Instead of using them to treat infection, biochemists use antibiotics for bacterial selection! In molecular cloning we can stick a genetic recipe into a circular piece of DNA called a plasmid vector, stick that into host cells (often bacteria) & convince the bacteria to make more of our gene &/or its protein product (when it’s the protein we’re going for we call this recombinant protein expression). We want to kill off all other bacteria, so that we only feed and work with the cells that have our plasmid. And we can do this by putting an antibiotic resistance gene) into our plasmid alongside our gene. If we grow the bacteria in media (bacteria food) which contains the corresponding antibiotic, we can kill off any bacteria which don’t have the plasmid, so only bacteria with the plasmid (and theoretically thus our gene of interest) can survive. 

originally posted 6/23/22, refreshed, expanded, & added video 4/3/22

For example, my pET Hector the Vector has a gene for Kanamycin (KAN) resistance → if we grow it on bacteria food spiked with KAN, only cells that have our plasmid will survive – it selects for the right ones. A few common antibiotics used for selection are Ampicillin (Amp); Kanamycin (KAN); Tetramycin (Tet) & resistance genes for them can work in different ways including chemically modifying the antibiotic to render it harmless. Pharmaceutical chemists toil away for hours in the lab trying to make modifications to chemical compounds that alter their activity, but nature’s the real expert and the antibiotic resistance genes in these plasmids usually were initially found in other bacteria. 

There are many antibiotic resistance gene/antibiotic duos to choose from, which is bad from a health standpoint since there are so many ways bacteria can evade drugs, but it’s great from a cloning standpoint because sometimes we need to stick multiple plasmids into a single host cell & we need to make sure that the host cell has ALL of them. We can do this by using multiple antibiotics, each corresponding to one plasmid. 

A few pairs I commonly use (note: there isn’t “one” antibiotic resistance gene for each antibiotic – there are lots (which can be a problem in medicine!) & they can work in different ways, but with molecular cloning we get to pick which ones we want to use, and these these are a few we commonly choose!

But first a note on naming – antibiotics are usually naturally produced by other bacteria to protect themselves. Ones made from Streptomyces bacteria normally get names ending in “-mycin” (e.g. streptomycin, kanamycin) and those from Micromonospora bacteria get names ending in “-micin” (e.g. gentamicin).

Kanamycin (KAN) & kan resistance gene (kanR)

KAN usually kills bacteria by binding to their protein-making machinery (ribosomes) & “tinkering w/the knobs” so that dysfunctional proteins are made. Ribosomes are RNA/protein complexes that put together proteins based on mRNA instructions by linking together amino acids (protein building blocks) that tRNA molecules bring them. 

At 1 end, the tRNA has the amino acid to be added & at the other end it has an 3-nucleotide sequence called an anticodon that matches a codon in the mRNA instructions. The anticodon binds the codon & the ribosome helps transfer that amino acid to the growing chain. This anticodon/codon matching acts as a “quality control” proofreading mechanism to ensure that the right amino acids are added in the right order.

KAN interferes w/this quality control so that the wrong amino acids get added → dysfunctional proteins get made → cells die. 

The kan resistance gene (kanR) makes an an enzyme called aminoglycoside phosphotransferase, which adds a (negatively-charged) phosphate group (PO₄³⁻) to KAN. This modified KAN gets a “shock” when it tries to bind the negatively-charged RNA of the ribosome it usually binds to (that whole opposite charges repel dealio) 

KAN is classified as an amino glycoside because it has amino (nitrogen-y) & glyco (sugar-y -OH) parts. The amino parts can get ➕ charged which helps them bind the ➖ charged RNA. Other antibiotics in this class include: streptomycin, gentamicin, & neomycin

Ampicillin (AMP) & beta-lactamase (bla) 

Instead of messing w/protein synthesis (translation), AMP interferes w/cell wall synthesis. Bacteria build protective cell walls out of peptidoglycan (sugar chains cross-linked by short peptides). AMP inactivates the transpeptidase enzyme (reaction mediator/speed upper) that does this cross-linking → cells can’t make strong walls → they “pop” (lyse)

beta-lactamase inactivates AMP before it can inactivate transpeptidase. It does this by breaking open AMP’s beta-lactam ring (a strained, 4-sided ring containing an amide (C=O next to an N). This prevents AMP from binding transpeptidase so the wall is safe (I guess you could say bla gives you a wall of resistance…)

An important thing to know when using it in the lab is that, unlike kanR, B-lactamase gets secreted. The plasmid/host team goes on the “offensive” – it sends out offensive players to inactivate the antibiotic opponent before it can even get to them. But this also protects other nearby bacteria that don’t have the resistance gene so you can get “satellite colonies” which are little clusters of nearby bacteria that don’t actually have the plasmid – the longer you let the plates grow, the more likely they are to pop up – so don’t leave your Amp plates in the incubator too long! 

