I Put CARROT Genes Into YEAST, Then Baked With Them

even though genetic modification often gets bad press it's actually pretty amazing in several previous videos we've looked at how the basics of genetic modification work and modified bacteria to fluoresce a wide range of colors we also went through many of the basic lab protocols and the construction of various pieces of lab hardware necessary to genetic engineering work so with that groundwork laid today we can finally tackle something a little more difficult if this is your first time learning about genetic modification I've linked to those previous videos below this is beta-carotene and this is a carrot when you eat a carrot which is full of beta-carotene the molecules broken down in your digestive system to produce something called retinol better known as vitamin A vitamin A is one of several essential vitamins which if you don't get enough you'll go blind and then quite likely die conversely if you get too much carotene you'll turn orange so you know balance is important now for most people getting in a fight of monnet is pretty easy just eat basically any vegetable once in a while but let's say for example there's been I don't know a global pandemic and so you've decided that you're just gonna bake sourdough and drink the tears of a passing sparrow to sustain yourself rather than going to the grocery store you've now got a problem well okay you've got a lot of problems but at least one of them is vitamin A deficiency but what if science could solve this for you and allow you to keep feeding your bread addiction while also giving you the vitamin boost you desperately need well it can and today I'm going to show you how on a more serious note vitamin A deficiency actually affects millions of people per year even killing about two million many of the people who suffer from this sort of malnutrition subsist largely on staple crops like rice which prompted scientists to engineer a strain of rice that produces beta-carotene which they called Golden Rice but for whatever reason when you'd make genetically modified plants people kind of lose their damn minds even though they're perfectly safe so getting the strain deployed to help those people has been very challenging but what if there was a different route what if rather than modifying the rice itself we instead modify yeast I think it would be much easier to convince people to bake with a bespoke yeast variety than it would be to get them to accept what looks like a whole new and even though only a few genes have been added in both cases in theory you can also brew with it and make a vitamin enhanced beverage though there's some problems with this which we'll discuss later so various researchers as well as an IgM team did exactly that and today we're gonna do the same what's nice is that you can theoretically use whatever yeast you want for this so you can choose strains that have properties you like and then just add the beta-carotene genes for example I like to brew with a type of yeast called D 47 which is known to be great for making things like wine and Mead or you could use baker's yeast which is obviously great for baking so how does this work well to go from sugar to beta-carotene there's a few chemical reactions that the yeast will have to do and by providing the right genes any yeast can be made to do them first they need to make a compound called F PP or farnesyl diphosphate now while this looks complicated it's actually one of a few baseline compounds that yeast and many other organisms already make large quantities of you see it's sort of perfect for making big or complex molecules by either rolling it up in specific ways or attaching different molecules to it or just sticking two of them together it can be used to make an absolutely massive assortment of biological compounds everything from squalene the oil found in olives and shark livers to various antimicrobial compounds flavor compounds and so much more point is yeast make a lot of the stuff already so it's readily available for making carotene and not something we need to bother messing with just feed the e sugar and we'll make it on their own to go from F P P to keratin though requires three and a half enzymes I say and a half because it technically takes four but you can shmoo two of them together into one chunky fusion that does both jobs at once the enzymes are CR te c RT b c RT i and c RT y though typically you fuse y and b together into one called c RT YB it'll become quickly apparent that biologists are [ __ ] at naming things so just go with it c RT e combines f PP with another readily available starting compound called isopentenyl die phosphate which adds an extra little chunk to it resulting in a compound called Geron older on old die phosphate which to me sounds kind of like Duran Duran but I digress from there CR TB also called f---eighteen synth dey's sticks two of those together into a compound called fighting from their CRT I does two steps where a bunch of double bonds get made and you end up with a compound called lycopene fighting is colorless but lycopene is bright red so at this point we start getting our first bit of color in the pathway from their CRT why finishes things off and curls the ends of the lycopene into rings and we've got our final beta carotene which is bright yellow if you think about carrots and their high carotene content you may have expected the compound to be orange but in reality carrots are orange because they contain high levels of both lycopene and carotene which combined the red and yellow colors to give the orange hue to the delicious roots in tomatoes on the other hand there's very high levels of lycopene but fairly low levels of carotene so they appear bright red there's also other compounds in the carotene pathway if you keep modifying it which are other shades of orange and red but they aren't important for right now the important part is that as long as you Express the four different enzymes in yeast or many