What are biochemists’ favorite action figures? 🤔 Transformers! 🤓 Come as a *shock*? Hopefully I’m *competent* enough to describe how in PLASMID TRANSFORMATION we can use chemically-competent cells and heat shock to get DNA into bacterial cells. 🤞 I achieve good *efficiency* in getting this information into your brains!
Most of the time lately, instead of being at this bench, I’ve been at the bench in the hot room, doing experiments involving radiolabeled things. But this bench has still been busy – in the mornings it’s a mess as I set up all the non-radioactive parts of my experiments – but it still gets visited on & off lots throughout the day because of what’s at the end of the bay! The water bath! And our lab needs it for transformations – so I often don’t know if people are coming my way for me or the bath!
In MOLECULAR CLONING we take a gene from one place and stick it into a VECTOR which serves as a “vehicle” to take that gene into cells. Often, we use PLASMID VECTORS, which are circular pieces of DNA that we put into bacteria.
The plasmid vectors are the vehicles but they need tunnels to get into the cells – but we don’t want those tunnels to be huge and always be wide open, or the cells’ contents could spill out, random stuff could go in, etc. So we need a way to control their size and/or open/closedness.
There are 2 main routes for artificial transformation – chemical competence + heat-shock & electroporation
With chemically competent cells, you use calcium to get the DNA right by the tunnels (adhesion zones) & heat shock to “open up an additional lane” (widen the pores), speed up the cars, and make the inside of the cell more attractive (less negatively-charged). With electroporation, you use electric current to “bore” temporary tunnels. We usually use chemical transformation, so that’s what I’m going to focus on in this post
Disclaimer: It’s not entirely clear how it happens mechanistically, but here are some leading theories
Regardless of the method, the tunnels have to pass through the cell membrane(s), which are like sandwiches made up of 2 layers of amphiphilic molecules – such molecules have one part that’s hydrophilic (water-loving) and another part that’s hydrophobic (water-avoiding)
The cells we normally use are different strains of e. coli, which as “Gram-negative” bacteria, have 2 of these double-layers to get through – there’s an outer membrane, then a “periplasmic space,” then an “inner membrane”, then, finally, you’re in the cell’s interior, the cytoplasm. At tunnels called zones of adhesion, these 2 membranes are fused.
All the membranes have PHOSPHOLIPIDS, which have a hydrophilic head with a negatively-charged phosphate group and a hydrophobic (water-avoiding) tail with lipid (fatty) chains. They orient tail to tail to form a barrier surrounding the cell. The outer membrane’s outer membrane also has LIPOPOLYSACCHARIDES (LPS’s) which, additionally have sugar chains (polysaccharides) sticking off them which can have even more negative charges.
DNA also has a negative charge because of the phosphate groups in its backbone. This helps it stay water-soluble, but it also makes it normally repulsive to the outside of the membrane (like charges repel). So we need a positively-charged (cationic) mediator.
Calcium chloride (CaCl2) provides a source of divalent (charge of 2) cations (positively-charged molecules) CaCl2 is the salt form we buy it in, but when we dissolve it in water, it fully ionizes, meaning that the Ca2+ shakes off those Cl- anions (negatively-charged ions)
The Ca2+ help hide the DNA & membranes’ negative charges so they don’t repel each other and, even better, Ca2+ can bind more than one thing at once, so it can link the DNA to the cell surface so that it’s right in place to rush in when the tunnels open further during the heat shock step.
When we heat the cells, the pores shed some of their lipids, so the tunnels get bigger, and the membrane “depolarizes” meaning that the inside of the cell becomes less negatively-charged & therefore more attractive to the negatively-charged DNA. Normally the cells maintain a membrane potential by pumping out protons (H+) so that the interior of the cell is more negative which wouldn’t be very attractive to negatively-charged DNA.
The heat also creates a thermal imbalance – the DNA outside isn’t “insulated” so it heats up faster – starts moving more -> cars speed faster through tunnel
Like I said, there’s still a lot of uncertainty about how it works, but I tried my best to find out how it works. And work, it (usually) does!
