 You're watching FJTN, the Federal Judicial Television Network. The Federal Judicial Center presents Science in the Court Room, a series of programs for judges on science and scientific evidence. Program 2, Recombinant DNA and Gene Cloning. This lecture by Edward S. Makarski Jr., Professor of Microbiology and Immunology at Stanford University School of Medicine, builds on his presentation in Program 1 of this series, Core Concepts of Microbiology. Hello and welcome. This lecture on Recombinant DNA and Gene Cloning will continue to expand core concepts of microbiology that have entered into the courtroom. In my first lecture, I presented an introduction to molecular biology, covering the way in which the four bases of DNA are arranged into a code and how they're replicated, that is, copied or reproduced. I also discussed the flow of information that gives rise to gene expression, including how an intermediate messenger RNA is transcribed from a DNA template and how this messenger is itself a template for the synthesis or translation of proteins. Proteins make up most of the structures and carry out metabolic processes central to life. The common language, or DNA code, makes it possible to recombine or take DNA from one organism and move it into another. Such recombinant DNA approaches have changed the way biology research is carried out and opened the way for a new industry, biotechnology. Biotech is a commercial child of recombinant DNA. Moving DNA from higher organisms, like humans, to bacteria, can provide an unlimited supply of such things as therapeutic proteins, insulin, growth hormone. Modifications of genes in organisms, such as the plant rice, has led to new variants that contain a more balanced source of essential amino acids. So today's lecture will deal with these basic recombinant DNA and gene cloning methods. The first slide summarizes the core concepts we'll try to cover. First off, and this is mostly from last time, the genetic code is universal. That is to say DNA and the four bases that make up DNA are common to all organisms. The arrangement of those bases, the order of those bases, dictate the differences in organisms. Bacteria, which are a simple organism, procad, exchange DNA in nature. And this is actually one of the first and really important principles that came up in experimental studies that allowed us to appreciate that you could move DNA from one organism to another. Bacteria exchange DNA and exchange, for example, antibiotic resistance markers. So they can acquire a resistance to an antibiotic in happens, in livestock happens, in therapies that are applied to patients in hospitals. Plasmids are DNA molecules that carry these resistance markers and naturally occur in nature. Gene cloning has allowed us to take those DNA plasmids and modify them or design them to carry or to transfer new DNA molecules into them. The bacteria that are most popular are called Escherichia coli. They were the first to be engineered or used for recombinant DNA and they're still the lab favorite. Human proteins, and I think this is the most important area, human proteins have been and continue to be expressed in bacteria as a way to create an abundant supply of important therapeutic proteins. So a human protein coding sequence can be taken and inserted into a bacterial context and allow that bacteria to then act as a fact. It tricks the bacteria into producing essentially a foreign protein, a protein that's of no use to the bacterium but can be of commercial use. So we'll go through these core concepts today. Next slide. So first, there's a natural tendency of bacteria to exchange DNA and again the most poignant example of that is antibiotic resistance. We all know that antibiotics that were useful years ago are no longer useful either in hospitals or in various settings in livestock because resistance has arisen. Well the resistance markers are carried on plasmids, say small DNA molecules. This movie is a cartoon as it were to illustrate the exchange of DNA from one bacteria to another. As you see, this armored bacterium who comes in from the right is suited up and can defend himself against an antibiotic and he just transferred something to the other bacteria and now we're going to slow that down and watch it in slow motion. If you watch closely you see sort of a white circle fly out of his head and hit one guy nearby and then that fellow also throws off a bunch of white circles that get transferred to everyone else. That's meant to depict the exchange of a plasmid from one bacterium to another. If we just go through it one more time we can appreciate that the process is colorful and actually occurs pretty much that way in nature. DNA exchange is quite free in bacteria so that one bacterium for example living in your gut in your intestine can exchange a plasmid with another bacterium that's also living in the same environment and that can give rise to resistance to commonly used antibiotics such as ampicillin. As we're watching the movie you can see a diagram of a plasmid off to the left there with ampicillin R, ampicillin resistance sort of written along its side. Plasmids are small DNA molecules, circular that can