How I Defeated My Arch Nemesis

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BL21 E. coli are by far the most obnoxious bacteria genetic engineers usually work with for 1 simple reason. They are maddeningly difficult to modify and load with new DNA. This wouldn't be an issue if they weren't also one of the best types for producing proteins en mass, and as such have application in everything from pharmaceutical production, to producing ingredients for foods. But after a few years of tinkering with it I finally found a protocol that reliably fixes this irritating issue, and makes it easy to transform them reliably.
Written protocol - github.com/thethoughtemporium...
Sebastian's spinner - www.tinkercad.com/things/5t5G...
Previous videos;
Modifying Bacteria - • Simple Bacteria Geneti...
Modifying yeast - • Making Bread Healthier...
Making a heat block/incubator - • From Beer Cooler to a ...
Growing spider silk - • I Grew Real Spider Sil...
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[@ExplosionsAndFire]

Science is temporary, grudges are forever xx

[@dangernoodle8813]

yellow bad.

[@captainchicky3744]

YOOOOOOOOOOOOO i love how all the science chanels are inteconencted

[@ieatdirtwasntavailable]

DO NOT DO BIOLOGY

[@jessepinkman1471]

Yellow

[@utsuhoreiuji6155]

Hi Tom, nice to see you here; As a greeting, here is the obligatory yellow chem bad (especially in a precipitation tittation) Good luck to your PhD!

[@alexbenavidez4500]

"You can fart DNA in their general direction and they'll take it in" That's a great fucking line of a biochemist.

[@MarkusAldawn]

The DNA equivalent of "yeah, I'll incorporate that into my belief system"

[@user-pr6ed3ri2k]

👍

[@waterunderthebridge7950]

6:06 It still surprises me how a lot of times in biology/medicine it’s just “We don’t care _how_ it works exactly. If it works consistently, we don’t question the process. Don’t anger the science gods with your impious insolence!”

[@knuckle12356]

Read up on mechanism of action for most every medication used today. The number of times that section begins with "while the _exact_ mechanism is unknown, it is suspected..." Translated: well, people used to slather this and smoke that, after which more survived than within the "control" tribe, which had neither poultice nor herb. So we refined the alkaloids. The health effects compounded. Then we changed a few methyl groups. People died. But THEN we changed these other methyl groups, and shit started popping off! Leading us to conclude that this shape is more gooder than these shapes. So we stuck with this one. Other than the copyrights, the basic drug remains unchanged after 300 years. Or 3000, etc. Kinda scary. Same with surgery and medicine. It looks like so much nonsense and butchery, but it takes a few decades to specialize this organ or that system, and lives are saved by these procedures. Babies are even operated on in utero!!! But it still looks like a kid searching a piñata for their favorite candies when they remove your intestines and just pile them up in a heap beside you... So much knowledge, so much history we build upon... but sometimes it sure doesn't look like it.

[@cristianruske2875]

I asked the same for my radio tech relative and he just said doesn't matter

[@thaumatomane]

@@knuckle12356 So, I am doing my PhD in the history of magic in the premodern world (I specialize in a sub-variety of European ritual magic). Anyway, one thing I always think is crazy is how confidently so many people say "I only believe in science." Which is ridiculous, science is a method of inquiry, not a belief system. When people say that they only believe in science, what they mean is that they believe in materialism - a historically constructed modern inheritance from enlightenment era philosophy. It is a cosmological system, just like Catholic theology, neoplatonic philosophy, or astrological theory (the 5000 year old complex stuff, not the silly tabloid/tiktok rubbish). One thing I think is valuable is to realize that materialism is not the baseline objective reality when all "beliefs" have been stripped away. It is just one of the many cosmographies by which people interpret the world. Edit: typos.

[@aragocz727]

Same goes for coding, if it works you don't question it, that's why programmers keep the 20 years old legacy code

[@mariomario1462]

​@@thaumatomaneuh no. Nothing you said Is remotely true. believing in science means you get results in life. Theology Is literal make believe. Maybe you should major in logic classes first before you talk nonsense

[@KateeAngel]

"Everything in biology is yellow". Me, a cyanobacteriologist: "Cool story, bro!"

[@DancingRain]

I see some very confused botanists and mycologists in the room too 😀

[@wormguts5950]

epic cyanobacteriologist meme

[@carsonfujita-turnbull4549]

Yeah, someone is gonna have to translate this into common speech in order for anyone but biologist to know what the joke is. If anyone could explain to me, I would appreciate it.

