[MUSIC] We're going to start this course with a brief history of recombinant DNA technology, a predecessor technology to synthetic biology. And I think it's important to sort of place synthetic biology in the broader scientific and historical context of the science. And that history isn't actually that old or that long. So it was only in 1944, that Avery, MacLeod and McCarthy demonstrated that DNA was the transforming principle. When they transformed one strain of pneumonia into another, it was more virulent. Following that experiment, though, there were still a few questions about whether DNA was it. But when Alfred Hershey and Martha Chase did their experiments in phage, The Hershey & Chase experiments, combined with the Avery and MacLeod and McCarthy experiments, really sort of locked it, that DNA was in fact the molecule of heredity. And then the next year, 1953, of course, Watson, Crick, and Rosalind Franklin determined the double helical structure of DNA. So even though it seems like we've known this information for a very, very, very long time, we haven't. In fact, its only been since the 1950s that we've really even understood that DNA was the molecule of heredity. In 1960, Simian virus 40 was isolated, I'll come back to that later, that'll become important in a little bit. Learned about regulatory control in '61. And then in 1964, the Epstein-Barr virus, which is a human virus, was identified as the cause of Burkitt's Lymphoma. This was the first time that we realized that there was a virus that could cause cancer in humans. Now we had known for a while that there were viruses that could cause cancer in non-human animals, but we didn't know that there was a virus that could cause cancer in humans. And keep in mind that at this time, cancer was a death sentence. We did not have good treatments or cures, so cancer was even more terrifying a diagnosis than it is today. And now there was a virus that could cause it. And then, in 1972 also important for our story, EcoR1 was isolated from E coli. I also want you to sort of keep in mind that at this time, there was, at NIH, the memorial labs. Which was, in fact, a lab dedicated to people who had died in the course of their science. Science was not activity without risk. This was not a time, as today, with many, sort of safety, personal safety equipment etc. There were no pipetman, you mouth-pipetted. That is, you used your mouth as the source of suction to move liquids from one space to another, for example, and that would include viruses. So going back to SV40, which I mentioned in the timeline, this was first isolated from monkeys in 1960. And it was cultured in monkey kidney cells. And we knew that virus-infected cells caused tumors in hamsters. So we had a cancer causing virus. Monkey kidney cells had also been used to cultivate poliovirus for the early vaccine. And it turns out that there's a pretty high rate of infection of monkey kidney cells with SV40. So we realized that some lots of the polio vaccine were in fact contaminated with SV40. Again, a virus that we knew caused cancer, not in humans, in hamsters. And this led to the exposure of almost 100 million recipients of the vaccine. So just think about what the atmosphere must have been like. This is also at a time when in the 1960s and early 1970s, where there were many instances in the press coming to light of scientists behaving badly. So this was at the same time of revelations about the experiments at the Jewish Chronic Disease Hospital in Brooklyn, where Dr. Salvin was injecting live cancer cells into elderly patients to see if they would be able to fight off cancer cells. There was an experiment at the Willowbrook State School in Staten Island, New York, where children with mental disabilities were given hepatitis in an attempt to track the development of the viral infection. And both of these were without consent of the individuals. And then of course in 1972, the Tuskegee syphilis study came to light, wherein poor sharecroppers from Tuskegee, Alabama were kept from getting treatment for syphilis. There were men who had syphilis, but the US government scientists kept them from getting treatments once treatments were available. And this experiment went on for decades. So the social situation at this time, not only was there a lot of social upheaval due to just sort of general, broader changes in society around race and foreign policy. This was also a time of distrust of scientists and worries about, for example, viral cancers. So moving to the beginning of their common DNA experiments, Paul Berg and his grad student, Janet Mertz Were interested in doing some experiments in bacteria, wherein they were trying to get a bacterial gene into the SV40 genome. Janet Mertz was then also interested in trying to replicate the SV40 genome in bacteria. Now, Mertz attended a workshop at Cold Spring Harbor in 1971. And she discussed at this meeting the proposed experiments. Following that meeting, Paul Berg got a call from one of the scientists who had been there, Robert Pollack, who basically told him don't use E coli as the bacteria. Because this is a bug that grows in the human gut, and it's just too dangerous. We don't know the cancer risk, given what was known about SP40 at the time. And Paul also believed that no one should be allowed to do an experiment like that sort of in secret, only to release to the world what they had done at a press conference. At which point, the whole community has to deal, and the society have to deal, with the consequences of those experiments. So following that call, Berg asked more members of the scientific community, and indeed found ambivalence among his colleagues, and he paused these experiments. So these are some of the early papers, wherein the group had successfully constructed Constructed the, DNA the way they wanted it to be. But they had not yet transfected that recombinant DNA into E coli. For folks who are not as familiar with the science as others, the basic idea, of course, with recombinant DNA is like cut and paste in your favorite text editing program. So you cut DNA at known palindromic sites, and then you use ligase to glue the DNA back together in new combinations. And you can introduce this recombinant DNA back into an organism, which will then replicate it for you. This was a huge advance over our prior techniques, such as doing breeding or random mutation and selection of those organisms that had the trait that you were interested in. This was the first that we could go in and specifically modify an organism in a way that we were interested in modifying it. So again, you have these palindromic sites. So the DNA, the letters read the same forward as they read backwards. The restriction enzyme cuts, leaving you often with these overhangs, also called sticky ends. And then you can paste a new piece of DNA into that site that has complementary sticky ends. That means you cut that other piece of DNA with the same restriction enzyme, giving it the same sticky ends. So then they will glue back together nicely. You can then transfect that into your organism of choice, and grow up colonies, to isolate that DNA. Restriction enzymes are site-specific cutters, as I mentioned before. At palindromic sites, they cut the same place every time. The same sequence, rather, every time. Usually four to six nucleotides, most are palindromes. Again, read the same way forwards as they read backwards. And the frequency of the site, so how many times a restriction enzyme cuts a piece of DNA depends on the frequency of the restriction site. And also related to that, the length of the recognition sequence. We have hundreds of these restriction enzymes that we've isolated from microorganisms. And again, they generate complementary sticky ends. You can use lots of different vectors. That is, certain pieces of DNA that you can use as the mechanism for getting the piece of DNA you want into the organism you want. Everything from plasmids, small circular pieces of DNA, up through bacteriophage, cosmid, all the way up to, for example, yeast artificial chromosomes. Which can accommodate much larger pieces of DNA. And these are also, the vector you use is going to be specific to the host in which you want to grow up your DNA or transfect your DNA into. Vectors, of course, need an insertion site. They need one insertion site. You need to be able to cut the vector and insert the piece of DNA you're interested in. They need a replication origin, so that they can make more of the DNA that you're interested in. Selectable markers, so you know which organism got the piece of DNA you're interested in. And vectors can come in single copy or high-copy sort of flavors. They can be used to isolate a particular gene or region of interest. To produce large quantities of protein or RNA. To improve the efficiency of biomedical production. Create recombinant organisms, GMOs. And potentially, in theory, to correct human genetic defects. And we are certainly getting there with genome editing and CRISPR-Cas-9 in the sort of more recent advances in this area of science. So at this point, the scientists, Paul Berg and Janet Mertz and others, had gotten to the point where they saw the possibilities of this science, right? They saw that they could create recombinant DNA. They saw the possibility of transfecting that into, for example, E coli, but they couldn't really judge the risks yet. They hadn't taken the next step of transfection, and there were many, many questions about potential risks, although the potential benefits were very exciting.