have a couple of genes but most importantly have a gene that encodes a protein or enzyme that can degrade a particular antibiotic such as ampicillin. So ampicillin is a drug, beta-lactamase is an enzyme that can break that drug apart and beta-lactamase would be encoded by an ampicillin resistance gene on a plasmid. If we go on, so this ability to have plasmids and select for bacteria that carry plasmids using antibiotics, antibiotic resistance is a core concept as well. It allows us to take a mixture of bacteria for example in a laboratory, introduce a plasmid into a select few of them and grow them out of a population because they're resistant to a particular antibiotic whereas the members of the population that lack the plasmid are susceptible to that antibiotic and this slide is meant to depict this process. So on the left on the top you see a bacterial cell sort of an oblong image with two DNA molecules shown inside of it. One is the bacterial chromosome, it's shown larger, it's actually a very large circle and the other a small circular plasmid. That plasmid has ampicillin resistance on it. It's been acquired by that bacterium but not by that bacterium immediately to the right. So when we apply ampicillin to a population that includes bacteria that have the plasmid or that lack the plasmid, only the ones that carry the plasmid will grow and propagate and in fact that's a key concept to be able to isolate a particular DNA molecule. In this case it's just a natural occurring bacterial plasmid but nonetheless it allows a clone of bacteria to be grown out as a colony and we'll talk more about that and essentially identified by a scientist and taken and used. So only plasmids that carry the antibiotic resistance marker will grow out when the antibiotic is present in the growth medium in a laboratory setting. And so that's used in practical terms to help a scientist grow out a particular bacterium. If we go to the next slide this shows a time course of E. coli, Ascherichia coli growth. These are actual micrographs, they're black and white images taken in a time sequence going from left to right in the three rows across. You see we start with three bacteria little rods on the upper left. As time goes by approximately every 20 to 30 minutes each of those bacteria divide. They're in the presence of nutrients that food that they can live on grow and so they divide. And by the end of a period of time shortly less than two hours I believe they end up forming a kind of colony of bacteria. This would still be microscopic, it wouldn't be visible to the naked eye but if we continue this process for another eight or ten hours if we go to the next slide we see that the colony now it's at lower magnification so you can't really see the individual bacteria but you can see they've piled up into a very large aggregate and eventually this reaches a size that can be visualized without any magnification on a growth surface. And so in the laboratory growing out colonies of bacteria have been used in many settings. I mean diagnostics, for example if you have a strep throat and you go to the doctor and you want to know if it's a strep throat they'll put a swab in your throat they'll streak it out onto a bacterial plate that has medium and look for a particular kind of bacterium by growing out colonies of that bacteria. In that case the bacteria isn't grown for resistance to any antibiotic it's just to isolate and identify the bacterium. In the laboratory we use the bacterial resistance markers antibiotics as well to identify and manipulate bacteria. What I'm leading towards is the ability to use these naturally existing plasmids in order to actually introduce foreign DNA into an organism like bacteria so that the foreign DNA can be carried along and studied or used in various ways. Next slide. So one of the first uses for plasmids in the laboratory came about 25 years ago and that is when these plasmids again that have the antibiotic resistance markers were found to have the ability to be opened with a kind of enzyme a protein that could recognize a particular base sequence and actually cut through the double-stranded DNA and leave open ends. What's shown here is the restriction enzyme which is the name for that protein ECHO-R1. ECHO-R1 is just a name of one of the kinds of restriction enzymes that are available and there are hundreds of these now available to scientists. ECHO-R1 recognizes and cuts through DNA at the site GAA-TTC and on the opposite strand is CTT-AAG their base pairs and that's what's shown on the upper left here. So after cutting the plasmid ECHO-R1 and taking any other DNA and exposing it to that enzyme ECHO-R1 sites are digested cut left open. Those two fragments can be brought together and recombined into a single now circular molecule that's a recombinant DNA molecule. It was not something that existed in nature it's something that was made in a laboratory made in a test tube and that can still be put back into bacteria and propagated in fact cloned the term cloning means taking an individual plasmid and inserting an individual new piece of DNA and isolating that as a pure clonal population. So very important to the process