[@wilmerreinholdsson1181]

@@carsonfujita-turnbull4549 the cell watching mfers are mad coz light vibes at the worng wavelangth n shit

[@inciteinsight2331]

@@carsonfujita-turnbull4549 Cyanobacteria are all blue. Their name means “blue bacteria”.

[@Pyriphlegeton]

4:26 "Sort of like Bioshock but less shooting Spiders and Lightning" *...for now.*

[@Drankoii]

Exactly my thought, lol. I hope to live to see bacteria that shoot fire and lightning.

[@sharpfang]

Electric Eel DNA will take care of lightning. Spiders - TRIVIAL.

[@beezmanit2683]

@@Drankoii hopefully it isn't sentient

[@Usual_User]

@@beezmanit2683 😂

[@Waluigi_Time778]

If the petri dish exploded with baby spiders I would be burning down the lab

[@ace-kz9id]

I might not understand this stuff at all but it is super intresting

[@jonathanodude6660]

i can never follow what the buffers are for or why one is used over another.

[@jonathanodude6660]

@@peterdodge8590 i have no idea what opamps are lol

[@DxGamer6767]

Same

[@jblazer9804]

Amen

[@gabrielsilveira8246]

S(h)ame

[@bryby216]

I'm a molecular biologist in an academic lab, and dude, I'm so thankful for your videos. It's easy to forget how cool what we do is, and instead just treat it like a grind. Thanks for making these videos, and thanks for sharing your protocol. It's refreshing

[@quote.d]

This

[@thersten]

Yes. Very true 👍

[@stijn2472]

This is one of the things I like about you. When you discover something, instead of patenting it or trying to sell a method you came up with to a company, you make it available to the public and I appreciate that. Thank you

[@thersten]

Bill gates says hello.

[@2ue]

Cant patent a simple change in methodology unless it was made proprietary anyway.

[@moonface550]

Obligatory "yellow chem bad"

[@TurboFryer1337]

ah, a fellow "piss chemistry bad" enthusiast

[@Dinnye01]

@@TurboFryer1337 and they are actually friends with the piss chemist

[@explosiverift2037]

@@Dinnye01 Nile Red?

[@22Mycraft]

@@explosiverift2037 explosions and fire, extractions and ire. Get chemist, highly recommend

[@TheRealDescartes]

*Y E L L O W*

[@VincentGroenewold]

I worked in science and doing things like this for over 10 years and 1 thing I learned is how everyone always copies protocols to the T. It may make sense of course, but it's never really a thing people think hard about it seems. I once started research on an entire new species for our work, got a protocol from another lab full of alternative approaches and no real explanation as to why. Turned out they had 1 step I recognized to likely be extremely in-efficient. So I just did it my way and presto, totally worked. That was when I concluded this probably happens a lot in labs. That protocol was in use for years as well.

[@calamariaxo]

Yeah, seen this myself in our lab, but it's not just in labs.. This happens everywhere. Humans are surprisingly happy to just follow instructions without thinking about what we're actually doing most of the time. Engineers and scientists might be somewhat better trained to notice these things, it's often the kind of magical *zing* that somehow improves your project or furthers science in some way, but we're still all human.

[@paavobergmann4920]

Yeah. In EM work, also. I think, usually your margins of error are unknown, or tight, and you have so many variables at play that systematically trying the parameter space to find the optimal protocol is not feasible (an EM fixation & embedding can stretch longer than a week, and you haven´t even started sectioning). Also, lab protocols tend to evolve like software code: stitched together from subroutines that used to work somehow for similar problems. So if it happens to work for you, or did work at least once for whoever published it, you stick with it, however inefficient or counterintuitive or downright weird it might seem. Better get "kind of ok-ish" results consistently than burning your PhD grant on optimization. You can start doing that once things stop working altogether. And you will, eventually.

[@kendarr]

@@calamariaxo agree I've seen some stuff in wood working that people just don't seem to know why it is the way it, and don't seem to try in change it

[@Max-cj8vm]

I used to work under a grad student who insisted that all steps of a Western blot must be kept protected from light

[@thersten]

That's pretty much how the peak of human technology is reached. It's a conglomeration of individual pieces of knowledge and technology. Ask how it works and very few people really know.

