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Overcoming difficulties

After this, Berg’s laboratory came to Janet Mertz, a new graduate student. Mertz is a rare genius who, like Lobban, studied undergraduate at MIT and earned degrees in engineering and biology. Mertz chose to join Berg Laboratories because she was very curious about Jackson’s recombinant DNA experiment.

Left: Paul Berg Right: Janet Mertz

However, Mertz has been thinking about a problem. What would be the result of inverting Jackson’s experimental purpose? Jackson’s experimental procedure is to insert the genetic material of E. coli into the genome of SV40. If the SV40 gene fragment is inserted into the genome of E. coli?

It turns out that inserting the viral gene into the genome of E. coli produces a huge technological advantage. E. coli carries a small DNA called a plasmid, which is identical to the SV40 genome. The structure of the plasmid is also circular and can replicate within bacteria. Mertz realized that if the SV40 gene was inserted into a plasmid in E. coli, she would have the potential to transform E. coli into a plant for the replication of foreign genes. As E. coli grows, the E. coli intracellular plasmid will also expand, resulting in a large number of foreign DNA clones.

In June 1972, Mertz left Stanford to attend a training course in animal cells and viruses in Cold Spring Harbor, New York. This course requires students to make simple reports on their future research plans. In the report, Mertz explained her research plan: constructing hybrids of SV40 and E. coli DNA and enabling them to expand with the proliferation of E. coli. Graduate students’ research topics generally do not attract the interest of experts, but when Mertz concluded the report, everyone began to realize that this was not a simple academic report. It was a long silence, followed by questions from the tutor and the students. The tide floods – Have you ever thought about the risk of building DNA hybrids? Will this cause harm to humans? Have you considered the ethical issues brought about by this technology?

After listening to Mertz’s report, virologist Robert Pollack immediately called Berg to discuss the technical risks of recombining DNA. In fact, Berg and Mertz’s experiments do have a very important risk factor: SV40 can induce hamster cell mutations to form tumors. Although there is still no credible evidence that SV40 may induce cancer in human cells, in the 1970s, scientists were not aware of the risk of the virus. This is indeed a question that deserves careful consideration. The author has described this risk in detail in the previous article on gene therapy. Because certain types of viral genome insertion sites are more specific, it activates proto-oncogenes to induce leukemia.

In addition, since E. coli can survive in human intestine, if Mertz and Berg really succeed in constructing E. coli hybrid DNA carrying the SV40 exogenous gene, if SV40 is really capable of inducing humans as scientists fear If the cell becomes cancerous, does this E. coli carrying the recombinant DNA actually induce cancer in human cells, causing a disaster?

In the next few weeks, Berg began to rethink the risks of the technology. But he still feels Pollack’s fears are superfluous. This experiment was conducted only in a relatively closed laboratory. There was no evidence at the time that SV40 could induce cancer in human cells. In fact, researchers are often infected with the SV40 virus during the laboratory work, but there have been no reported cases of cancer caused by the virus. In response to outside doubts, Berg even proposed to drink the SV40 virus he had cultivated to prove that the virus does not have a cancer risk.

Although Berg believes that there is no problem with the security of the SV40, he also has to be careful with the outside world. He consulted with authoritative oncologists and microbiologists to assess their risks. Dulbecco is also confident in SV40, but can scientists really accurately predict the unknown risks of a technology? Berg is very clear that for any scientist, it is quite common for the misjudgment in the scientific research process and the prediction of the experimental results to fail. However, this time, unlike previous scientific research, if his prediction of the danger of SV40 is wrong, then the catastrophic consequences of the technology must be something he cannot bear.

Since it was impossible to completely determine the risk, Berg decided to take a compromise approach and only operate on free DNA without introducing hybrid DNA into living cells to avoid the risk of inducing cell cancer. At this time, Mertz gave Berg a big surprise. Her experiment ushered in a major breakthrough. Prior to Berg and Jackson’s cutting and connecting DNA requires 6 steps, extremely cumbersome. But Mertz found a shortcut – using EcoRI can reduce the steps for cutting and joining DNA in two steps, making the experiment extremely efficient. The enzyme was provided by Herb Boyer, a microbiologist at the University of California, San Francisco (UCSF).

Boyer was born in Pennsylvania in 1936. He has a special preference for biology since childhood, and Watson and Crick are also his idols. Boyer joined UCSF as an assistant professor in 1966 and has since been focusing on the purification of DNA cleavage enzymes.

