truncated, there is a backup to make sure that life goes on. But when screening for essential or nonessential genes, that gives rise to a number of false positives.

If you delete a gene, and the cell continues to survive, you assume that the gene was nonessential. But if you then delete the BACKUP gene, the cell might suddenly die.So which gene was the essential one; the first, the second or both?All of that redundancy took an enormous amount of time to sift through, using a variety of cutting edge Tn5 mutagenesis and DNA synthesis. And there are also the quasi-essential genes. These are genes that aren’t absolutely necessary to survive, but for whatever reason are very necessary if the goal is to grow or reproduce efficiently.

There is also the trade-off between genome size and growth rate.It took the team several rough years to reduce the genome to 516 genes (only nine fewer than the original M. genitalium) and several more to finally reduce the genome to the Syn3.0’s “working approximation of a minimal cell” at 473 genes. So Syn3.0 actually only becomes the compromise between small size and researchable growth rate.

So what makes all of these genes essential? We’re still not sure. To be clear, this isn’t an absolute result. Being essential means different things depending on the environment in which something lives. Certainly, Native Americans in the Amazon jungle have different essential requirements than the Inuit do in the Polar circle. So is the case for bacteria. Free living bacteria require a wider range of redundant and novel genes to cover a wide variety of unknown circumstances. That’s just life. But bacteria such as M. genitalium live in an isolated and closed environment, allowing them to lose genes over time that are no longer necessary to their existence. Put those bacteria into a harsher existence and you’ll probably find that they’re no longer able to adequately cope.

As a species, humanity enjoys priding itself on its adaptability and tenacity under stress. Certainly we have developed ways to survive, and even thrive, under even most of the harshest conditions that we have found on our planet. But at the core of that, we must remember that we do not do it alone. We adapt and thrive based on the DNA, the cells and even the bacteria within us that together allow us to conquer an ever widening arc of unknown circumstances; further proving that “what is essential” is just as elusive an idea as “what is independent”.


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Category Code: 79105 79102 79101

Posted by Karen on March 16th, 2016  ⟩  0 comments

If you find yourself questioning the difference between various luciferase or luciferin types, this guide will set it all straight. Find out what to look out for when shopping for luciferin, why you should choose one type over another, what advantages exist between different luciferins, the importance of purity, storage and more.

Luciferin/Luciferase ultimate shopping guide



GENERAL CONSIDERATIONS

1. Solubility Considerations:

If you just type D-luciferin into our search bar (or any search bar), you’ll get options that include D-luciferin potassium salt, D-luciferin sodium salt and you might see D-luciferin firefly, free acid. So what is the key difference between all three D-luciferin types? Well, for potassium and sodium luciferin there isn’t much difference at all. The two products can be used interchangeably in experiments. However, the sodium salt version is slightly more soluble than the potassium version. And solubility is where the differences really rest, because when it comes to the free acid form of D-luciferin, you’re going to run into some issues. The free acid form is not like the water soluble salts. It requires pH adjustments in order to be made soluble.

Therefore, when it comes to choosing between the three, solubility should be a major consideration.

Note: Why would anyone choose D-luciferin firefly, free acid? Some researchers choose the free acid version despite solubility issues because it contains more photons/milligram than D-luciferin sodium and D-luciferin potassium salts.




2. Assay Considerations:

The type of assay you’re working with is going to influence your luciferase choice, which in turn will influence your luciferin choices. So why luciferin over coelenterazine, vice versa or both? Let’s look at it this way:

appropriate luciferases for assays - luciferin shopping guides



Other things to consider within this topic are some characteristic differences between FLuc (Firefly) and RLuc ( Renilla) such as ATP dependence, peak emission and protein half–life.

luciferin product shopping guide - luciferin table of characteristics



Finally, you need to consider situations that may or may not optimize your experiment. For example, in very small volumes, the FLuc/RLuc dual-luciferase assay is not ideal due to well constraints. Another example, which would lead you to choose coelenterazine instead of D-luciferin has to do with its size. When referring to the chart above, you can see that Gaussia luciferase and Renilla (compatible with coelenterazine) are considerably smaller than available firefly luciferases. In some cases, the advantage of working with a smaller tag is that you can reduce the possibility of interference with your protein of interest, which the tag is attached to.

