Hacking Your Own Fluorescent Yogurt

There is a common conceit among we DIYbio enthusiasts, namely to suggest that one could opt to create “glow-in-the-dark yoghurt” using DIYbio-oriented techniques as a nigh trivial matter. Indeed, this conceit led to my recently being queried by twitter and email about the possibility; where are the guides and how-tos, if it is so trivial? While a conceit it may be to suggest that glow-in-the-dark yoghurt would be trivial, that's not to say it's at all out of reach to the dedicated biohacker. Here, I will lay out a suggested course of action based on the available literature.

Firstly, let use choose our definitions. What do we mean by “glow in the dark”? There are two commonly pursued strategies to choose from; fluorescence and bioluminescence. The former means that the bacteria will glow some colour when exposed to blue or ultraviolet light, usually green. The latter means that the bacteria will literally glow, emitting their own light from within provided they have enough energy from food.

Though bioluminescence is without doubt a cooler trait, for a variety of practical reasons, fluorescence is a more practical choice. Until, that is, one has more experience in yoghurt hacking and more money to burn on ambitious goals! So, here we will explore the transformation of a yoghurt bacterium with a variant of “Green Fluorescent Protein", which renders organisms fluorescent under blue or UV light.

So, now that we know what we want, what are we working with? Yoghurt is most often composed of a co-culture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, shortened hereafter to L.bulgaricus (its former name). The two species work in concert, providing nutrients and cofactors to one another and producing acids and bacteriocins that prevent invasion of other unwelcome species. Together they digest and remove much of the lactose content of the milk, and produce the polysaccharides and volatile molecules that give yoghurt its distinctive mouthfeel and flavour.

Of these two, either could be engineered to fluoresce, though the construction of the DNA and the methods needed to get it into the cells would differ. For this example, let us say that we have chosen to engineer S.thermophilus (chosen at random; I haven't even researched L.bulgaricus, and it may indeed be a far easier bet for this project! Expect an update later if so.).

A quick literature hunt on DuckDuckGo and Google Scholar reveals that the most popular method for engineering S.thermophilus is through induction of a state called “Natural Competence". In this process, cells are encouraged to activate a natural system of DNA uptake by exposing them to starvation or stressful conditions. This system is composed of multiple regulatory systems, and involves proteins that actively bind and absorb DNA, and proteins that splice this DNA into the cells’ chromosomes if it matches them at least partially. Not all strains respond to the usual methods used to induce competence, and some strains are considered nontransformable even though they are known to contain a seemingly intact competence gene array.

This poses a problem to DIYbioers, who may not know whether their chosen strain responds to induction of competence. However, a deeper search through the literature reveals a more universal method. This method uses an artificial pheromone called ComS(17-24) (AKA Shp316 which consists of the amino acids “PYFAGCL”), which mimics the natural induction pheromone required for competence to develop. Interestingly, even in strains where competence cannot be induced normally, addition of ComS induces competence provided the necessary genes are intact. Therefore, this seems a safer bet for DIYbioers seeking hacked yoghurt!

You might be thinking at this point, isn't that an awkward thing to get? Firstly, the alternative involves starving the cells in an extremely awkward “chemically defined medium”, the ingredients for which are likely to be expensive if desired in moderate purity. Secondly, the pheromone is a short peptide (a very small protein), and there are several companies offering cheap peptides; $2 per amino acid seems to be the usual price. For the ComS peptide (“PYFAGCL”), that would amount to $14, although expect to pay tax and refrigerated shipping sufficient to raise the price to perhaps $40 (or more?). For that, you will probably get quite a few transformations done!

Methods employed for induction of competence (see methods in this paper for an example) through ComS seem to bear a few artefacts of prior methods, including the use of chemically defined medium and prolonged incubation times with DNA prior to the addition of ComS. The use of Chemically Defined Media was originally necessary to induce competence by starvation, but with ComS added artificially this shouldn't be needed anymore. Although I have not tested either the referenced methods or my suggested updates, here is a protocol that ought to work based on my understanding of the process and the function of ComS:

  1. Grow overnight culture in a rich broth at 37C.
  2. Overnight culture washed twice in same volume of broth; (to isolate cells between washes, centrifuge at 5000g for 9m at room temperature).
  3. Re-suspend in one volume of 50% diluted broth.
  4. Dilute 1/30 in 50% diluted broth, aliquot to 300ul Volumes.
  5. Add 1uM ComS(17-24) and 25ng linear DNA with 1kbp+ complementary ends to target site.
  6. Incubate for 5h at 37C.
  7. Plate on selective media and incubate overnight at 37C.
  8. Choose colonies and PCR-verify.

