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Unformatted text preview: 2.27.07 Bacterial Genetics • Transformation: transfer of DNA into a bacterium from its environment o Movement from the donor to the recipient o Way it happens in nature allows the recipient to pick up a fragment, a piece of DNA that is actually not heritable o Looking at the diagram you can see the combination that will allow that fragment to become a part of the recipients chromosome, now it will be a part of the circular molecule with an origin of replication, and this DNA that has been taken up will be heritable o Foreign DNA has to have homology with some portion of the recipients DNA o RecA a protein is bound (in multiple copies) to the foreign DNA and allows for a swap if the sequence is homologous, and the foreign DNA displaces a strand of the recipient DNA because of the base pairing between foreign and recipient, then an endonucleases cuts at either end of the displaced recipients DNA and it is degraded o If the foreign DNA was merely homologous and not identical to the strand it replaced there will be mismatches along the length here and its okay because it won’t stay this way, it won’t be long before replication and each one will serve as a template and one of the daughter molecules will have the new DNA that came in from the donor in combination with other DNA (alleles at other genes) that it already had One daughter cell will get the new recombinant DNA and the other daughter cell will get the other copied strand that was just as the recipients original DNA was The two daughter cells will actually be genetically different and adds to the variation of bacteria that is present o Recombination is not efficient, many times DNA will be degraded when it enters and there has to be a hole in the recipient’s cell wall so that is rare as well, but Griffith saw it happen and rare things happen often in bacteria o Transformation is a technique used often in the lab but you don’t like its inefficiency so you optimize each part of it to greatly increase efficiency overall – this refers to E. coli because CaCl2 is used specifically for E. coli The foreign DNA introduced into the cell isn’t a fragment but instead it is integrated into a plasmid Plasmid makes the foreign fragment circular so it has an origin of replication The cells cannot be transformed without damage in the cell wall, these cells are called competent (if they have damage in their cell wall they are competent to be transformed) Competent cells are rare in nature, but in lab we make them competent ahead of time (must make them competent by growing them in Calcium chloride ahead of time) Now the plasmid DNA mixed with the competent cells, you see that the plasmid slips inside to the cell, but there is another barrier, the plasma membrane, when you mix the two together in the tube in aqueous solution you warm the tube a few degrees in order to open up pores in the plasma membrane Plasmid slips inside, this is temporary, take it off the heat, the pores close right back up...
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