(Thorne, Inglese & Auld. 2010)





3. Luciferin Types to Consider:

Sodium, Potassium and free acid luciferin were already mentioned previously along with their pros and cons. However, other varieties of luciferin exist such as DMNPE-caged luciferin and L-luciferin. Why would anyone use these – especially L-luciferin? We gathered some important information to answer that question.

a.DMNPE-caged luciferin: Our product description suggests that it can be used to measure intracellular functions (inside the cell) since it readily crosses cell membranes. But what does that mean exactly? For starters, this would suggest that it’s useful for in vivo settings, but then what’s wrong with using D-luciferin potassium or sodium salt? At neutral pHs, D-luciferin runs into some permeability issues. DMNPE-caged luciferin, therefore solves that obstacle by optimizing the approach. Due to its ability to cross the cell membrane, it’s going to allow for the most efficient use of the product.

b.L-Luciferin: L-luciferin is the chiralic sister to D-luciferin and produces no light. While the Lembert paper suggests that it can produce weak levels of light, it is thought that the real reason behind that is the racemization of the chemical back into D-luciferin. So if it’s useless in producing light, what do researchers use L-lucferin for? According to “Bioluminescence: Fundamentals and Applications in Biotechnology, Volume 2,” L-luciferin is helpful in quenching light in dual assay systems. This occurs because L-luciferin acts as a competitive inhibitor, and therefore actually helps increase the Km for D-luciferin, boosting the upper limit for the assay.




4. Coelenterazine Variant Considerations:

We have a fact sheet which highlights information about the three coelenterazine analogues: coelenterazine, coelenterazine h (not available) and coelenterazine 400a. While any of the three are useful in an experiment, this sheet describes reasons why you might want to consider one over the other. 







PURCHASING CONSIDERATIONS

5. Quality & Cost Considerations:

When price shopping for a product, you’re going to encounter a lot of noise. You may find yourself getting a little cross-eyed while perusing offers and sales. Some of them are absolutely worthwhile. However, as you’re weeding through all of these deals, trying to find the best one, be sure you remain vigilant and critical without losing sight of what’s important. Ask yourself about the purity and publication rate. Both are indications about how well you can trust a product. Is a COA available? Can you try a sample? 



6. Purity Considerations – How Much Does Purity Actually Matter:

In an earlier article, we explore this topic in greater detail, teaching just how important purity can be, but I’ll pull out one of the more important points in this section.

Deeper into the article we began to talk about what a 99.7% purity really means and compared that to 98%. A 1.7% difference, after doing the math proves to have 0.02 g of potential contaminants. While that doesn’t sound too significant, we find that it equates to 0.8 g per liter, and if these little guys had a molecular weight of 1000g/mol, the concentration would be 800µM!



7. Considerations for What Impurities Might Exist:

Since we're on the topic of purity, it's good to have knowledge of what that means exactly in terms of luciferin. One impurity would be the small presence of L-luciferin, which is a competitive inhibitor. Dehydroluciferin is another inhibitor which can greatly impact the results of your experiment. While you can shop for high-purity luciferins, it is important to properly store and handle the products because it is believes that improper storage can result in the formation of dehydroluciferin. 








STORAGE & HANDLING CONSIDERATIONS

8. Proper Storage of Luciferin:

Essentially, you need to protect your luciferin from light, air and moisture. Oxidation of luciferin is catalyzed by light and oxygen and exposure can rapidly render it useless. Our COAs suggest storing the product desiccated at -20 ˚C in order to maintain the longest shelf life.


9. Proper Handling of Luciferin:

Once you have opened it and are ready to use it, allow the bottle to come fully to room temperature. And before resealing, it must be sparged (removing dissolved gases such as oxygen) with nitrogen or argon.



10. Stock Solution Considerations:

Your prepared stock solutions can be used immediately. And with proper storage, you can keep your stock solution for up to a month. To properly store your luciferin stock, aliquot it out and store it at -80 ˚C. 








