How We Got The DNA From This Extremely Rare Ciliate

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To study organisms at the genetic level, we need their DNA. Which means that we need to be able to wade through all the bits and pieces lying within their tiny bodies to pick out something even tinier-something we can’t just dig out with a shovel. So how does James manage to get the precious DNA from Legendrea loyezae and the other ciliates he’s interested in studying?
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[@ke9tv]

When I saw the previous episode on Legentrea, I thought, "I wonder if James has managed to isolate enough DNA from these to run a PCR and sequence." When I saw the title of this one, I cheered! PCR stands for "pipette, cry and repeat!"

[@keku52]

lmfao, accurate acronym. My classmates (bachelors) trying to isolate STEC (Shiga toxin producing E. coli) and MRSA (Methicillin-Resistant S. aureus) have been crying how many times they have to repeat many times to extract enough genetic material for amplification and confirmatiin

[@Michaelroni-n-cheese]

Me too on being a gay lil dandy. I am very gay and will always be that way 😅

[@MeetAnEcoregion]

7:58 Looking at old research with new eyes. I like that notion very much.

[@dianagibbs3550]

I remember being in a microbiology class in 1996ish and hearing about this new technology PCR, and how it was going to change the world. It has, and continues to do so. So cool.

[@Bryophytan]

These days it seems like CRISPR/cas9 is going to be an equivalent, if in a field skewed in a slightly different direction

[@dianagibbs3550]

@@Bryophytan I was thinking that too!

[@osmia]

James, you have such a fascinating job!

[@ColinTimmins]

That "little guy" really stands out if you know how to look. Thank you for giving us all a look at the things we would have surely missed, every day, right under our nose.

[@Srcsqwrn]

I'm only a little sad the little cilliate didn't gete to live out it's life. But the discovery was amazing, and being able to find it's siblings in genes is amazing!

[@gtbkts]

Same

[@janetf23]

Same here too

[@eg-fi3ju]

you sound like a vegan it dosent have a brain its literally a single cell thats like being sad that your skin dies

[@Srcsqwrn]

@@eg-fi3ju ok boomer

[@ivy_47]

@@eg-fi3ju what are your thoughts on pro-life philosophy?

[@1234j]

Well DONE, James! And your colleagues. Thank you for tracking the huge yet tiny stars and filming them for us, and thank you, all those on this channel who serve us up fresh knowledge and insights, one drop at a time - sometimes via the freezer. Cheers from England.

[@TheTm9090]

Such humble beginnings and now we are making videos covering them. Absolutely mind blowing

[@shgstewart4674]

It's a rather pretty little organism. Nicely done!

[@bugpal]

Do you amazing people have any plans or ideas for a documentary length feature that can be shown at IMAX Theatres, for example? That would truly be a sight to see.

[@misilagata]

Me encantan vuestros videos. !Encima en español! Mis viajes desde el sofá al microcosmos es una aventura apasionante.

[@Taomantom]

always learn something new here thanks!

[@jimmyjames2022]

Congratulations to James and the research team for their work on this rare Legendrea ciliate, one of the coolest ciliates ever!

[@trevinbeattie4888]

The thought of a tiny microbe taking a bath amuses me. :D

[@yajuvendrasinghrajpurohit7888]

James your are amazing and I love this channel

[@finch600]

I had to clean a mite to mount to be seen under an electron microscope when I was a volunteer. Using a pipette to clean something is so bloody finicky.

[@dixietenbroeck8717]

*Poor little critter!* At least we can say "it devoted it's life to science" - _SOLELY_ for the benefit of a *COMPARATIVELY **_ENORMOUS,_** (and UNKNOWN) **_ORGANISM!_*

[@WAMTAT]

Not often you watch a KZbin video on the cutting edge of science.

[@WAMTAT]

Amazing work James.

[@glennk.7348]

Can you also add the speed of filing to the data on screen? I was amazed to watch your 800x slower video and wondered how you even track some of these animals!

[@LunarForte]

They're all in real-time unless otherwise stated

[@alfredwaldo6079]

This is truly a journey to the microscpe moment

[@Streetsy]

This channel is really interesting. Love your work.