other organisms they can be made to produce beta-carotene now that's all fine and well but how do we get those genes and how do we get them into the yeast well luckily thanks to those previous researchers and the iGEM team I mentioned earlier the genes for doing this are readily available I got them from a company called add gene which is sort of like a github repository but for bits of DNA and there's another company called Carolina biological which also carries them as a DIY kit and I've linked to both below in this case they come as a plasmid which is a small circle of DNA that contains all the genes we need as well as some extra genes that make it easy to select for the modified yeast once we put the DNA into them so all we have to do is take this DNA put them into some yeast and they'll immediately start making carotene let's quickly go over the DNA map and see how this works you can see the various CR T genes but there's also this one extra gene called th mg one technically this extra gene isn't necessary and without it the yeast will still make carotene but having it helped speed up the process of making one of the precursors for fpp so you get higher yields of carotene as a result I won't get into that pathway though as this is already complicated enough as is but the point is more fpp means more care Burdine so having it is good the other important features I want to quickly point out or this one marked can are this one marked amp R and this one marked Ori the can are gene codes for resistance to an antibiotic called g418 which we'll be using to select for the modified yeast later amp R is ampicillin resistance which is also a gene for resistance to an antibiotic but this one will only run when the DNA is in e.coli when you just want to make copies of the DNA so you've got more to physically work with we put the DNA into e.coli because they're easy to modify grow quickly and are more importantly easy to get DNA back out of basically there the photo copiers of the DNA world so this amp R gene and also the Ori are there for that purpose the Ori is literally the spot where that copying starts for more about this process and the specifics of how it works check out my video on genetically modifying bacteria to make the plasmid last a long time and importantly make it so we can grow the yeast without using antibiotics when we want to bake with it later there's a similar bit of code to the ori but a yeast version it's the section marked cen /a RS this turns the plasmid into basically a tiny artificial chromosome that will get copied over as the yeast divide there are also these little tags called loxp sites which can be used to fuse this DNA with the yeast core genome making the mod truly permanent but that'll have to be a topic for another day as it's a really involved process okay so we know what's in the DNA now we just need to get it into some yeast now this is actually the part where I've been struggling don't get me wrong I managed it as you'll see but for the biologists watching I'll be asking for some help in a moment if you've got a better protocol than the one I'm gonna be showing for this protocol I'll need a few things first are two different types of growth media one with and one without antibiotic mixed in for the one without I'm gonna actually need this in two forms liquid broth as well as solid petri dishes the antibiotic one I just need two solid dishes I'm not going to go over making media as I've covered that several times before I'll just say that I'm using malt extract broth and malt extract agar for the antibiotic plates there's 200 micrograms per milliliter of g418 in the media in terms of the tools I'll need an inky Eider set to 30 degrees Celsius a mini centrifuge some inoculation loops and a Bunsen burner pipettes and tips some sterile one and a half mil and 15 milliliter tubes and though not strictly necessary some sterile individually packaged swabs are also helpful now the only other thing I need is a solution which will make it easier for the yeast to uptake the new DNA there are a lot of variants of this but the one I went with is called plate solution this is a mixture of polyethylene glycol 3350 lithium acetate tris hydrochloride and EDTA the last two are for buffering and making sure the solution stays stable and the former to make the e cell wall more permeable I can't tell you exactly how this works because like many things in biology no one actually knows we just know that it works and then one final ingredient is a little bit weird but also mandatory which is short single-stranded DNA again no one's really 100% sure why this is necessary but it seems to have something to do with the yeast natural defenses against foreign DNA and is believed to clog up that mechanism and allow the DNA we want to enter the cell in this case I'm using single stranded DNA derived from salmon sperm and no I didn't jerk off a salmon this is something you buy from a biological supply company pre-made into single-stranded form before using it though it needs to be boiled for 10 minutes and then quickly cooled to make sure that none of the single strands have stuck back together I do this by just taking a small aliquot from my main stock tube and adding it to a little PCR tube then heating it to 98 degrees in my PCR machine which I've got preheated then I just stick it in some frozen peas which I use as my reusable ice bucket rather than bore you with the tedious process of mixing the plate solution I've just included a recipe in the description everything was measured out and mixed and then filter sterilized once everything was dissolved as I've showed how to do in a previous video this makes it sterile and ready for use I also prepared 100 millimole lithium acetate solution separately in the same way which we'll be using to wash my yeast speaking of which for the version I'll be showing here I use the D 47 line East which I mentioned earlier but any strain of sacrifice you service EI should work for this if you want you