BONUS INFO

11. Considerations For Other Luciferases:

In this article, we focused almost entirely on firefly and Renilla luciferases. However, there are other luciferases available for the lab such as Gaussia and click beetle. So we will spend just a little time talking about these luciferases:

a . Click Beetle Luciferase: When talking about what click beetle luciferase is, do not confuse this with “beetle luciferin.” In this case, click beetle luciferase is exactly that, and is derived from Pyrophorus plagiophthalamus. It is particularly useful for dual color assays. Here are the specs for click beetle luciferase:

i. Molecular Weight: 60 kDa (matches firefly luciferase)

ii. Peak Emission Wavelength: Red: 613 nm, green: 537 nm (based on click beetle type)

iii. Substrates: D-luciferin (does not have to be “beetle luciferin”) and ATP

iv. Protein Half-Life: 7 Hours

v. Uses: Primarily for dual color assays, allowing for simultaneous monitoring of a variety of events due to their different color emissions.

b.  Copepod Crustacean (Gaussia): This luciferase type was mentioned earlier. It is derived from marine copepod and produces stronger flash signals than firefly luciferase and Renilla luciferase. Here are the specs for Gaussia luciferase:

i. Molecular Weight: 20 kDa

ii. Peak Emission Wavelength: 460 nm

iii. Substrates: Coelenterazine (no ATP dependence)

iv. Protein Half-Life: 6 days in cell media

v. Uses: It can be used for live cell assays, and its small size offers an advantage by reducing the interference with the protein of interest.

c.  Ostracod Crustacean (Vargula/ Cypridina): This is another marine version and is also known as the sea firefly. It isn’t nearly as common as firefly luciferase and therefore information about this particular type is far more limited:

i. Molecular Weight: 62 kDa (matches firefly luciferase)

ii. Peak Emission Wavelength: 465 nm

iii. Substrates: Vargulin also known as Cypridina luciferin (no ATP dependence)

iv. Protein Half-Life: 53 hours in cell media

v. Uses: Its sensitivity and detection of small amounts of luciferase activity is one advantage as well as its ability to be multiplexed with other luciferases. It is used for promoter studies, drug screenings and signal transduction pathway analysis.

         (Thorne, Inglese & Auld. 2010)





12. What About Beetle Luciferin:

Beetle luciferin is just plain D-luciferin. Our fact sheet on this topic goes into more detail. Ultimately, there is no difference between firefly luciferin, synthetic firefly luciferin or synthetic beetle luciferin. And when it comes to working with either firefly luciferase or click beetle luciferase, you simply need to use D-lucferin as your substrate.



There you have it - everything you need to know when shopping for lucferin/luciferase and more. If you have other pointers, feel free to comment them below.



References:

Lembert, Nicolas. "Firefly luciferase can use L-luciferin to produce light." Biochemical Journal 317.Pt 1 (1996): 273.

Luque-Ortega, J., Rivero-Lezcano, O., Croft, S., Rivas, L. (2001). In vivo monitoring of intracellular ATP levels in Leishmania donovani promastigotes as a rapid method to screen drugs targeting bioenergetic metabolism. American Society for Mircrobiology. Doi: 10.1128/AAC

Nakamura, Mitsuhiro, et al. "Construction of a new firefly bioluminescence system using L-luciferin as substrate." Tetrahedron letters 47.7 (2006): 1197-1200.

Ohmiya, Y. (2014). Applications of bioluminescence: Cell based assays and imaging. Photobiological. Retrieved March 11, 2016 from http://www.photobiology.info/ohmiya.html

Thousand, G., & Marks, R. (n.d.). Bioluminescence: fundamentals and applications in biotechnology (Vol.2). New York City, NY: Springer.

Thorne, N., Inglese, J., & Auld, D.S. (2010). Illuminating insights into firefly luciferases and other bioluminescent reporters used in chemical biology. Chemistry & Biology, 17(6), 646-657. Retrieved March 15, 2016, from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2925662/


    
              Karen Martin
GoldBio Marketing Coordinator


"To understand the universe is to understand math." My 8th grade
math teacher's quote meant nothing to me at the time. Then came
college, and the revelation that the adults in my past were right all
along. But since math feels less tangible, I fell for biology and have
found pure happiness behind my desk at GoldBio, learning, writing
and loving everything science. 



Category Code: 79104 88251