[@obieobrien5883]

James must’ve been super excited to find this one!

[@Syntaxxed]

Thank you all so much. I will be doing a bachelor of physics or math. I really like that I can still be updated on biology and chemistry through channels like these.

[@cantsay]

Thank you for sharing James amazing journey with us. What an amazing discovery, I'm sure his mind it blown, and will keep exoding in his head for months.

[@speckledjim_]

Absolutely stunning footage, so crisp and clear. i cant help but think that this little guy is similar in appearance to a cuttlefish or squid

[@Bryophytan]

It's the quality you get with ridiculous expensive microscopes.

[@clairejeansonne9800]

Comments for the very, very clean algorithm,

[@THELITTLERIVERNERD]

Love this video! Would love more videos that dig into the biochemistry & genetics of the microcosmos!

[@vernonbrechin4207]

Thank you for answering some of the pre-PCR preparation questions that I had. I admire the patience it takes to engage in all those repetitive processes, especially that of cleaning the minute organism. Beyond that step I wonder why the DNA code is not damaged, or scrambled, by the physically disruptive steps that follow the 'washing.' Is the organism washed in distilled water, or water containing some nutrient minerals? It seems to me that even this washing step would not rid the subject organism of parasitic organisms that might be in it, such as viruses.

[@TheRedKnight101]

James looked specifically at the 18S ribosomal RNA sequence which is only in eukaryotes. Only that sequence was amplified so any DNA from bacteria, archaea, and viruses would not be amplified. That is why it was important for the cell to degrade any DNA from other ciliates it may have eaten because that DNA could be amplified if they weren't careful.

[@vernonbrechin4207]

@@TheRedKnight101 Thank you for your clear and thorough explanation.

[@HesderOleh]

@@TheRedKnight101 I figured that it was 18S barcoding based on the way it was described, which made me wonder why not get the whole genome?

[@Raydensheraj]

@@TheRedKnight101 The Carl Woese method....what a freaking genius the crazy old man was....

[@nefeli2737]

Good episode. I'm in search of this microbe now.

[@ppartsx]

James explained to me months ago on r/microscopy how he gets the dna from singular microbes so i had an idea already how it was done before watching :]

[@TedToal_TedToal]

Thanks! So, did you sequence its whole genome (and how big is it, btw) or just some part such as ribosomal DNA?

[@personzorz]

These days there's no excuse to not get everything

[@fasferg2999]

@@personzorz Using PCR it is much easier, cheaper and faster to just focus upon the 16s Ribosomal DNA than try a single cell full genomic sequencing. The 16S rDNA is typically varied enough that you can determine the taxonomy all the way to family level.

[@TheRedKnight101]

Whole genome sequencing while cheaper than it was just 10 years ago is still fairly expensive. Also you need more than one cell's worth of DNA so you can sequence the genome multiple times to ensure there are no mistakes in the sequencing process. Considering the rarity and size of the organism it's amazing they managed to get enough DNA for PCR. For determining taxanomic classification 18s is usually enough to get a rough understanding of the taxonomy. While whole genome sequencing would provide more information than was needed to answer what it is related too.

[@HesderOleh]

@@TheRedKnight101 You can de novo assemble a whole genome from a single cell now a days. It is kind of sad to have that opportunity missed, but honestly unless someone was going to annotate that data, it would be hard to justify the cost.

[@travcollier]

@@HesderOleh Whole genome sequencing from single cell is really quite difficult, and even harder to get the long reads or mate-pairs needed to do a decent assembly. There are 'in between' options which amplify a lot of common genes simultaneously instead of just 16s, and then you sequence all those using the same equipment as you'd use for whole genome. That might have been an option here, but it is a lot more expensive. Anyways, this stuff is still quite tricky, even when you have a lot more DNA to start with. FWIW: I do genomics/bioinformatics on mosquitoes.

[@DracarmenWinterspring]

I wonder, for such a rare find, is it worth trying to give it the time and environment to replicate, if only to have a "backup" in case the DNA extraction fails? Or is there too much of a risk of it dying or losing track of it before it has a chance to reproduce?

