Wedding dress selection during antibiotic selection! You might be a nerd if, during your sister’s Zoom wedding dress shopping, you get distracted by panicked texts from colleagues asking you which antibiotics they were supposed to have used. And, your cousin might be a nerd if she then asks you which antibiotics were mixed up. In her defense (not that you need to defend neediness! It should be a thing of pride!), she’s a doctor. But she’s used to using antibiotics to treat patients’ infections – and antibiotic-resistant bacteria are her foes. For biochemists, we’re used to using antibiotics for bacterial selection, relying on antibiotic resistant bacteria – but bacteria that are very specifically resistant to a single antibiotic.
That resistance is conferred to them by a gene that’s on the same circular piece of DNA (plasmid) that contains a gene we want the bacteria to have. If we grow the bacteria in media (that bacteria food we were talking about yesterday) 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. But if you spike the food with the wrong antibiotic, your bacteria will get killed off too… As a labmate learned the hard way yesterday…
On your marks, get TET, go! In the game of plasmid-containing cells versus antibiotics, different bacterial teams take different strategies.AMPICILLIN-resistant teams go on the offensive. KANAMYCIN- & TETRACYCLINE-resistant teams play defense, but with different strategies. Yep, girls (including scientists) can use sports metaphors too & when we’re playing this game of ANTIBIOTIC-BASED PLASMID SELECTION, we need to make sure we know what players are on our team so we can rig the tournament so our team always wins! Let’s take a look at the players.
A lot of times, molecules are so useful that they become “tools” and scientists don’t stop to think about how they actually work (no judging here – I know we’re all busy and we can’t know everything! kinda like that having to walk to school uphill in snow both ways cliche – or using a calculator – it’s not that “scientists these day” are “lazy” it’s just that instead of figuring out how to use antibiotics and antibiotic resistance genes to make sure that only the bacteria we want grow as our life’s work we build upon someone else’s life’s work and use it to spend our time tracking down other questions – like how does the protein that’s in the plasmid with the antibiotic resistance gene that we’ve stuck in the bacteria to make it for us actually work.
But sometimes it’s important to know how the molecules are actually working so you know what’s compatible with what – what might mix up your results, what molecules are doing the same or different things, etc. – and sometimes knowing is just cool! So today I want to take you “behind the scenes” of the molecular dramas being played out during antibiotic selection
In MOLECULAR CLONING we stick a gene 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 need to put together a plasmid/host cell “team” to carry out this DNA/protein-making work. We “assemble the team” in a process called transformation, where to stick the plasmid into host cells – often through “heat shock” – you take chemically-weakened bacteria, drown them in DNA you want to put in, stick them briefly in warm water to open up holes in the bacterial membranes & let the DNA rush in, then stick them back on ice & let them recover. More here: http://bit.ly/2Jj7L47
But not all the cells take in the plasmid & other bacteria can sneak in, so we need to SELECT the “right” teams – those w/host cell AND plasmid. The host cell provides needed machinery (& genetic instructions for making that machinery) & the plasmid contains the instructions for what we want it to make, so we need BOTH
It might seem like the host cell part of the team is providing all the molecular “players” but not so! The plasmid provides ANTIBIOTIC RESISTANCE GENES whose protein products protect the cell from ANTIBIOTICS so that the host cell can do all that work. Different plasmid vectors have different antibiotic resistance genes & we use knowledge of which one(s) our plasmid has to design the tournament so that our team always wins! We choose an opposing antibiotic team that only the right team can beat. All the other bacterial teams will get knocked out.
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’is 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 setting up the tournament w/multiple antibiotic opponents. In the initial cloning stage we usually only need 1 antibiotic, but later, when we’re making bacmids (which will let us make an insect-specific-virus we can use to infect insect cells so they’ll make our protein) we’ll need multiple
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) http://bit.ly/2tcW4ky
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 AMINOGLYCOSIDE 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) http://bit.ly/2WSXfmL
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! more here: http://bit.ly/2tcIAVT
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)
- AMP targets cell wall synthesis
- AN & TET target protein synthesis (translation)
the resistance teams
- B-lactamase & kanR 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-resistant team *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 codons & going from mRNA to protein (TRANSLATION): http://bit.ly/2TBFLsU