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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! blog form: http://bit.ly/transformheatshock 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.). Continued in comments