AMP is classified as a beta-lactam antibiotic because of that B-lactam ring. Others in this class include: penicillin, methicillin, oxacillin, amoxicillin, cephalosporins, monobactams, & carbapenems

Tetracycline (TET) & TetA

Like KAN, TET binds the ribosome & interferes with protein synthesis (translation). BUT it does so differently. KAN doesn’t stop proteins from being made, it just “distracts the quality control department” BUT TET *does* stop proteins from being made bc it prevents incoming tRNA (carrying next amino acid to be added) from binding → cells can’t make proteins → cells can’t grow

TetA makes an efflux pump, which drives out TET that enters the cells. This is a very different (yet effective) mechanism than kanR & B-lactamase, which both inactivate their antibiotic opponent by chemically modifying them

TET gets its name from its chemical structure – it has 4 (tetra) fused rings (cycline). It’s classified as a polyketide antibiotic – a KETONE is a —C-(C=O)-C— & TET has several (poly) of them. Others in this class include: chlortetracycline, and oxytetracycline (naturally occurring) as well as methacycline, minocycline, and doxycycline (semisynthetic)

Chloramphenicol (CAM or Cm) & CAT

Another example of a ribosome-targeting antibiotic we can use is chloramphenicol. I had originally had it in a separate post because it has additional features we can take advantage of – we can use it to increase plasmid copy number in certain strains. And you can learn about that here: 

But basically, certain plasmids (like those with the pMB1 or ColE1 ORI (such as those in the pUC, pGEM, & pBR families) don’t require new proteins to be made in order for the plasmid to be copied. So, when chloramphenicol stalls translation (which it does in a cool sequence-specific manner) it doesn’t mess with the copying. 

But that won’t work if your cell has a chloramphenicol resistance gene. Often this resistance gene is some type of chloramphenicol acetyltransferase (CAT), an enzyme that inactivates Cm by acetylating it (adding a CH3CO), which prevents it from binding the ribosome.

To summarize:

the opponents 

  • AMP targets cell wall synthesis
  • KAN, TET, & CAM target protein synthesis (translation)

the resistance teams 

  • B-lactamase, kanR, & cat chemically modify the opponent to neutralize the threat
  • TetA ships out the opponent

how the games play out – different teams use different strategies 

  • AMP-resistant team goes on the offensive, shipping out the AMP-inactivator B-lactamase
  • KAN & CAM-resistant teams *figuratively* “takes out” the enemy antibiotic by “tackling” it & inactivating it whereas the
  • TET-resistant team *literally* takes out their enemy antibiotic by shipping it out of the cell (note that tetracycline resistant teams are shipping out the antibiotic itself, whereas the AMP-resistant team ships out the antibiotic-inactivator!)

Other ways bacteria can be resistant to antibiotics include modifying the “targets” to protect them – instead of changing *antibiotic* so they can’t bind the target, change the *target* so the antibiotic can’t bind it! For example, mutations in the targets (like the sites on the ribosome where Kan or Tet bind) change the shape, charge etc. of the binding pocket so the antibiotic no longer “fits”

More on the sequence-specific chloramphenicol stalling I talk about in the video: 

Crowe-McAuliffe, C., Wilson, D.N. Putting the antibiotics chloramphenicol and linezolid into context. Nat Struct Mol Biol 29, 79–81 (2022).

Syroegin, E.A., Flemmich, L., Klepacki, D. et al. Structural basis for the context-specific action of the classic peptidyl transferase inhibitor chloramphenicol. Nat Struct Mol Biol 29, 152–161 (2022).

Tsai, K., Stojković, V., Lee, D.J. et al. Structural basis for context-specific inhibition of translation by oxazolidinone antibiotics. Nat Struct Mol Biol 29, 162–171 (2022).

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