can start with regular baker's yeast by just dissolving a few pillow of yeast from a store-bought jar or packet into some sterile distilled water then using that solution streak it out onto a malt extract plate to get individual colonies I've also shown how to do this in a previous video then put the plate into an incubator for a day or two to grow this plate can then just be stored in the fridge until it's needed the night before I did the transformation I prepared one milliliter of malt extract broth in one and a half mill tube and inoculated it with one colony from the plate this will give lots of actively growing yeast to work with now the yeast need to be washed and all the growth media needs to be removed to do that I used my new micro centrifuge the yeast were spun down at 6,000 RPM for about 30 seconds to pellet them and then the liquid was removed and replaced with one milliliter of lithium acetate solution this was vortex to redistribute the yeast into the liquid before spinning it down and again removing the liquid next I added 10 microliters of the single-stranded DNA and 5 microliters of the carotene plasmid which was about 1 microgram worth of DNA total then I added 500 microliters of the plate solution and gave it all a mix the tube was then transferred to my incubator for 1 hour in theory the yeast are already up taking the new DNA at this point but to really make sure it gets in there I used a technique called heat shocking basically this means warming up the yeast a little more than their usual growing temperature sort of stress them out and make the cell membrane more permeable I did this using the heat block I made in a previous video which was set to 42 degrees Celsius unlike bacteria where their heat shocked for no more than a minute or two yeast are much larger and more resilient so the heat shocking lasts for 20 minutes also unlike bacteria where they need to be quenched immediately in ice afterwards Easter just spun down and the plate solution is removed and replaced with one milliliter of malt broth before the yeast are simply resuspended at this point because I'm using antibiotics for selection I need to give the east time to start expressing the new genes so the tube is transferred to the incubator and they're allowed to grow for three to four hours once they've had time to grow and start expressing the new antibiotic resistance genes they can be plated on the antibiotic plates without this incubation period they would just die instantly as soon as they hit the plate in this case I plated 150 microliters of yeast solution and spreaded around with a sterile swab the plate is then placed into the incubator and left alone for a day or two to grow now here's where I get to show off my absolutely legendary transformation efficiency only one single colony grew which means that of all the millions of yeasts that hit that plate literally one single cell survived so not an ingrate home about but the nice thing with bio is you only need one that single cell grew over the few days into literally millions more and formed a nice plump bright yellow colony which I promptly named Frederick I then took a sample of Frederick and streaked it out onto a new plate which now can serve as the basis for all future experiments and be expanded further as needed just as a quick comparison here's some plain yeast and the modified yeast side by side I think the difference is pretty clear now since my transformation efficiency is such garbage I'd like to ask for your help if you work with yeast let me know what your improved protocol uses in the comments down below and I'll pin whichever one ends up working the best once I test them out one quirk of this particular plasmid is that the journey from fpp to carotene isn't exactly smooth and the system gets a little bit gummed up at the phyto in stage it's great at making phyto in and any like a peanut makes is quickly turned into carotene but the phyto into lycopene step is weirdly inefficient so you end up with a really large reserve of the colorless f---eighteen and sort of medium levels of the carotene but very little lycopene which is why it's so incredibly yellow the iGEM team I mentioned earlier got around this by adding an extra copy of the see RTI gene which helps speed up the conversion of phyto into lycopene and so they ended up with nice orange colonies as a result that's a bit too much to cover in this video so I hope to revisit that process at some point in the future if I ever manage to replicate it now that I've got some functional golden yeast let's put Frederick to work and actually make some stuff first up some basic sugar wine here I prepared 400 milliliters of sugar solution which I added a small pinch of wine nutrient to this is a mix of minerals and nitrogen compounds that'll help give the yeast and add in boost so they aren't just growing off of pure sugar but you only need a tiny amount so it won't tint the solution and any color will come from the I oughta cleave that mixture to make it sterile and then inoculated it with a big scoop of the yeast using a sterile swab and then I just capped it with an airlock and let it grow for a few weeks and when all was said and done there was a nice cake of vibrant yellow yeast at the bottom but the liquid was still basically perfectly clear this is what I meant at the beginning about there being issues with brewing with this turns out carotene is hilariously insoluble in water so even if the yeast pop when they die almost no carotene ends up in the solution so if we want that vitamin boost we're gonna have to take a different approach conveniently a recipe I demonstrated on my second channel the taste Emporium comes to the rescue which by the way if you haven't already you should absolutely go subscribe it's all the science goodness you've come to love but with a delicious edible twist and some of the previous videos I think I've ever made links in the description while everyone was on their lockdown sourdough kick I made