“Rich Broth” in this case is usually an exceptionally rich broth called “M17”, which includes beef extract, yeast extract, pre-digested protein, glucose, and a few salts and phosphatase inhibitors, sometimes even with lactose added for good measure. This is almost without doubt overkill. A broth consisting of skimmed milk powder and a bit of Yeast Extract (without added salt) will certainly suffice. Let's say 20g Skimmed Milk Powder and 3g Yeast Extract in 1L deionised water.

You'll note that in the above protocol, the DNA is required to have 1kb+ complementary ends to chromosomal DNA. That's because the uptake of DNA is only the first part of the process; the DNA has to integrate into the cells’ own chromosomal DNA in order to be maintained for longer than a generation or two. To achieve this, you have to provide DNA that has flanking complementary regions long enough to encourage a process called “homologous recombination", wherein the cell exchanges part of its normal DNA for the provided DNA. In order to select for the subset of cells that do exchange their DNA thusly, it is common to use antibiotic resistance genes, so that non-transformed cells can be killed.

Once cells have been selected, antibiotics are probably not necessary in order to ensure the DNA is inherited by daughter cells, as it is part of the chromosome. Since antibiotics are not needed once the gene is established, it would be responsible to remove the cassette afterwards, even if it introduces additional complications. This is too complicated for a “beginner's guide to hacking yoghurt”, but should be strongly considered; I'd suggest an inducible Cre/LoxP system, and would be happy to consult and assist anyone wishing to attempt this.

For our project then, we want DNA that resembles the following:

A diagram showing the format of the desired DNA
Colourised and labelled sections of the DNA, presented with the unnamed section of target DNA. This assumes the simplest scenario where no "cleanup" mechanisms are included to remove the resistance gene.

The promoter (Prom) should be a constantly “on” promoter, termed “constitutive”. The terminator (Term) is a region of DNA that prevents the gene from transcribing beyond its normal context, which could cause unintended interruptions of cellular functions; unhappy bacteria could result, and the gene could end up unstable. The antibiotic should be chosen to avoid medically significant antibiotics such as ampicillin; this is a civic responsibility matter, as otherwise your yoghurt could end up assisting dangerous pathogens in becoming resistant to medicines. Ideally your antibiotics would be self-excising once they become unnecessary, leaving a yoghurt containing only harmless fluorescent proteins and nothing else.

The homologous regions (Flank1 and Flank2) you choose are down to where in the chromosome you wish the DNA to end up. There's a few million letters of DNA there to play with, and quite a few unnecessary genetic bits you could replace if you chose to. There are probably some retroviral genomes hiding away in the average S.thermophilus genome that you could deliberately replace, for example. However, a design consideration to bear in mind is the presence of native restriction enzymes; enzymes that chop up DNA when they detect certain “words”. In S.thermophilus, there are quite a few such enzymes, and the most common “words” they target are: CCWGG; CCCG; GGCC; CCGG; GATC; GCSGC; GCNGC; CCWGG.

Although native S.thermophilus DNA won't be cut thusly, due to the protective effects of the same restriction systems, any newly added DNA will be, and the efficiency of transformation will suffer greatly. Therefore, choose target DNA that doesn't contain these words, or contains as few as possible. Also make certain to screen the newly designed DNA for these words, modifying as necessary to remove them. Most codon adaptation tools will offer to exclude sites, and manual removal from other DNA may be required. If you don't know what you're doing manually editing promoters and terminators, opt first to use different promoters/terminators rather than risk failure of the DNA entirely.

Once you've identified the target sites, order PCR primers to amplify the two flanking regions using PCR. You'll need a good enzyme like Pfu or KOD rather than old-school “Taq”, because taq can't reliably amplify large enough regions. Splash out and treat yourself to a good kit, or homebrew some from a Pfu-producing strain.

Then design and order your DNA: choose a GFP derivative of your liking, there are plenty to choose from. Alter the codons in the protein to minimise those that S.thermophilus doesn't use, so that it expresses well. Don't bother with a “best codon” optimisation strategy, as these strategies often fail to achieve the expected results. Just use tools like JCAT (downloaded locally, rather than the online tool) to identify which codons are least desirable.

To do this, find the S.thermophilus genome in NCBI, download it, and feed it into JCAT so that JCAT can build a profile. Then provide the desired GFP derivative to JCAT, and let it calculate which codons are least optimal; replace these so each codon is at least above the 50% margin. JCAT is designed to automagically make all the codons near-perfect, but manual alteration of just the limiting codons is all that's needed. Sometimes, having all-perfect codons can be counterproductive, for reasons not fully understood yet.

