Bacterial transformation - chemically competent cells & heat shock

[@thebumblingbiochemist]

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: bit.ly/antibioticselectionscience 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.” more on cloning checking: bit.ly/colonychecking 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 more on molecular cloning: bit.ly/molecularcloningguide more on other transfection methods: bit.ly/transfectionmethods more on minipreps: bit.ly/minipreps thermo fisher has some nice educational resources: www.thermofisher.com/us/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/molecular-cloning/transformation/bacterial-transformation-workflow.html www.thermofisher.com/us/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/molecular-cloning/transformation/competent-cell-considerations.html    more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com

[@luukdanen209]

hi, I am doing research into the heat shock transformation or E.coli. I was wondering if you might have more literature about increasing the zone of adhesion during heat shock?

[@thebumblingbiochemist]

I don't sorry, but good luck!

[@linda-ll4fj]

Love you guys

[@thebumblingbiochemist]

Just me - but thanks!