a video showing how to make a simple sourdough starter and then use that to make some delicious rustic bread and I'll be modifying that process here the nice thing with bread is you end up actually eating the yeast itself so any of the carotene they produce will end up being eaten first I poured off most of the sugar wine into a separate container for storage and then save the yeasty sludge at the bottom using that liquid I made a mix of flour and yeast sludge in a clean jar I also added a couple drops of lemon juice to make sure that the mixture is slightly acidic to discourage bacterial growth this was allowed to grow for a day or two to make sure the flour was chock full of golden yeast and very active then on baking day I added a fresh batch of a little bit of flour and a little bit of water to the mix to give it some food and get everything really active then waited four to eight hours to get it good and bubbly then I just followed the usual recipe as if this was just a regular sourdough starter 150 grams of starter 250 grams of water 25 grams of olive oil 10 grams of salt and 25 grams of sugar are all added to a bowl and mix thoroughly then once it's all combined 500 grams of all-purpose flour was added and the mix was needed until smooth this is then covered in aluminum foil and placed somewhere warm for a few hours to double in size once it's doubled in size the dough is turned out onto a well floured surface just to coat it gently in flour and then it's transferred to a bread which I lightly greased with some olive oil in advance this is then covered with a towel this time and again allowed to rise until it's doubled in size once that's done I made some slashes in the top to give it a place to expand while it bakes and has then placed into a preheated oven at 375 degrees Fahrenheit or 190 degrees Celsius for 10 to 20 minutes until it's golden brown and that's all there is to it when I cut a slice as I expected the bread doesn't look the neon yellow you might have hoped but the iGEM team baked a similar loaf of bread for their experiments and through chemical analysis was able to detect very noticeable levels of carotene in the loaves made with the modified yeast so you'll definitely get the vitamin boost you need big question though is how does it taste honestly the same as any normal bread that's the best part of this it looks the same tastes the same but it's full of extra vitamins if this stuff was sold in packets to bake with you'd never know the difference and that's what's so beautiful about it in theory it doesn't have to stop with just vitamin a several other vitamins can be engineered into yeast in a similar way vitamin C for example has also been covered by the iGEM team and have linked to their research below if you want to check it out all in all this is a beautiful piece of engineering that could legitimately have a real impact on people if used in the right circumstances for huge portions of human history surviving on mostly just bread has been an unfortunate reality but I think it's awesome that a little modern science can help make the lives of those people better at literally zero expense I was actually so thrilled with this whole project that I had my fantastic artist friend NAT make these awesome t-shirts so if you want to join the biologist baking club there's some links below where you can pick one up before I wrap things up I need to quickly thank the sponsor of this video expressvpn as you may have gathered from the name they're a VPN service which helps protect you while you're on the internet even though most sites these days are encrypted so people listening in can't see what you're doing on a website they can still see what websites you're visiting you may not think this is a big deal but you can figure out a lot about people just from their browsing habits ISP is in the US and UK can even sell that metadata and if you're using public networks like a university dorm cafe or airport the administrator can see that data too personally I always use a VPN especially when I'm going on late-night internet rabbit-hole trips as I can end up in some pretty weird places VPNs get around this by acting like a middleman so all the ISPs in admin see is you connecting to the VPN from there your connection is sent off to the website you're actually interested in not only does this protect your privacy but it also unlocks blocked content as well to the website your traffic looks like it's coming from wherever the VPN server is so if you're in Canada like me for example you can view content that may only be available in the US and Express VPN servers are secure and don't log any of your data so you can rest easy while getting the best speeds available best of all it's super easy to use and in just one click you're set up and online find out how you can get three months free by heading to express VPN calm / thought Emporium or clicking the link in the description down below if you enjoyed this trip into the weird and wonderful world of bioengineering I've linked my earlier videos below and I've got many more coming up so you should definitely subscribe and if you think yellow yeast are cool just wait till you see the versions I'm working on that produce everything from spider silk to egg whites and even deer milk and many of those are being designed on the live streams I now do twice a week so if you want to learn how to design projects like this I recommend stopping by finally I need to say a very special thank you to my amazing patrons channel members and supporters on cofee and stream labs your support is what lets me work on these amazing projects and I can't thank you all enough if you'd like to help keep the flow of science videos coming there are some links below and of course if you'd like to keep up with these projects and see how they're doing then be sure to head over to my other social media pages to see updates and snapshots long before they end up in videos that's all for now and I'll see you next time