So let’s look at we do it in practice. And we do it a lot, so we get a lot of practice! (And this is the protocol I usually use, but the exact times, etc. depend on the types of cells you’re using, volumes, tube types, etc.)
Competent cells are competent to take up DNA but not very competent to survive… they’re really “delicate” so you have to treat them with TLC
To avoid unnecessary freeze-thawing, you usually make little “single-use” aliquots of them – each tube has the perfect amount for 1 transformation.
You will have to thaw them at least once, and when you do so you want to do so “gently” – on ice. The cold temperatures also help keep the calcium stuck to the membrane where we want them (the molecules don’t have the energy to go looking for better alternatives)
Once they’re thawed, you can add the plasmid and stir gently or gently tap (don’t pipet up and down as that’s too harsh)
Then it’s off to a bath for the heat shock. We stick the tubes in a 42°C hot water bath for 40s. My bench is one of the most popular places in the lab because it has the water bath. We do a lot of transformations, so at any given time there’s often someone standing there, looking up at the clock and waiting for those 40 seconds to pass by.
Then it’s right back to the ice. That heat shock was pretty traumatic for the bacteria, so you need to help them recover. You give them some liquid food – this can be LB (basic bacteria food) or a “richer” food like SOC (this has glucose so that the cells burn glucose for energy and leave those amino acids & lipids alone so they can go towards protein-making & wall-fixing)
Importantly, this food does NOT have antibiotics. The plasmid you put in has an antibiotic resistance gene so that you can select for it -> not all the cells will have taken in the plasmid, but only the cells that have will be able to grow in the presence of the antibiotic. So we’ll use that antibiotic when we grow them on plates, but first we have to give the cells time to make the thing that makes them resistant.
Additionally, since they don’t have to spend energy fighting for their lives from antibiotics, they can spend energy fighting for their lives from all those holes in their membranes! They can use this recovery time to help repair their membranes
So, in the OUTGROWTH step we add antibiotic-free media and let them grow for ~30min-1hr. The time they need depends on the antibiotic you plan to use. Antibiotics like kanamycin and chloramphenicol inhibit translation (protein-making) so the cells need a longer outgrowth or else they won’t be able to make the proteins they need to inactivate the antibiotics. Antibiotics like ampicillin that don’t stop protein-making so don’t need as long to recover. More here: http://bit.ly/2IxPOM8
During this time, even the untransformed cells can grow, but they’ll die off once we plate them. Once they’ve repaired their membranes & started building up their resistance, we take them from suspension growth (liquid-based) to plate growth – we spread the liquid (full of bacteria) over an LB-agar plate containing that antibiotic – the agar is a sugar that forms a gel filled with LB food spiked with antibiotic
Now, since the LB food is spiked with antibiotic, only the transformed cells should be able to grow. And when they do, they do so by copying all their DNA (including the plasmid you put in) then dividing. So you end up with lots of cells from each original transformed cell, and these “families” will appear as globby “dots” called colonies.
The better the “transformation efficiency” the more colonies you’ll see. This efficiency depends on a lot of things like how competent the cells where, how big the plasmid was, whether the plasmid “still needed help” etc.
Sometimes, the plasmids you put in are “pre-made” and have already grown in bacteria (then been taken out & purified) but other times, they’re things you’ve just pasted together (ligation products) or even plasmids with gaps the bacteria needs to fix through homologous recombination (like SLIC products). These are “harder” because you have to have the engineering go right AND the transformation go right. So you should expect lower transformation efficiency.
You can isolate the plasmid DNA from these colonies to use and, especially if you’ve just cloned it, send it for sequencing to check for “typos.”
Note: In addition to this “artificial competence”, some bacteria have “natural competence” – In nature, some bacteria can take up *linear* pieces of foreign DNA (such as that released by dead, exploded bacteria) through a form of transformation that relies on DNA receptors