[@afroninjaman1]

The superstition comment got me. I've always said genetics is half black magic. In my capstone project lab the grad students had special P10 pipettes labelled "Golden Child" and "Vampire" because respectively the former made reactions generally and PCR specifically work like magic, and the latter drained the life out of your experiments.

[@NicRobertsNerd]

What an amazing thing to be living in a time when credible science procedural tutorials are available for free on KZbin. Thank you so much for posting this.

[@janrace6466]

3:35 Ooooooh, so THAT's why the university of biology in my country keeps a herd of sheep!

[@andrewseung2692]

Lol

[@noahhuffstutler2686]

As a computer scientist, can confirm that fresh sheep blood helps make our programs work.

[@firestorm5371]

@@noahhuffstutler2686 child sacrifice also work

[@noahhuffstutler2686]

@@firestorm5371 I've tried it before, but the Devil is sick of my shit. IIRC his last words to me were "You are putting me through hell."

[@janrace6466]

@@noahhuffstutler2686 And that explains why the university of computer science is right next door.

[@joeydubbs763]

Explosions & fire & Ecoli?..... Taco Bell?

[@Alpha13Wolf]

I was waiting for that joke :)

[@jeravogel]

Hotel?

[@danielshin4526]

@@jeravogel trivago.

[@SGT_Fon]

Was thinking White Castles... but same idea 💡

[@zenzetsu]

Me: subscribes to The Thought Emporium for his physics videos with little knowledge or interest in biology The Thought Emporium: releases biology video Me watching the biology video anyway: I like your funny words magic man

[@nmraven-gx9gt]

Opposite direction for me lol

[@davidgrigoryan6207]

Me an Electronic Engineer: Ah yes, E. coli

[@shadowghost-lk1sg]

If anything electrical engineering is pretty close. Lots of theory and terminology in genetic engineering are taken from electrical engineering; the engineered sequences are called circuits and a lot of the papers even use IEEE formatting. Its actually really interesting stuff.

[@meltossmedia]

@@shadowghost-lk1sg first them, now neuroscientists. It's the ultimate "You can copy off me, just change it a little"

[@jacky9575]

@@meltossmedia as a neuroscientist, I confirm, literally we have biostatistics, advanced mathematics and bioelectricity as subjects

[@_-_-Sipita-_-_]

Electronic coil

[@karigucio]

the advert i got before this vid was about getting rid of ecoli from the water. Quite on point

[@Amipotsophspond]

yeah the word detecting advert is funny sometimes. Video Title:why the new kia is a death trap! Advert: buy the new kia!

[@junigearx7452]

Ahh.. Foreshadowing i like that.

[@jafizzle95]

This is my favorite channel where I don't know what the hell is going on.

[@lasinhouseinthetrees1928]

same i just go "Mmmm broth chicken broth"

[@Quroe_]

You've made bioengineering somewhat understandable to me, a mechanical engineer. I hope I get to collaborate and effectively communicate with a bioengineer well enough later in my life and make something cool because of you.

[@kdevlogs5550]

I've always been interested in all types of engineering, and biology has always been mind blowing to me from the dumbest mechanical perspective because, by some miracle, a tiny protein made some mechanism and those mechanisms became bacteria, and now we're here. crazy how complicated even the tiniest little organism is

[@lucashuttebraucker6255]

Hey there, just a few tipps: When you prepare your cells fresh every time you have a big margin of error. I would suggest to prepare competent cells in advance supplement the buffer with 15% glycerol and freeze them. There are lots of viable protocols available. Important is just to work on ice the whole time, and keep the cells always cold. Best at -80c but -20 is sufficient for a week or two. Also I recommend to transform one prepared aliqout with a standardized plasmid and calculate the theoretical transformation efficiency. Then you can transform cells within 2 h. Also for plating the cells I personally prepare always 2 plates, one with 100 ul of the cells in soc media and one with the rest one with the rest. Finally if you can't or don't want to prepare cells ahead, you can use TSS transformation, it is fast, simple and idiot proof. You can use e coil directly from the plate. Just the efficiency is not that great.

[@blackychan7335]

I understood almost nothing but still enjoyed every minute

[@jonathanodude6660]

@Niculae George his bacteria wasnt getting genetically modified so the step that kills unmodified cells killed everything. this is how you make the GM bacteria survive

[@LordSniggles]

@@petertran6785 this guy gets it. Ignorance does not equal lack of intelligence.