of recombinant DNA is the need to be able to manipulate DNA by digesting it into circular sites, defined sites with restriction enzymes. Restriction enzymes like ECHO-R1 that have a six base recognition site occur naturally in all DNA they're not special things just the fact that DNA sequence exists about once every 500 bases will be an ECHO-R1 site so there's actually quite a lot of ECHO-R1 sites in everyone's DNA in all organisms. Besides being present naturally though transchemists have figured out ways long ago to make synthesized DNA and so ECHO-R1 sites can also be synthetically prepared by chemists and placed onto larger DNA molecules so there's a couple of ways to actually end up having ECHO-R1 sites available to make an insertion into a particular plasmid the plasmid then becomes a cloning vehicle or cloning vector. Next slide. So why clone DNA? Well one of the earliest reasons was to simply have an abundant supply of any DNA it's still a very important reason to have it in fact the organization of a complex genome like the human genome cannot be done without cloning that large DNA we talked about that being 3 billion bases our genomes are 3 billion base pairs that's an enormously large amount of DNA it comes in 23 different chromosomes and in order to study that it's been cloned, it's been cloned in fragments anywhere from a few hundred base pairs to hundreds of thousands of base pairs and the largest clones that are available in fact some are almost a million base pairs but the value of having the clonal pieces is they can be then ordered or mapped into a particular arrangement so we can actually study them in sequence determine the nucleotide sequence of each of the regions of the human genome and so the human genome project that is to say the sequencing of one set of 3 billion base pairs is all possible because DNA can be carried in relatively abundant levels in bacteria and finally we're going to go into how this whole process is important because it allows a scientist to express a foreign protein such as insulin in a foreign organism, a bacterium but that insulin the protein that would be expressed is just like it's human origin human original it's just like the natural protein and again this has to do with the universality of the code the universality of the fact that you can express any DNA segment as long as you give it the right controls and of course what I mean by expression is a process of transcription of a DNA template into an mRNA intermediate and then the translation of that messenger RNA into protein so in a recombinant DNA setting the transcription would start at bacterial control sequences and transcribe through the foreign protein encoding sequence and then the messenger RNA would be read and translated into a foreign protein so DNA can be cloned in for a few bases to hundreds of thousands and this is a very versatile system of using particularly E. coli for this purpose if we go on now to look at a little more about the restriction enzyme and again staying with E. coli1 is our example so restriction enzymes are said to cut a site on the DNA in the case of E. coli1 on the bottom you can see that I've got the double helix cartoon expanded out and there's only six bases shown well that six bases GAA TTC with its respective base pairs would be present on a DNA molecule exposing that DNA molecule to E. coli1 would leave what's called a staggered cut that is to say a cut that would release the two ends but leave single stranded base overhangs in the case here it's AATC AATT on the top strand and TTAA on the bottom strand those so-called sticky ends are what can be used to recombine any other E. coli1 cut DNA into a site so the fact that there's an AATT or TTAA allows other pieces that end the same way fragments that end in the same sort of cut site to be used they're complementary or compatible ends and so recombinant DNA the recombination in recombinant DNA is indeed the annealing or the sticking together of these ends that are common on a variety of different DNA fragments on the one hand a plasmid that might be opened and the other some DNA fragment say from humans carries the insulin gene on the other together as one the process of sticking together requires some enzymatic steps and those are well established in most biological laboratories these days next slide so one of the key things we've already talked about is that plasmids allow bacteria to grow out as colonies and so if you insert a series of fragments in this case in this example on the slide there are four different fragments in our mixture of DNA that's going to be cloned and what's important is it doesn't matter if that's four fragments, four hundred fragments or forty thousand fragments after they're introduced into the plasmid all of those individual variants can be isolated because bacteria take up only one plasmid and then they grow as a colony and so out of that mixture of four as shown here or forty thousand individual fragment clones inserted into the plasmid clones will grow out on a bacterial culture dish as shown in this cartoon now those colonies are a lot bigger than they normally would be relative to the culture dish but