Interviewer: Lydia Morrison, Marketing Communications Writer & Podcast Host, New England Biolabs, Inc.
Interviewee: Bill Jack, Senior Scientist at New England Biolabs.
Topics include: Molecular Biology, Molecular Cloning, DNA Cloning, Gene Cloning, Synthetic Biology, Polymerase Chain Reaction, Gene Therapy
Hi, and welcome to the Lessons From Lab & Life podcast. I'm your host, Lydia Morrison, and I hope that our podcast offers you some new perspective. Our podcast today focuses on molecular cloning. I'm joined by Senior Scientist Bill Jack from the research department at NEB. Today we'll discuss where the concept of cloning began, how it works, and where it's headed in the future.
Hi, Bill. Thanks so much for joining me today.
Happy to be here. Thank you, Lydia.
So we're here to discuss molecular cloning, and I think when people hear the word "cloning," they think of a lot of different things: sheep, sci-fi movies, and who knows what else. Could you tell our listeners, what is the definition of molecular cloning?
We really have stolen that definition from the botanists, which is ... if you go to the dictionary, a clone is an exact copy of something, similar to taking a piece of paper, walking over to the copy machine, pushing the button, and having a copy come out. In plants, you can think of it very easily. I'm sure all of you have gone out and taken a plant and split it and put it in two different locations. That's really exactly identical information there in those two plants that's been put in those two different places. We've taken that to mean cloning on a molecular level, meaning to make copies of specific pieces of DNA, and that's really where the molecular biology of cloning comes in. Maybe we can talk in a little bit about some of those other sheep and science fiction and Star Wars drones and other kinds of cloning, but for a basic definition it really is just a molecular copy of pieces of DNA.
Who invented molecular cloning?
There are several advances that came in to allow molecular cloning to occur, but it's basically Stanley Cohen and Herb Boyer that are credited as being the first molecular cloners in making recombinant DNA. This was really preceded by several advances. Stanley Cohen's lab had been instrumental in showing that you could take small, autonomously replicating DNA circles in bacteria and easily transform them or put them into other bacteria. They were also able to identify selected markers on those plasmids, such as antibiotic resistance, which allowed not only the transformation but also the strong selection for any bacteria that had obtained that plasmid. Later on, the advance of the introduction of a few key enzymes: first of all, DNA ligase, which glues pieces of DNA together; and the second, restriction endonucleases, which cut DNA in specific spots and leave sticky overhangs that allow ligation to occur. Those two enzymes really revolutionized the ability to be able to take a foreign piece of DNA, stick it into these plasmids, these autonomously replicating circles, and put those back into bacteria and have not only that plasmid replicate but also that inserted piece of DNA.
So that's really the underlying scheme of what constitutes molecular cloning, namely just making more copies of a specific piece of DNA.
What is the purpose of molecular cloning?
Molecular Cloning has allowed us to be able to take very small and selected regions of DNA and make multiple copies of that. With the multiple copies, it's made it much easier for us analyze, for example, the DNA sequence, the content of those, the genes that are contained within those pieces of DNA. It also has allowed us to move our technologies into making the gene products from those particular pieces of DNA. All those kinds of analysis really have been enabled just because we've been able to make multiple copies that makes it obviously a lot easier to be able to analyze those DNAs.
Why does molecular cloning require a host?
In the simplest form, we could probably do many of the things in a test tube. We can amplify DNA very well today with a polymerase chain reaction, PCR. However, bacteria have already been doing this for millennia, and so they're very good at it. They're very efficient at making the DNA. The bacteria are easy to manipulate, easy to extract high quantities and quality of DNA from the bacteria. In fact, the fidelity of DNA made in bacterial hosts is much higher than we can ever do in the test tube.
So, for these reasons, it's a matter of convenience for us to use bacteria to do that. That doesn't mean that there won't be specialized situations in which we don't want to make the DNA outside in a test tube, but really the bacteria are such a good manufacturing machine that it seems infeasible for us to use any other method on a large scale.
Where are we with cloning today?
The roots of cloning go back into the seventies, and so we have a good forty-some-odd year history of doing cloning, and we've certainly grown in appreciation of the things that we can do with cloning, the techniques, and the speed with which it's done. I've been in the field for a little over 35 years, and it's remarkable to me to see the speed at which things can be done now, in terms of making clones and analyzing the clones and producing the clones.