[@michable100]

Well it will not have been alone in the environment it was found in. So if you mess up you can always go back and try to catch more.

[@basedlordprime]

Very cool to see it swimming in the water drop.

[@mineovernutter]

Can you add James' paper to the list of sources in the description? I think it is important to put it in there.

[@rot_studios]

Wondered about this lately, thank you so much for making a video about it

[@ronaldhowell2005]

Thankyou for sharing such important information and video, awesomeness !!!

[@ptarmigan1356]

Can I ask how James decided to use freeze thaw to lyse the cells? How did he know how many cycles would be enough? For comparison I have heard of using lithium acetate and SDS to lyse yeasts, followed by precipitation of DNA by ethanol. Also what is the structure of these organisms like, eg do you know if they have a cell wall? How different are different ciliates, what sort of things could James assume about these organisms before knowing from sequence data which family they belong to?

[@HesderOleh]

One reason to use freeze thaw for lysis is that you aren't introducing any buffers or chemicals that could interfere with analysis techniques. For 18S rRNA barcoding one cell is enough, but actually now you could even do whole genome with one cell (even de novo assembly). They don't have cell walls. I don't know much about cilliates, I did research on algae.

[@seahamdesigner]

I have to come back to these videos, as its information overload for me. Always amazing though!!!

[@you2angel1]

WOW °~.🌟.~° You guys should be quite proud!

[@Mei-oe6sg]

I’m so appreciate these videos, thanks a lot 🙏 ❤❤

[@carsonclayson7063]

It just hit me that vibe-wise Journey to the microcosmos is Hank's Anthropocene reviewed

[@stevenkarnisky411]

As a non-biologist, I sort of understand the DNA harvesting process, except for the "amplication" process. Is it simply gathering more bits of DNA from the original organism and adding them to the original sample, or does it involve getting the DNA samples to replicate somehow, like bacteria in a petri dish? I wouldn't think the second is possible, but what do I know?

[@ptarmigan1356]

The PCR amplification produces more copies of (certain regions of) the DNA. You mix your ‚template‘ DNA with DNA building blocks and enzymes that can read the template and produce more of it by joining the building blocks together in the correct order.

[@FBIandre123]

As a portuguese watcher and lover of the calm and charismatic voice of hank green, is a little bit annoying having to change the voice in begining of every video.

[@whizthesugoi]

It's an awesome feature though

[@KitsuNoir]

KZbin loves telling you what you prefer. I love when they choose a random language when English is unavailable for captions. Like, no, just because there's no English subs doesn't mean i can read Spanish or German suddenly, just turn it off till i see another video. Developers, don't assume stupid things, like that everyone speaks the same language or can only speak one. Or assume we can only speak one instead of simultaneously assuming contradictory ideas.

[@1234j]

Just hop forward a few seconds when you start the video, so you get all the soothing and none of the normal - just don't tell Hank.

[@whizthesugoi]

@@1234j no no, they mean the auto translate audio feature that is auto selected if you're from a portuguese or spanish speaking country

[@belstar1128]

They just use text to speech i tried it in Portuguese because i am learning Portuguese. but i can't stand the text to speech voice i thought they would have a system where they can have multiple soundtracks for different languages. but with an actual human voice over but i will sick to the original for now.

[@bitterlemonboy]

So... Where's the DNA of L. Loyezae? Can we download it somewhere? Or is it only available on patreon?

[@Bryophytan]

Genbank NCBI

[@ooooneeee]

They only did PCR of a part of the ribosome, not a whole genome sequence.

[@bitterlemonboy]

@@ooooneeee Then they should have titled the video "How We Got The DNA Of A Part of The Ribosome of This Extremely Rare Ciliate"

[@JeevasJerico13]

This channel is so underrated

[@fatihozdemir6564]

Awesome Lang translate and Amazing video

[@RJFerret]

Anyone find online images of "epispathidium papilliferum"? Mine turned up other epispathidium but not those.