Channel: The Thought Emporium

Views: 504,684

Keywords: baking, yeast, genetics, vitamin a, retinol, beta carotene, carotene, genetic modification, gmo, genetic engineering, biology, molecular biology, biohacking, biohack, hack, brew, brewing, bake, bread, sourdough, molecular gastronomy, science, education, tutorial, stem, steam, engineering, biochemistry, enzyme, protein, plasmid, dna, rna, metabolism, expressvpn, vpn

Id: DHNPnO5UOYQ

Length: 21min 48sec (1308 seconds)

Published: Mon Jun 29 2020

Reddit Comments

[u/padizzledonk]

"And no, before you ask, i didnt jerk off a salmon..."

I am not a scientist and this video is dense af but it is highly entertaining

[u/Desmeister]

Is this that guy who cured his own lactose intolerance and will possibly give himself cancer in the process

[u/gratefulyme]

Psh, forget vit A, get us some of that yeast that produces psilocybin...

[u/WanderingOnward]

I like this a lot, but I have a question. Is “shmoo” a technical term? I laughed so hard at that

[u/WayneCarlton]

this guy genetically modified himself to not be lactose intolerant using crispr i think. he's crazy.

[u/onahotelbed]

Little secret: the yeast that you buy at the store has been engineered to make compounds that improve the bread you make with it. In fact a lot of commercially produced bread is made with engineered yeast.

[u/TacoDoc]

I tied my shoes today.

[u/bombayblue]

Remember when we already modified rice to produce Vitamin A but then Greenpeace killed it?

I’m sure the tens of thousands of people suffering from Vitamin a deficiency every year are thankful.

[u/RealFunction]

i was expecting yellow bread.

i leave disappointed, but well-informed and entertained.