How is molecular cloning used in synthetic biology?
Synthetic biology, in my own definition, would be putting together multiple genes and pathways to be able to study how different proteins interact. Much of molecular biology and biochemistry has been necessarily based on study of individual enzymes: purifying the individual enzymes, looking at their properties, and trying to analyze them in terms of what their structure might be and how they might perform their individual activity. However, the field increasingly has been turning to looking at multiple complexes of proteins together and how they act. The ribosome or the DNA replication complex would be two examples of that. Certainly those have been studied through the years, and yet once we have a number of proteins together, the analysis has been much more difficult to appreciate what the individual components do in order to make that entire complex work.
I think our visualization processes today, in terms of advances in X-ray crystallography, cryo-EM has made great advances at being able to look at large complexes, and now the ability to make multiple proteins at the same time and have them assemble in complexes gives us an ability to be able to look at that in even more detail. So I believe that one of the uses of synthetic biology will be a turn toward broadening our scope, being able to take individual enzymes and look at them in complexes. My hope is that a lot of that can be done actually in vivo, within cells, so that an entire metabolic pathway can be constructed inside a cell. We can let all those components be expressed and come together and perform their action, and that we will develop better techniques for analyzing the complexes as they operate in vivo. I think that's one element of synthetic biology that I'm excited about that I still think has a lot of development to do.
The second one is using the steps of synthetic biology to make precursors or drugs of interest. A lot of our drugs come from natural sources, at least the original ones do. The antibiotics are a prime example of that, coming from bacteria. And those are all made from multiple steps within the cell. The precursors are built up one step at a time. It's been found that the synthetic biology's been able to take those individual enzymes, put them together on an expression construct, and therefore replicate a lot of what has had to been done in the vat of chemicals within an organic chemistry lab. And that's not to say that this will take the place of all organic synthesis, but some of these precursors are exceedingly complex, and particularly before we understand the detailed structures of those, using these pathways and using bacteria as factories to make at least precursors, if not the final product, is also a very attractive element of synthetic biology.
That's interesting. So, how does molecular cloning enable the production of a transgenic organism?
There we're going to a different realm, because the cloning that I've been talking about is just individual pieces of DNA being put together. The cloning that we talk about when we're talking about Dolly the sheep, or something else, is really at a different level. It's easy to think of cloning in terms of bacteria, because as they reproduce, they essentially just take everything, split it in half, and divide. So each bacteria is essentially an identical copy of the parent cell.
Once we introduce sex, we've got problems, because-
It gets complex.It gets complex, and in that case, all the progeny have a contribution from both the mother and the father, and it's not always predictable how those things will go together. So even if we take purebred strains and strains that are identical and mate them together, we still have some differences between them. That's as close as we really get with higher organisms using sexual reproduction to having clones that result out of the mating.
In the case of Dolly the sheep or other transgenic animals, we actually are able to take the same eggs and split them up, let them divide, and take individual eggs that have identical contents, and put them into multiple animals. In doing that, we're able to make identical copies of the same animal. That's really what we're talking about with transgenics, animals, being able to make identical copies, because we start with identical starting materials that haven't been mixed around by some sexual process.
How is molecular cloning involved in gene therapy?
Gene therapy involves the change or alteration of genes that are already in the body, and so the molecular cloning is probably not the best term to really refer to that. We would certainly use the principles of molecular cloning to develop molecules that could be used to go in and guide the alterations in the genome that would be there. In a larger sense, it's the machinery that drives that ability to make those genetic changes and target those to the regions that we're interested in without targeting other places themselves. So, it is important because it is easier to manipulate the individual DNA molecules in a test tube and then use them in bacteria. It's easier to examine those, make sure we have exactly what we want to introduce into the higher organism, and introduce those, and then go through and very carefully screen and analyze whether we've made the specific changes we want.
That becomes feasible when we think about some simple genetic diseases, things like sickle cell anemia, which is ... its genetic basis is a single change in the DNA code that is encoded in the genome. If we could go back and just correct that one single base, it certainly has the prospect of curing that disease. Again, we are doing those sorts of things with animals. The application of that to humans is still a little ways off. We're still proceeding very cautiously, because we want to make sure that the changes we make are very specific and are not broadly introduced into the genome, where it might cause other deleterious effects.