[@katha-studio]

Good information 😋

[@hherpdderp]

We must avenge the Loyazea

[@mrwalk6171]

This would be a perfect organism for single cell whole genome sequencing. Practically what the technology is for, hope this can be revisited one day (i.e. another is found)

[@janwodarek4133]

Loved this video, as a future real scientist, present amateur scientist I would love to see more videos about the procedure and method. As I was always drawn to protozoans (now protists i guess) I hope I could one day become another Polish master of microscope. Thanks for the vid :)

[@timothygreer188]

Can wait to read the published paper

[@husaynbootwala1729]

If this organism is so rare and has been barely studied, how did you manage to make primers to use in the PCR process? Was the primer a common sequence found in all ciliates?

[@sobbski2672]

I'm assuming they used universal primers targeting the 16S ribosomal subunit

[@husaynbootwala1729]

@@sobbski2672 but would that be enough to get the entire genome? Or would the 16S subunit only be able to amplify some portion of the DNA?

[@ptarmigan1356]

It would amplify a specific region which is quite conserved between organisms. The sequences are similar enough so that you can compare them and see how closely related they are.

[@husaynbootwala1729]

@@ptarmigan1356 but if we wanted to sequence the entire genome, then what would be done?

[@mineovernutter]

@@husaynbootwala1729 We would either need to culture the ciliate to get more cells and more DNA. I dont think we are able to culture these cells however otherwise james would have probably done that in this video as a kind of insurance. Or we could maybe do some single cell studies which are quite expensive, and since we dont know which primers are needed to cover the whole genome you would likely not get good enough coverage to get a sequence.

[@Sy-dn9bq]

why exactly did the narration imply the pandemic is past tense?

[@cgomes12]

Congratulations on the amazing job! Which segment of the genome you used in the PCR? Have you considered whole genome amplification and than sequencing of the entire genome?

[@ladycormix4456]

So why freezing instead of centrifugation? Is the ciliate too small?

[@stonefish1318]

This was an incredible inside behind microsience! Pls more of this kind

[@mediocreclementine7649]

I'm going INSANE how tiny is that micropipette?!?

[@lavinleitrim44]

The narrator reminds me so much of the one for Tale Foundry, very similar way of speaking.

[@sloth0jr]

Yeah, PIA - don't feel comfortable with a VPN service whose marketing slides don't understand the format of an IP address.

[@AlienAnthony]

I have a personal collection of algea I've collected from a few years back. I've lost the original sample from the dirt I got it from but if I wanted to submit a sample could I? I have 3 bottles of it. The first was a sample of the water of the first dirt sample. And each bottle after is from the previous one.

[@Beryllahawk]

As with the microcosmos, also ourselves. Don't neglect to visit your own "familiar ponds," because in us, too, there is always hidden something more.

[@supreetsahu1964]

Anyone know the song at 5:50?

[@tatoverde9312]

PCR was abused during the last 2 years. The inventor made it very clear that being + does not mean you are infected or contagious. The use in this experiment make sense. However for diagnosis is faulty to say the least.

[@pjssjr]

I thought you’d describe a case of horizontal DNA transmission between ciliates and humans!

[@heartstronggarden5134]

💕🤘🏼🔬 just chillin'

[@HNIW]

Gratulejszens myster Dżejms 😁 Pozdro 🇵🇱👍 Powiedz gdzie jest to tajemnicze jeziorko 😉

[@Eline_Meijer]

1 Legendra Loyezae was harmed in the making of this video

[@jaikumarsagar]

Bro, what do you use to slow down these microbes? I am facing some difficulty in observing some of these fast swimming microbes under the microscope

[@nightthought2497]

Calling PCR a pandemic management tool is like calling a hammer a house building tool

[@TheRogueWolf]

I noticed that this video did _not_ feature a microscopic consent form! Scandalous!

[@inkuii]

Out of curiosity, how long did the cycling take?

[@brianedwards7142]

You're just being cili.

[@hallowacko]

Curious: Are there microbes isolated in caves that can be talked about? I started thinking about like, the family tree of eukaryotes, and how something can wind up super rare and only in a couple ponds - like how did they get there? is that the origin of a whole limb of the tree of eukaryotic life? surely not, maybe they just got isolated there or something? Do microbes get isolated? How did eukaryotes get to all these bodies of water?

