TB or not TB – I recently had that question asked to me – well, actually they asked “TB or LB?” They were asking what kind of bacterial growth media (microbial cuisine) I was planning to use to express a protein. Protein expression is just one of the things we use bacteria for in biochemistry – we also use them a lot for making copies of DNA for us. And we have to feed the bacteria in return for getting them to work for us (I mean, it is only fair!). We call their food MEDIA and there are different types to meet bacteria’s different tastes. These include Terrific Broth (TB) and Lysogeny Broth (LB). Let’s get to some media influencing!
but first, in case anyone’s wondering, I chose TB this time (and am expressing a protein called TEV protease – and teaching a new grad student how to do it so I’m happy – also really happy she joined!) 🙂
We don’t give them 5-star cuisine, basically we want to spend as little money as possible while still giving them the nutrients they need. At a minimum, we need to give them a source of energy, something they can break down (catabolize) to make ATP – such things can be sugars, proteins, fats
In addition to breaking things down, they need to be able to make things like proteins and DNA (do the anabolic part of metabolism). This requires nutrients that provide the elements needed like carbon and nitrogen.
They can’t make everything they need, so we also need to give them anything they need that they can’t make themselves. If they can make something themselves, we say they’re AUTOTROPHIC for that thing. If they can’t make something themselves, we say they’re AUXOTROPHIC for that thing – being auxotrophic is only a problem if you’re in conditions where you need that thing (we’re technically auxotrophic for making coffee but unless you’re someplace that you need coffee to live…)
If they have the same making ability as their parent, we say they’re PROTOTROPHIC for that thing
Sometimes, we feed them in liquid media, when we’re growing them in suspension – we can grow them in broth in culture tubes or flasks, usually at 37°C with shaking and plenty of breathing room
Other times, we embed this media into a gel made of agar (a sugar chain) – they don’t eat this sugar chain, it just gives the gel structure so the bacteria can grow on top of it as colonies and the gel can kinda hold a stock of food for them. http://bit.ly/2MfWDmg
cool fact I learned – apparently the first gelling agent tried (by Robert Koch in 1881) was gelatin. But the bacteria ate it, and it also melted too easily. The wife of one of Koch’s associates, Angelina Fanny Eilshemus suggested he try agar and it worked well. The bacteria didn’t eat it, and it stayed solid (and didn’t kill the bacteria 😛 )
A lot of the time the media we put in there is LB which stands for LYSOGENY BROTH – sometimes initials for Luria, Lennox, or Luria & Bertani get credit for the name, but it really stands for LYSOGENY BROTH and its recipe was first published (by Giuseppe Bertani) in 1951. He was using it when studying lysogeny (a process where a bacteria-infecting virus called a bacteriophage (“phage”) inserts its own DNA into a bacteria’s DNA & bides its time until conditions are right for entering the lytic phage where it cuts itself out, makes lots of copies and bursts open the cell) http://bit.ly/2HLuB1S
Its recipe is really simple and sufficient for lots of bacteria. There are 3 main components (though 2 of those components themselves have a lot of components.
- TRYPTONE -> this is a mix of peptides formed by the digesting a protein called casein with pancreatic enzyme -> this provides amino acids the bacteria can use to make new proteins
- YEAST EXTRACT -> this “autolysate” of yeast is basically just whatever happened to be in yeast (organic compounds including vitamins, trace elements, etc.) – and if it was good enough for the yeast…
- SODIUM CHLORIDE (NaCl)(table salt) -> allows for osmotic balance, transport, etc.
A few of the major LB formulations are the “Miller,” “Lennox,” & “Luria” versions & they differ in the amount of salt they have. Miller & Bertani drown the bacteria in NaCl (10g/L) whereas Lennox just uses 5g/L and Luria just 0.5g/L -> such low salt recipes are good if you’re using a salt-sensitive antibiotic
in the original paper, Bertani also added glucose, but most later recipes leave it out. Sometimes though, you want to give the cells glucose so they’ll use that for energy instead of the peptides, which you want them to use to make proteins. Similar to why endurance athletes eat those sugar gels on the trail so they can get energy without going into their protein & fat stocks, after we heat shock competent cells to open up pores in their membranes to let DNA in, we often give them SOC media afterwards, which has glucose, so they can use the other nutrients to heal themselves
SOC stands for Super Optimal broth with Catabolite repression and it’s SOB (Super Optimal Broth) with glucose added. SOB (aka Hanahan’s broth) has more peptone, amino acids, & peptides than LB. And it has magnesium sulfate to supply the magnesium ions DNA Pol needs to copy DNA.
So SOB & SOC are good for DNA-making. For protein-making (expression) you might want to try TB (Terrific Broth). Terrific Broth (TB) is more nutrient rich than LB, so sometimes it will give you better yields of protein or DNA in less time. It also has phosphate to act as a pH buffer to prevent cell death.
Another common type of media is 2X YT means media with 2X as much yeast extract as usual LB, which can be good for growing lots of cells for a long time.
All of these are types of “non synthetic” or “chemically undefined” media because they’re not made from precise, purified chemicals. Tryptone and yeast extract vary from batch to batch, so so will your LB
Problem is, if you want to go the pure synthetic route you need to make sure you give them everything they need (so hopefully they only need a few things!) Often, synthetic, “defined media” has Glucose or glycerol to provide carbon, and ammonium salts or nitrates for nitrogen. an example of a defined medium is Minimal salts (M9) medium: M9 medium contains essential salts and nitrogen (sodium phosphate, potassium phosphate, ammonium chloride, and sodium chloride). And you can add in other stuff the cells are auxotrophic for so they can grow.
But you don’t want everything to grow -> selection media contain things like antibiotics etc. that suppress the growth of things you don’t want to grow, “selecting for” the things you do want to grow. we design plasmids to have an antibiotic resistance gene (along with our gene) so we can spike the food with that antibiotic and it can still grow, but other stuff can’t http://bit.ly/2tcW4ky
There’s also differential media – this allows for “screening” as opposed to “selection” – you don’t keep things from growing, but you change how they appear – for example, we use X-gal for blue-white screening http://bit.ly/2MxNPs2
After I put a plasmid with my gene into bacteria and get colonies, I pick a few of those colonies and put them in liquid LB (with antibiotic) to grow overnight to make lots of copies of the plasmid, then I can purify out those copies and send them for sequencing to check for typos before l enter the “expression prep” part.
Regardless of what media you use, you need it to be sterile. So you autoclave it (stick it in a really hot, high pressure dishwasher) -> make sure the bottles aren’t sealed tight or they’ll explode (thankfully I haven’t made this mistake) – and don’t re-tighten the lids until the bottles have cooled of or the lids will get stuck (I *have* made this one). Another mistake not to make -> don’t add antibiotics before autoclaving, or you’ll inactivate them. We usually don’t add it until right before we’re ready to use it.
In undergrad, I made all my own media, but here, we use so much of it, we have a “media-maker” lab technician who’s amazing and makes & sterilizes our bacterial growth media