[@thersten]

I don't really know why anyone who hasn't studied biology would watch this..... But just study biology and you'll get it. 🤷👍

[@GetBonked]

@@thersten the same reason people watch any other educational video. To learn lmao. You dont know things until you introduce yourself to them. The more you watch, the more you understand.

[@thersten]

@@GetBonked he literally said he understood almost nothing. That's not how educational videos work and that's not how courses work either. If you didn't understand like him then you're missing more than a few prerequisites. LMAO

[@-w-.]

Now that E&F is taking a break, I'm glad that someone decided to take his place.

[@pas.]

What's E&F? Asking for a friend of a friend.

[@-w-.]

@@pas. Explosion & Fire, the mad chemist TTE mentioned at the start

[@kamilag2791]

Bold of you to assume there's only 1 position

[@twothreebravo]

My thought exactly.

[@bur1t0]

Is it a one year break? Or is it just his usual upload schedule?

[@NachozMan]

I've been learning Micro Biology for college this semester and I'm starting to understand what you talk about a bit more in your videos now! Really cool!

[@AdalizMColon]

This was fascinating! Thank you for giving me an inkling again of why I loved and studied microbiology. I never got to work in the profession but still love it. Also, the last note, where it read "Use loop, not swab," brought back how much I hated using a loop in my college lab courses. I was so bad, I always broke the agar. Again, this was great!

[@LagnajeetPradhan]

This was my protocol for transforming BL21s when I was doing protein engineering and crystallography. I believe I also used to add MgCl2 besides the CaCl2 for making the cells competent. I guess there's no need to do that since ur protocol clearly works very well. I worked with BL21s for a long time. Never knew that it could be someone's arch nemesis. 😊

[@yeduprasad]

I think it's a bit of a sensationalist take on the differences between various E. coli strains. I agree with you, BL21 is a perfectly uncomplicated strain to work with.

[@ekaterinaburakova8629]

That's what he's doing here: just regaining cell competence that is lost upon storage or just over time. This is indeed never a problem when you work in a well equipped lab.

[@skegfod51]

I was just about to type this up, but yeah Ive always added MgCl2 when making compitent cell. Ive never had a problem making compitent BL21 with a high transformation efficency. BL21 is very well behaved compaired to some of the other strains i have had to work with!

[@cybersentient4758]

I like your funny words magicman up

[@cybersentient4758]

I like your funny words magicman

[@sealpiercing8476]

You know it's gonna stop working because you put up this video, right?

[@MaxSeidel1]

Not if you sacrifice a goat.

[@powerup3005]

@@MaxSeidel1 🔪🐐

[@Ashley-1917]

Its definatly going to be fixed in the next patch.

[@suryanshtagore7180]

staring at them while growing threatened them was my superstition

[@sealpiercing8476]

I knock on wood literally every time I state that something important is going to work the way I want it to work.

[@sirgareth87]

KZbin: Are you interested in microbiology? Me: No KZbin: Well here's a video on bacteria. Enjoy! Me: But I.... KZbin: I SAID ENJOY!!!

[@Reddotzebra]

Like we used to say back when I was in molecular diagnostics "The definition of insanity is attempting the same thing over and over and expect different results. In this lab that's Tuesday."

[@skublicsimre8047]

ah yes explosions and fire my favorite crackhead chemist

[@hugovandenhoek1032]

He now works with state secrets. I'm not sure if this is ridiculously good, bad, or both.

[@ExplosionsAndFire]

@@hugovandenhoek1032 look, it's either good, or bad, or both, and I also haven't figured it out yet

[@05Matz]