[@Cor97]

This ciliate measures about 50 um, correct? On my screen it measures 12 cm. So its magnification is 2400, right?

[@Bryophytan]

Maths checks out but the ciliate (L. loyezae) is usually 70 um up to 120 um in length

[@kgv9902]

It's mind blowing knowing the tentacles are part of the single celled cilliate, i still don't understand how the tentacles move, it ain't got no muscles...

[@thea_kober]

I think they're probably moved by the cell's actin filaments or microtubules, like cillia and flagella

[@Bryophytan]

@@thea_kober Contractile vacuoles in tandem with cytoskeletal filaments, basically like puppet strings and balloons!

[@pfischer08]

Acho que é ótimo ter conteúdo assim em português.

[@upasananandi1271]

Ok, so here's something that I've been mulling over for a while: How exactly are the primer sequences for the PCR decided in these cases? As far as I know, the primers not only have to be gene sequence specific, they've also got to be unique to that gene alone and ideally, shouldn't form dimers. That alone creates crazy complications when it comes to designing them. I'm aware that what I know of is sort of the tip of the iceberg, if not entirely wrong, so if someone could offer some clarity, I'd really appreciate it.

[@azureabyss538]

Such a cleanly and evenly posed question, it broke my heart to see it didn't get to see the light. Make sure you pose it in places where it receives appropriate attention.

[@anandg5843]

@6:15 🙏

[@Aimee42]

What do you think of Astrophage?

[@sharkmeldon]

What a trip. 😁

[@st1220music]

0:43

[@CloudColumncat]

Since we are made of cells, it is amazing that we live and are affected by how small and simple cells are. Is it because they have a sense of duty that life is maintained in my body? Is it my will? Maybe my body is like a city. All infrastructure is built using resources by life, and continues to accept, exchange, and survive. In such a daily life, trillions and hundreds of billions of lives arise and die in my body, and new ones are born and maintained. Life is truly a strange thing.

[@kaltkalt2083]

But what i really wanted to know was how people get the DNA out of one tiny little speck? I figured PCR is used to get a lot more of that DNA.

[@9a3eedi]

The whole process of moving the organism to a new drop of water sounds like homeopathy :p

[@sdjhgfkshfswdfhskljh3360]

Is result open source?

[@websurfer5772]

Can you show us proof of the DNA molecule?

[@MaryAnnNytowl]

I was sad that such a rare little critter had to die for science. 😭

[@belstar1128]

Rare ? there are more of them than there are humans.

[@DekkarJr]

Ciliate: Can I has dat back pweez :3 :3 ;3

[@Haplo-san]

I didn't get why freezing and thawing it increases the amount of DNA. It's just a single organism, doesn't it die when you freeze it? And if it dies, how DNA keep replicating itself? I have dozens of questions about it.

[@ptarmigan1356]

Freezing and thawing was just the step to get the DNA accessible by breaking open the cell. It was then amplified by PCR in a separate step to make more copies.

[@MrBoegela]

i just imagine that some alien takes us up for research and cleans the hell out of us before freezing and thawing and heating and cooling for science soup

[@Quad5Ny]

Audio tracks are still random. They do not default to English/Users Language NOR do they save your preferences, each video When played will pick a RANDOM audio track confusing the viewer!

[@felvinperez957]

Actually the answer of this question us very simple. Universal intelligence that if you believe that universe itself is a conscious universe and life on this planet including us human is mere illusion from the conscious universe where we as pary of its spark..

[@alchemi8085]

Life would be simpler if I were a microbe.

[@DrewishAF]

Spoiler alert: They murdered it

[@caiden-_-]

He should tried to get it to reproduce if it is asexually to have one to keep while the other dies

[@whizthesugoi]

Wooooow

[@michealwestfall8544]

RIP.

[@liamomalley5779]

You should be worried about this employee... he puts subjects into isolation, forces them to defecate (probably on camera) transfers them to a clean room and then makes his kill by blowing them apart with ice crystals and cold exposure.... just sayin..