it's done this way so you can see that it's a pile of bacterial cells normally they're only about a couple of millimeters across but they're big enough to see selected and used so in the process of introducing these plasmids into E. coli is called transformation of E. coli or transformation of the bacteria and the bacteria grow again on a nutrient auger that's a source of growth it's a sort of solid surface on which they can pile up that contains an antibiotic that the plasmid has a resistance marker to a resistance marker helps us identify the plasmid and then we look and see which DNA fragment might be cloned into it so that's the principle of cloning next slide now besides being able to just clone DNA into bacteria and have it be a place to carry foreign DNA a very important development is that bacteria can be manipulated to produce foreign proteins so bacteria go through life just as we do expressing their DNA as genes expressing their genes and gene products and making proteins so plasmids have genes we've already talked about one ampicillin resistance beta-lactamase is a gene on a plasmid that gene is expressed in E. coli because it has all the appropriate gene expression signals we talked very briefly about these sorts of signals yesterday I'll talk more about them today the E. coli bacteria were the first and remain the most common to be used in a laboratory and the important control elements besides the protein coding DNA sequence that's moved from humans the most important control elements are called promoters that are places in the bacterial genome or in a plasmid genome or actually in bacteriophage genome bacterial virus genome that have been adapted to be able to control transcription or expression of an RNA intermediate in bacteria that can be used to translate that foreign protein then in a context that's completely foreign to that original gene let's say human insulin gene which would not be expressed in E. coli without having those manipulations so before I go on I need to just stop and digress here and show you what a bacteriophage is the control elements that are used by scientists commonly come from bacterial genomes from plasmids and from bacteriophages and we really haven't talked about bacteriophages up to now so the next slide is a movie that depicts one of the most commonly used bacteriophages that are in fact E. coli it looks sort of like a lunar lander if we just stop the movie for a moment it looks like a lunar lander it's very small as most viruses are relative to the host cells that they infect and here the host cell the bacterial cell is below these three bacteriophages that have landed on the surface of that bacteria now what's going to happen here is the bacteriophage which is a virus carries its own genome and in this case it's a DNA genome as well is packed inside of a protein shell which is that upper part of the lunar lander the collar and those legs that it uses to land with are all machinery that it uses to simply inject its genome into those bacteria so bacteriophage like these get around in nature by finding a bacterium and injecting DNA into the bacterium if we go on with the movie we'll see what happens then so the bacteria inject their DNA is expressed because it's recognized by the bacterial machinery you get proteins you get progeny bacteriophage there there are only about a dozen in fact normally when one bacteriophage infects a bacterial cell you get thousands of progeny one of the things viruses do very well and so here we go again the bacteriophage has landed they inject their DNA the virus DNA is expressed the viral proteins are made assemble into progeny viruses and then they destroy the bacterial cell and are released to go off and do more damage the important point here though is that these bacteriophages have very strong promoters and control regions that have been used in the laboratory to direct expression of foreign proteins just as some of the promoters from the bacterial genome and from plasmids have also been used to get a recombinant protein there are some principles involved first off you can't just use any plasmid the plasmid has to be modified so that it can be used as an expression vector what's called an expression vector an expression vector has one additional component at least compared to the cloning vehicles we've talked about up to now the extra piece is a promoter region here shown in blue that is adjacent to the site into which a human gene let's say like insulin is going to be inserted so the human gene is going to be inserted into that plasmid again using a restriction enzyme like EcoR1 to make the recombination but it's going to be put into a context where there's a bacterial promoter that allows it to be expressed and we're going to talk a little more about that before we come back to exactly what happens then in any case the plasmid is still maintained just like a plasmid we've talked about before but now it's got an insert adjacent to a promoter so that as the bacteria are grown this foreign protein this new protein can potentially be made in E. coli so if we go now and step back and look at the way in which bacterial genes are arranged they have