@@hugovandenhoek1032 Wait, really? That sucks. Secret "science" isn't real science. It's why I always have such mixed feelings when cool new technology is announced and then I find out it's a publicity stunt from somebody like DARPA and won't actually be used for non-war purposes for decades, if ever -- an organization officially dedicated to world domination (OK, technically "maintaining USA global dominance") is a bad poster child for science, no matter how much people try to conflate the two (perhaps they watch too many James Bond movies?). Governments will use new technology only to undermine each other or otherwise play zero-sum or negative-sum games until it is eventually independently discovered by too many people to keep it secret, rather than allowing it to be developed on and benefit all of humanity.* Only in the openness of daylight and peer review can true science exist. Open Access is the only true scientific publishing, though for the other 'non-proprietary' stuff, at least there's Sci-Hub to make the best of a bad situation that shouldn't exist in the first place. *(Not that monopolistic corporations are any different in that regard -- they just borrow the state's apparatus for violence and have a time limit on how long they can be dicks about it due to patent expiry [unless it's considered a pharmaceutical in the USA, in which case it never expires as long as you keep up on the "change of process" paperwork]. DIGRESSION ON PATENTS FOLLOWS: In my opinion, patents should be a central registry that is automatically licenced by the government on the holder's behalf for a fixed maximum royalty rate, and you can negotiate with the holder [if they can be contacted] for a better deal if you want. Nobody should ever hold a true monopoly, or be unable to use something because they can't figure out who owns the rights or if they even still exist. It would also help small inventors get money out of patents without the need for a legal team, marketing, negotiations and the like, as well; and discourage the filing of large, intentionally vague, over-broad patents in favour of a greater number of specific, clear, well-documented ones relating to different parts of a process -- becoming a money-making building-block for other companies' processes, rather than 'area denial' to prevent competition. You might even WANT to publish guides helping other companies implement your patents, as it makes them more likely to use them rather than find a way around them or buy from someone else. Patent enforcement, royalties collection from users and receipt of funds by patent-holders would all be easier, more transparent, and more efficient if a single centralized government body (rather than hordes of legal contractors and sub(sub(sub))contractors backed up by the overworked court systems) handled it -- as would taxation of patent income (such as to fund more research), which is probably another reason why incumbents would be against such reforms. From what I've heard, both Intel and AMD can only exist today by violating each other's patents flagrantly (as you can't build a modern X86 CPU without technology from both companies) and settling the inevitable lawsuits out of court by 'trading' dropping lawsuits against each other over and over again. This is silly, and prevents any third company from competing in that space because they don't have the patent arsenal to 'trade' settlements and the big two aren't licensing their patents at any price. I'm sure this goes on in other fields as well. Mandatory licencing changes that to a monetary barrier to entry that can be easily quantified (such as to potential investors) even if all negotiations break down -- they'd end up with a significant amount of their profit siphoned off to the big two in royalties depending on how many patents they need to use, but if their own technology is revolutionary enough, they can get a shot at trying it out and might still end up profitable (and/or used in turn BY the big 2 after the independent offering fails, still granting the new inventor a revenue stream). If a technology is truly world-changing, the barriers to implementing it need to be as low as possible, and if we're determined to give the first-to-file on a new technology a cut of revenue from all products even vaguely related to that invention (instead of, eg. funding R&D through government prize programs or commissioned research), this is the least damaging way to do it.

[@sharadthecurioustardigrade1533]

@@ExplosionsAndFire it's in a superposition of all of them (quantum shit)

[@theowainwright7406]

ok

[@haydenclaussen7155]

BL21 E. Coli: *nooo u cant just spin me to get me to accept DNA easier* ThoughtEmporium: *hehe spin go brrr* bro you should get an award for this, bc this is incredibly smart

[@SouthernHerdsman]

this comment should be pinned ;)

[@ReapingMiner]

This is common practise in molecular biology labs across the world. ;)

[@Relatablename]

@@ReapingMiner I really liked how he plated the entire cell pellet though. I haven't seen that done during my time in the lab (as an intern).

[@ReapingMiner]

@@Relatablename yeah, i usually grow a liter to OD 0.45, wash, and then ultimately resuspend the pellet in like 20 ml transformation buffer or so; with a little DMSO and make ~60 tubes of 300 ul aliquots. Easily enough for 540 transformations and will last me 2-3 years. Unless my colleagues "borrow" my cells or if i come across many difficult transformations.

[@Relatablename]

@@ReapingMiner Sounds pretty efficient. We were in a medical research lab so cultured stuff was a minority of our work. Our trials were mouse based, so lots of BALF samples, RNA extraction from lungs, qPCR and western blotting. Maybe that explains why I hadn't seen it before? Idk, but the mouses were pretty cute.

[@FantomZap]

I really can't tell you enough how much I love your videos. Every single one is immensely informative and relaxed. Thank you so much. Also as per your question, I'd like to see a multicellular organism modified in a unique way. That would be so cool although I know its much more expensive and labor intensive.

[@Eterrath]

It's the first time in highschool that I learned about DNA, Escherichia Coli and this mythical field called genetic engineering. I was looking up on KZbin about DNA and stuff to prepare my notes better and oh well, here I am now. But I really like your sense of humor and how you were so open minded about sharing your recipe with everyone. So, liked and subscribed! Best of luck.