Absolutely. In thinking along those same lines, what are the current challenges or limitations of performing cloning today?
We have a prospect of putting together more and more pieces within a clone.
The early days of cloning really took two pieces of DNA, joined them together in a circle, and used them.
Today, we have a variety of techniques that would ask us to put together not just two pieces but maybe five, 10, 20, or even more. And the advances in cloning have come down today to be able to make that practical.
For example, the Golden Gate assembly system, if correctly designed, has been demonstrated to be able to assemble up to in excess of 20 pieces, with greater than 90% efficiency of getting the correct clone.
It's amazing, and it's really based on fundamental work that's been done on the biochemistry of the joining reactions between DNA pieces, so that those can be intelligently designed to be able to come together in a desired way.
The other thing that is of interest is to be able to put together longer and longer pieces and have those stably maintained. It's a little bit separate from the problem of putting together multiple pieces in that when we put together long pieces of DNA, we need to make sure that they're maintained stably within the host that we put them in, and there appear to be some limits in bacteria as to how big a piece can be stably maintained, partially based on how many copies of those individual plasmids, or circles of DNA, that there are in the cell. There's a need in the field to identify ways of putting together and stably maintaining longer pieces of DNA, so those genes arrays that I talked about can be manipulated and put together well. It's a problem of manipulation outside of the cell, as well as inside the cell, and I think that there are a lot of advances now that are coming forward, approaches people are working on, that will help us to solve that problem.
In forward thinking, where do you see cloning technologies heading in the next 5 to 10 years?
They should continue to advance as they obviously have. Just when we think we've reached a peak, someone comes up with a new technique, which is even better, faster, and more efficient at assembling pieces. The continual need for having assemblies of larger number of pieces, and longer pieces, will continue to drive that innovation. I believe that the emphasis will be on, as I said, putting together many pieces and will be increasingly on the fidelity of synthetically manufactured pieces of DNA and being able to make sure that those are produced with high quality.
We've already seen a real revolution in the way that we work and manipulate DNA sequences that we want. It's become much more cost-effective now to order large blocks of DNA and have them made synthetically than it is to try to go out and to find them in their native sources and clone them. This has also revolutionized the ability to take environmental samples and being able to pull particular DNA sequences out of those. For example, there have been many expeditions that have gone to various sites in the world, pulled out DNA from all the organisms existing in that particular environment, and then done random sequencing of those. Hasn't required any culturing of organisms in order to get those sequences, yet once you have that sequence, you're kind of stuck, because you don't have the original organism to go back to, to try to clone that gene out of.
With synthesis methods, we can go ahead and synthesize that gene and see what that gene does, even though we don't have the original organism there with us to work with. So it does give us an ability to move beyond what we could normally do, because I think it's been estimated, oh, I'm going to miss the number here, but something like less than 10% of the organisms in the oceans can actually be cultured in the laboratory. So we really have a very small view of what the entire world looks like. Synthetic biology allows us to look at and recreate those pathways, the enzymes that occur in those organisms, and I'd look for that to be an even more important element of our studies going forward.
That's interesting, and I would think that that would be really important for conservation efforts, as well, to be able to categorize and catalog the organisms that you might just have a very small sample of, or a single sample.
A lot of the conservation efforts to date have been trying to preserve identifiable organisms from the marine environment. The Ocean Genome Legacy at Northeastern University, which had its foundations at New England Biolabs, have been on that task for twenty, twenty-five years. But being able to pull out those genetic resources from those organisms that we can't cultivate certainly is a reservoir that we just really haven't tapped. Very important.
Thank you so much for joining me today, Bill. This has been interesting conversation. I love hearing that the application of science, even when you're talking about molecular cloning in a lab, is really being applied outside of the lab to field research and conservation efforts all over the world.
Thank you for inviting me, and I share your enthusiasm and excitement about seeing things that we consider to kind of basic research really having an application and making a real difference in solving people's problems and helping us to have a more holistic view of the world and conserving its resources. Thank you.
Thanks for joining us for this episode of our podcast. As always, check out the transcript of this podcast for links to additional information. Hope you'll join us next time for a discussion about the intersection of art and science with Scott Chimileski, who's a post-doc at Harvard Medical School and co-curator of "Microbial Life: A Universe at the Edge of Sight," currently on exhibit at the Harvard Museum of Natural History.
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