 

In November 1972, Berg was still struggling to weigh the risk of viral-bacterial heterozygosity. Boyer had already travelled to Hawaii to attend the microbiology conference that year. After finishing the long morning meeting report, Boyer ran to the beach in Hawaii to enjoy a sweet afternoon. Also in the evening, Boyer met Stanley Cohen of Stanford University.

Cohen is an expert in the field of plasmids. Prior to this, Boyer knew this person and read Cohen’s article, but he never met him. After dinner, Boyer, Cohen, and another microbiologist, Stan Falkow, strolled along Waikiki Beach in Hawaii to discuss plasmids and chimeric DNA. Boyer and Cohen both know about Berg’s recombinant DNA research. Cohen also knows that Mertz, a postgraduate at Berg’s lab, is working at Stanford University’s microbiology lab to learn the techniques of transferring chimeric DNA to E. coli cells.

Their discussion turned to Cohen’s work. Cohen had successfully isolated some E. coli plasmids, and he had been able to purify some of these types of plasmids very efficiently, making it easy to transfer plasmids between E. coli strains, and he Some of these plasmids were found to carry resistance genes for certain antibiotics (such as tetracycline and penicillin).

What happens if the antibiotic resistance gene is cut from the plasmid and transferred to another plasmid? Is it possible to make these E. coli strains carrying resistance gene plasmids resistant to antibiotics so that they can survive in an antibiotic environment and E. coli without the plasmid is killed by antibiotics?

This flash of light is like a neon light in the night, illuminating the beach in the dark night. The other two people also realized the value of this idea, because in the Berg and Jackson experiments did not find a suitable way to identify which bacteria or viruses obtained exogenous DNA. Cohen’s antibiotic-resistant plasmids are able to efficiently identify recombinant DNA, because only E. coli, which acquires an exogenous DNA (antibiotic resistance gene), can survive in an environment with antibiotics. The recombinant DNA was successfully constructed.

Cohen worried that Berg and Jackson’s experiment had another serious problem – the efficiency of constructing chimeric DNA was too low. If the success rate of constructing heterozygous DNA is only one in a million, it is difficult to screen successfully constructed recombinant DNA even if there is a better screening system at a later stage. However, Boyer’s EcoRI solves this problem very well. Mertz has successfully optimized the construction of DNA hybrids.

After a long silence, their thoughts did not stop. Boyer has been able to efficiently purify the restriction enzyme used to construct hybrid DNA, and Cohen has been able to isolate the appropriate plasmid. The experiment of constructing recombinant DNA seems to have been very simple. It seems that an experiment can be completed in one afternoon. After the re-ligation of plasmid DNA cut by EcoRI, a part of the plasmid may be able to form a recombinant plasmid DNA molecule, and then used. Antibiotic resistance can screen for E. coli that has successfully acquired the foreign gene. With the proliferation of E. coli, the exogenous gene will also be amplified and the recombinant DNA will be continuously cloned. Cohen whispered: That is to say .. . . . . Before he finished talking to Boyer, he said: Yes, this should be feasible.

In fact, Cohen and Boyer’s experiment has another very important advantage: the biohazard of the experiment is less controversial. Unlike Berg-Mertz’s virus-bacteria hybrid experiments, Cohen and Boyer’s hybrid DNA contained only bacterial genes, so Cohen and Boyer believe that their theoretical biological harm is at least theoretically smaller.

Left: Stanley Cohen; Right: Herbert Boyer

In the winter of 1972 and the spring of 1973, Boyer and Cohen had been busy carrying out experiments to construct DNA hybrids. Although Berg and Mertz at this time already knew that Cohen from the same school was conducting research on recombinant DNA, they still did not conduct experiments to introduce chimeric DNA into living cells.

In February 1973, Boyer and Cohen had begun to introduce chimeric DNA into cells. They used restriction endonucleases to cleave both bacterial plasmids and transfer specific genetic information from one plasmid to another, after which the DNA fragments were fully ligated using a ligase to form a chimera. The chimera cultures bacteria after they are introduced into the bacterial cells. After the bacteria are cultured overnight, the exogenous genes in the cells are continuously replicated as the bacteria multiply. Their experiment was successful and a new technology that could change the world was born.

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