[@Tommy-pv1vh]

6:10 just like a cadillac northstar v8 nobody knows for sure how or why the engine is running but as long as you don't look too closely at it it will work

[@TheAechBomb]

until it just doesn't for no reason

[@Dinnye01]

@@TheAechBomb superposition. It both works and doesn't, until you start checking explicitly. Macro wuantum phenomena.

[@SoI_Badguy]

Just keep putting oil in it lmao

[@dominicsurette2890]

EXPLOSIONS AND FIRE, YEESSSSSS

[@seannoe750]

I love troubleshooting that goes like this. Something that seems daunting at first becomes obvious when you perfect your protocol.

[@RangerAce]

Oh my god thank u for sharing ur protocol with everyone! Also as a student who's done tons of bacterial transformations, I love seeing seeing u work with such mastery on camera :)

[@129chrisseb]

Avoid like the plague, says the biochemist with several petri dishes before him. . .

[@shadesmarerik4112]

>.< so true

[@PancakeInvaders]

Thanks to your videos, I've been motivated to follow biology and chemistry courses on MIT open courseware, and I now understand way better the things you talk about in your videos and streams about genetical engineering (plasmids, promoters, etc)

[@thorvaldspear]

14:00 nice Tom Scott reference

[@andoan4510]

This is both fun contents and helpful stuffs. I am trying to do a similar thing in my undergrad research lab but with a mammalian cell line, though different, there are some pretty helpful stuff to learn here while enjoying the content. Please don't stop making videos :)

[@latitzouri]

The problem is that you're too interesting, each time you publish something I redo a marathon

[@Broetchen98]

The comparisson with the goat reminds me on a Lab at my university, where you shouldn't wear a hat. Because everytime someone wears a hat, the experiment did not work.

[@lewan1905]

These are the kind of things that I love about working in a lab. These completely absurd and random rules that get established because something went wrong one to many times or something worked once and nobody wants to change it.

[@Lorentari]

I have a possible improvement to your plating technique to avoid having only a big smudge of cells that you talked about around 12:45 - On a single plate, plate your bacterial in one quarter of the dish. - Take a new inoculation loop and drag a sine-like wavy line from the inoculated quarter an adjacent quarter. - Take another inoculation loop and drag a new wavy line from quarter 2 to quarter 3 - repeat for 3 to 4. This way you will have made a natural dilution of your culture, and assuming you had enough for the first quarter, one of the quarters should have an appropriate amount of cells for your continued work.

[@TotallyNotJoe_]

This honestly gives off the same vibes as a relaxing cooking channel

[@ShamelessDuck]

I've only just a week ago re-discovered E&F channel and binge watched lots of his videos. And now my favorite biology channel posts a video that starts with E&F's grudge towards yellow chem. I love it!

[@morkovija]

Oh boy!The wait is over! Smother us with delicious bacteria comrade!

[@heh2393]

Uhhh, mom! The neighbour's kid is behaving weirdly... again!

[@RonakDhakan]

Biologists getting their fun by naming bacteria 'Turbo".

[@paavobergmann4920]

biology is nickname heaven

[@michaelclark4876]

@@paavobergmann4920 Joke nickname heaven even. Especially the names or mutant phenotypes and the genes producing them in many model organisms. Rutabega and Dunce mutants for memory defect mutants, cheapdate for ethanol sensitivity, ether-a-go-go for making flies shake and dance from ether. The gene gets named after the mutant. So dunce encodes a serious, stoodforcan probably guess what the name for the cell cycle regulatory kinase called YAK means. We kept pulling kinase after kinase out of screens for a while. It got old.

[@MegaScienceguru]

To think i started following this guy when i was a teenager and he was making a fuser in the basement with an old vacuum cleaner and now we're here. My hats off to you dude your videos never disappoint

[@o_--]

14:03 do I spy a tom scott reference?

[@randomgooy7456]

Wait how is this comment 3 hours old

[@Theinatoriinator]

@@randomgooy7456 channel member / patreon

[@randomgooy7456]

@@Theinatoriinator oh

[@sohamsengupta6470]

Yes indeed. I'm just trying to figure out if it's also a covert little jab at the ad itself which he's doing

[@criszis]

@@merrillgeorge1838 the website is "gay pirate assasins", a reference to tom scotts vpn video.

[@matterwiz1689]

3:46 Is it alchemy? Or is it surface plasmon resonance?

[@PranavViswanathan]

all the references in this comments section make me so happy. nah its probably ghosts.

