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Prokaryotes

Prokaryotes

Cloning

In this section, we’re going to give a refresher on cloning and point out just how much of it depends upon bacteria. We’re going to make a little cloning list to point out all the ways this is true.

Cloning list
  1. Me… I’ve always wanted a twin…
      
  2. My dog… I don’t want to share her with my twin afterall…
      
  3. Goose that lays the golden egg…
Okay, wrong cloning list. Let’s try that again.

Cloning list
  1. PCR the gene you are interested in so you have a pure sample of lots of copies of it
      
  2. Get some plasmid to plop it into
      
  3. Cut (aka digest) both the PCR and plasmid DNA to give you sticky ends so you can…
      
  4. Glue (aka ligate) them together to make a circular plasmid
      
  5. Put the DNA into E. coli bacteria (aka transformation)
      
  6. Express the protein in E. coli
If any of this is unfamiliar, we suggest you go back to either the section on PCR or the section on expressing a "super ape" protein in bacteria.

Each of these steps depends upon tools developed by people studying bacteria.

Cloning List Item #1: PCR Depends Upon a Bacterial Polymerase

If bacteria didn’t live in extreme environments, we wouldn’t have PCR. PCR depends upon a thermostable DNA polymerase. In PCR cycles, the reaction mix is repeatedly raised to near-boiling temperatures in order to disassociate the DNA strands and enable a fresh cycle to begin. DNA polymerase from humans, or mesophilic bacteria for that matter, would be obliterated by this process. It is only because bacteria did the work for us of evolving a thermostable polymerase that we are able to do PCR at all.

Cloning List Item #2: Plasmids Come From Bacteria

Bacteria carry some of their DNA on plasmids, which are small bits of DNA. While the E. coli genome is 4,639 kb (kilobases) long, plasmids are typically less than about 15 kb long. Plasmids tend to contain DNA for surviving in specific environments, such as antibiotic resistance genes.

Cloning List Item #3: Restriction Enzymes Are a Primitive Bacterial Immune System

Restriction enzymes, which are essential for cloning, come from bacteria. Restriction enzymes are used in bacteria as primitive immune systems. Like us, bacteria are susceptible to viruses, except in bacteria these viruses are called bacteriophage. Bacteria produce restriction enzymes to cut foreign, viral DNA.

Bacteria are able to differentiate their own DNA from foreign DNA by adding a small chemical modification to their genomic DNA. This modification is called methylation. Methylated DNA isn’t cut by restriction enzymes. Methylation means the addition of a methyl group (-CH3) to something—in this case genomic DNA. Eukaryotes methylate DNA too, but they do this in order to control gene expression, not to protect it from restriction enzymes cutting.

Cloning List Item #4: Ligase

The DNA ligase used for cloning is actually from a bacteriophage, rather than a bacterium. While bacteriophages are viruses, they are bacteria viruses we know about from investigating bacteria, so that still counts.

Cloning wrap-up (or "Bacteria Are Important")

So in order to clone a gene, you use special bacterial DNA called plasmids, you PCR, digest, and ligate with enzymes from bacteria (or bacteriophage) and, finally, you replicate and express the final DNA construct in bacteria. In summary, a lot of the things we can do in biotechnology depend upon our past investigation of bacteria. The study of bacteria is important for understanding symbiotic interactions as well as pathogenic ones, and finally for developing the biotechnological tools of tomorrow.

Just for Fun

And because, why not, here’s a Weird Al song about being a clone.

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