Once you have the protein as optimised DNA, attach a constitutive promoter; you can probably find one by searching the literature, nicking it from a critical metabolic gene, or a virus that predates the species. Also add a shine-dalgarno sequence to your protein coding sequence, just in front of the start codon. If you can't find an appropriate Shine-Dalgarno, the complementary sequence to the last few nucleotides of the 16S rRNA (changing Us to Ts) in S.thermophilus should be ideal. That is, after all, what Shine-Dalgarno sequences usually seem to entail; target sites for 16S rRNA binding.

Finally, add a terminator. Terminators should be fairly species independent, as their function is down to their RNA structure forming as they are transcribed. Therefore, you can probably use a good, well-tested terminator from the parts registry, even one from E.coli. However, if you can find a nice B.subtilis terminator, stick with that; it's more closely related to S.thermophilus than E.coli. Best be on the safe side?

The resistance cassette can be built likewise, but it's easier to just find one of the plasmids commonly used for studying S.thermophilus genetics, of which there are several, and to copy/paste the DNA corresponding to the resistance gene out of one of these. Be careful to ensure that the annotated DNA includes a full gene and not simply the coding sequence for the resistance protein; your resistance gene does need to be transcribed in order for it to work! So, if the “resistance cassette” starts with ATG and ends with TAA, be careful to include a wide margin of DNA around it, perhaps 150bp to either end to be on the safe side. If you actually have a plasmid matching one of these in stock, or can source one, just get it and PCR the DNA out to save money.

Once you have the desired DNA for expression of the GFP protein and the resistance protein, you need to flank it with the intended target sequences. In order to achieve this with the PCR-amplified target sequences, you need to use another round of PCR to assemble the two flanking ends to the custom designed DNA; for this to work, there must be primer-length regions of complementarity between the amplified flanking regions and the designed DNA.

In order words, add some DNA to either end of the insertion cassette that matches at least 20bp of the inner ends of the flanking DNA. Then, order your DNA and wait a month or so. You'll be paying about 28€c/bp; that's what I was charged by Epoch, at any rate (whom I can highly recommend).

When the DNA arrives, the procedure goes like this: use PCR to amplify the flanking ends of the target site, and to amplify your intended insertion cassette. Use another round of PCR to fuse the “left” flank, the insertion sequence, and the “right” flank together. Use a third round to amplify from this the finished DNA, by using the upstream (“forward”) primer of the “left” flank and the downstream (“reverse complement”) primer of the “right” flank. This will certainly call for a good enzyme, as you'll be amplifying in excess of 3kb of DNA.

This PCR product can then be directly added, along with 1uM of ComS (which you should also have received in the post by now), to your washed, diluted overnight cells as described above. The cells should, if all goes well, be induced to active competence, absorb the DNA, and include it in their chromosomes. When you apply antibiotic selection after 5/6 hours of incubation, you'll kill any cells that didn't undergo this transformation; there may be very few cells indeed that survive due to correct transformation.

With any luck, you'll see colonies on the agar the next day; try flashing a blue LED at them and see if they fluoresce the desired colour! You can filter out blue using an orange filter, which can be as contrived as a sweet wrapper. If you use UV light, you'll probably have a clearer view of any fluorescence, because UV doesn't appear very bright if it's visible at all.. but don't leave the light on too much or you'll kill your cells! Definitely avoid using UV sterilisation lamps; stick with the sort you use to check banknotes, which use lower frequency light which is less harmful.

If you get your desired glowing cells, congratulations! Reunite them with L.bulgaricus, and try brewing up your first batch of fluorescent yoghurt! If not, try again and don't worry too much. The transformation frequencies with S.thermophilus are really low, so perseverance may be called for.

However, in the end of the day, it mightn't work at all, and there might be no foreseeable reason why it didn't. If so, you'll have joined the ranks of biohackers worldwide who've tried only to meet with unexplained failure. I hope the project didn't cost you too much to attempt, but don't throw it all out; share that DNA with others, see if they can improve the procedure; perhaps a different insertion site? A slight modification to the integrated DNA? A different transformation procedure? Most importantly of all, share your failure; mankind learns most from failure, and not enough failures are shared in science. Be the difference, speak up and tell your cohort what failed for you!

If you want to try this, get in touch with me first for more in-depth advice on some of the details. Before you commit, bear in mind that this project will cost a lot of money at current costs per nucleotide! Unless you can access the DNA for a green fluorescent protein and a resistance cassette (which would save you a lot of synthesis money), you'll have to order these from scratch, and that won't be cheap. At least a few hundred Euro. However, the reward is something you can share with others and boast about for years to come; your own fluorescent GMO yoghurt. Very 2012.

 Share, if you like. Fediverse sharing is preferred, though.