[@matterwiz1689]

@@PranavViswanathan the ex&f fanbase is on withdrawal. The last video came out pretty recently but we know it's gonna be a while...

[@jefflyon2020]

I like how this scientist describes his area of study.I usually watch astronomy or chemistry videos but this guys personal experiences mixed with procedure is great! Thank you for such interesting video content, I may be ignorant regarding knowledge of biology but love to learn, and i ALWAYS come away with new understanding of our world around us no matter how big or large, close or distant, hot or cold, or safe or toxic.

[@roborichi2307]

Happy to see that you got over this hurdle! Hope you get over more soon and fast!

[@dsblocks]

"the biggest issue with these is: they were ONLY selected for speed, this means they're terrible at just about everything else" also accurately describes horses.

[@michaelhicks8603]

Thankyou for referencing possibly the greatest chemistry channel on KZbin!

[@simonsays5552]

Most of this was over my head, and I’ll probably never use any of this in my day to day. I loved every second of it!

[@piegolem2266]

Love the E&F clip & reference. Both amazing channels.

[@jim2725]

Its funny how i watch these video's, while the closest thing to actual lab equipment I have is a $20 microscope.

[@CamStLouis]

Man I wish I had known this approach back in my college days (2010-14). My old bio prof will love this, I'm going to get in touch and see if we can't amp up that disappointing glow gene demo with this method.

[@PaulMenden5659]

This is interesting. I haven't had many issues with transformation efficiency of BL21 (I used Origami B) My trafo protocol is: 200uL chemically competent cells + 1uL Plasmid (from a miniprep) Incubate on ice for 30 minutes Incubate at 42°C for 1 minute Add 800uL LB medium Plate 100uL on agar containing an antibiotic (I used Amp). I didn't incubate the cells in LB before plating on antibiotic, but it works. Variation with more recovery time: After the 42°C step Transfer all 200uL to 2mL LB Incubate at 37°C for 45 minutes (agitated) Plate 100uL on agar containing an antibiotic.

[@justsomeperson5110]

Awesome! Thanks for sharing!

[@michaelclark4876]

You might be surprised how often even academic labs are like this. Though often it's is less completely homemade and more vintage stuff kept working with duck tape and epoxy. When I was doing a post-doc, I did built an elevated plus maze and open field from scratch using acrylic sheet. Saved a few thousand that way.

[@slowlylosingmysanity9726]

The container and apparatuses you guys use just look amazing am im really curious what are those and what they do, im planning on collecting em for some reason lol

[@kendarr]

Right? They look so cool lol

[@Djoodibooti]

GLOW IN THE DARK BABY, HERE I COME!

[@theovermuffin1027]

I do a very similiar protocol for quick and dirty E. coli transformation. Only differences: 0,1 M NaCl is already pre-cooled No washing of the pellet with NaCl Heat shock done at 42 °C for 45 s I only add 200 µl of LB0 for recovery, do the same incubation and then plate twice, once 10 µl and once 100 µl (to buffer out too little or too many colonies) Rest is stored at 4 °C over night in case I switched up the antibiotic resistance :D For spreading I love glas beads, best spread I have ever seen

[@lequix9787]

Love the honest description of how VPN works and the Tom Scott reference at 14:03 (The traffic is still encrypted without VPN though)

[@mmcharchuta]

My internet's low, but i just have to set the quality to the highest to appreciate your nice shots!

[@ttld678]

I can’t believe we have a scientists such as yourself who’s also willing to share such amazing breakthroughs that’s revolutionize Proteins synthesis processes

[@damianvdm6527]

I don't comment much but watching this video I really was blown away by how easily you share such valuable knowledge. Just know that people like you keep the world spinning

[@bippybips2840]

Totally trying this ASAP... you're the best :)

[@namAehT]

"They all smell like butthole" is probably the best line I've ever heard you utter. Ok, "Fart DNA in their general direction" is pretty good too

[@e2rqey]

Ayeeeee!!!!! Great to see someone else into E&F. Hopefully you guys can do a collab somewhere down the line!

[@ExplosionsAndFire]

What would we do? Is there like a bacteria/explosives cross over? Hmmm

[@kriegh94]

@@ExplosionsAndFire idk, the only thing that comes to mind is "methane biorreactor". But now i think it, thought emporium has made videos about other cool stuff appart from microbiology and genetic engineering. Like exploding water with ultrasounds or a freaking fusor if i recall correctly.

[@Serachja]

that's very interessting, very cool video and congrats to your success with your efficiency improvment!

[@tanteidaiko]

In my lab we plate it a little differently. Similarly, we pellet down the bacteria. But instead of picking up the pellet, we remove access LB, leaving about 50uL of LB. Then, we resuspend the pellet in the 50 uL LB. The 50 uL suspension is then drawn and plated. I find this method much more effective as we do not leave any pellet inside the tube.

[@nedisawegoyogya]

That's good progress. Most procedures are so rigid to me. I know this is because it's so hard to make things right. But sometimes that cost obvious change like this.

[@SomeGuy-yl1bx]

*Mentions Explosions&Fire* Ah, I see you're a man of culture as well.

[@kadenherzog9727]

Some of my favorite youtubers, a mad chemist, an explosive guy, death ray tech, and God to bacteria

[@PoIiwag]

"How do defeat bullies" Google: tell an adult Bing:

[@devinnelson2504]

The explosions&fire love is great to see. I’d love to know what advice you’d pick up from crime pays but botany doesn’t lol

[@Ethereal.18]

Any new updates on synthetic spider silk beverages 😂

[@teteteteta2548]

It’s silky smooth

[@mozkitolife5437]

@@teteteteta2548 Here, take all my likes.

[@hermanni1989]

Its really cool to learn about the different tools and tricks like the syringe filter.

[@A2dy]

I wish I worked for Qiagen, Thermo, ependorf, VWR, etc. so I could hook you up with all the goods to make your work easier! Love this type of content.

[@devilgene7330]

Next video "how I got raided by the FBI because of a thumbnail

[@Mchacz8008]

Amateurs: Ice Pros: *PEAS*

[@FilterExel]

Ahhh, you've found the ease and magic of the CaCl2 method. I used that to make some Rosetta(DE3)PLysS cells competent to about 1x10^7 cfu back in the day. It really saved me. Before that, my competent cells were all incompetent.

[@georgejones5019]

I'm in IT and going for cyber security. I stumbled upon your channel because I follow other science channels and sciences contribute or benefit from computer science as well. This is oddly interesting to follow.

[@greenonions785]

I loooooooove explosions and fire! You just earned extra respect from me

[@leemon2677]

No idea what is going on in the video but youre basically the Half Blood Prince of microbiology

[@louis1001]

I love your open source approach to science.

[@christopherperry8389]

The way you explain this stuff makes me feel capable of (given infinite time, patience and materials) modifying DNA in my own house. Great work!

[@hgbugalou]

Explosions and Fire is my favorite youtube chemist, though nurd rage and nile red and close. He is aussie though so advantages.

[@genericalias2179]

this video: *exists* me, an artist who probably won't ever ever do anything with this information: interesting

[@f.g.4309]

Everytime, after I done PCR site saturation mutagenesis. I always struckle on no colony form. I use both BL21 and NEBstable for my experiment thank you very much for your dedicated attemp to find a new efficent transform protocal ❤.

[@grinreaperoftrolls7528]

Congrats on the breakthrough, my dude!

[@BobofWOGGLE]

0:25 "The media is yellow" have fun when this line gets taken out of context in a few years

[@thingyjim]

I LOVE THE PEAS! Gunna try bringing a bag of peas into my lab and see how it goes with health and safety!

[@edmclaughlin4923]

Excellent video Sir. Thank you!

[@throughcolouredglasses9300]

I cannot get over how solid the science and protocols are, like this guy is legit - yet all the "I bought this" and "I built this cheaply myself" -equipment gives it the distinct vibe of just some dude in his garage fiddling with a hobby he taught himself from youtube tutorials

[@bobfrediii2131]

Programmers nemesis is the semi colon ;

[@OkuriLucy]

Damn you

[@JGnLAU8OAWF6]

Not if you write in Python

[@danielnewton2390]

The frozen peas really caught me off guard!

[@AxelLazuli]

This was super interesting! Thank you for sharing!

[@ayselsozer67]

I successfully transformed BL21 E. coli with a pBAD/His containing IDH1 gene (both mutant and wild type) for brain tumor research. Also once transformed pGFPuv (green fluorescent) into BL21 cells. A lot of the steps here are the same as what I did in the lab. Amazing tip about swabbing with solid loops (i did with sterilized toothpicks worked fine as well). I second this protocol. What you're doing is inspirative and amazing! can't wait for more extraordinary contents