a sort of hallmark arrangement of both promoter and operator that we haven't spoken about at all yet and a ribosome binding site and let me just go through that using this bacterial operon and the term operon is also new this bacterial operon which has three genes so bacterial genes are often set up into arrangements where there was one promoter one place where the RNA polymerase starts making a messenger RNA the DNA shown here on the top strand on the top line and the messenger RNA shown just below it so the process of transcription of messenger RNA is starting from a promoter and proceeds to go through multiple genes so that the messenger RNA has more than one gene carried on it in each case the messenger RNA is translated into individual proteins and so on the messenger RNA there are several boxes in between each gene which represent a place for ribosomes remember ribosomes are the protein synthesis machinery they start protein synthesis and so mRNA to be read and turned into a protein synthesis requires a ribosome to come in and start to read it and that's the starting point for reading and in front of each of those genes gene Z, gene X and gene Y there's a little ribosome binding site box on the messenger RNA and each of those genes gives rise to an individual protein they're shown as a kind of oblong image so an operon controls the expression of a series of genes all the genes of an operon are regulated by the same conditions and some of the most well understood operons in bacteria control things like how the bacteria respond to nutrients the presence of a particular sugar for example in the growth medium or in nature bacteria like E. coli live in nature they live in various places and they look for different nutrients in the lab they can be grown on different sugar sources two sugars that are used commonly are a sugar called glucose which we also can use as energy source and a sugar like lactose scientists have adapted these regulatory control regions to control the expression of for example human insulin in bacteria in a recombinant DNA sense so if we go to the next slide it tries to put that together and this slide is actually just a modification of one we saw three slides ago so if we look at insulin as an example we have the promoter operator region which is the blue region taken from a natural bacterial operon and in fact in the first case where this was done it was the lac operon and it was the lac promoter operator so that insulin the insulin cDNA once it was inserted into the expression plasmid which is shown in a top series of three circles to make the final plasmid expression vector on the right when that was introduced into E. coli and when the E. coli were grown under the appropriate conditions to induce expression and that would be lactose in the presence of lactose would induce this expression insulin was made and could be collected from cells from these bacterial cells that would not naturally have ever made that protein so it's a very important step in commercialization of recombinant DNA because at this point in time which was the late 1970s there was really no real understanding of the kinds of practical possibilities although it was well understood that DNA was a universal code the demonstration that you could express a human protein in bacteria was a major hallmark in biology in molecular biology and since that time any number of different therapeutic proteins have been expressed in this same fashion we go to the next slide so expression of recombinant proteins in bacteria takes advantage of naturally occurring control regions taken from bacterial genomes from bacterial plasmid genomes or from bacteria phage genomes the bacterial viruses the elements that regulate these bacterial genes can be used to control expression of any foreign gene it's not guaranteed to get a lot of it but you can certainly set it up and express any foreign gene under those control elements if it's made in sufficient quantities in bacteria that recombinant protein can be purified and in fact the basis of all the commercially viable proteins that are made this way is that they're made in high enough quantities to be purified and available in pure form and indeed the real benefit here is that they're identical to the protein that was originally natural to humans let's say and so recombinant protein can be used to replace the natural human protein for example insulin to treat an important disease, diabetes and it's real important because it's really important to follow that the reason the recombinant protein approach is so valuable is that it is the natural human protein in the case of insulin before the availability of recombinant insulin collected from pig pancreas porcine pancreas was used and that protein while it's similar to the human protein is not identical people who continue to receive this pig insulin throughout their lives to keep their diabetes under control eventually they had a response to that foreign protein because it wasn't the same as their own insulin it had a different amino acid sequence and eventually they would respond to it their immune systems would reject it and that would cause problems so that it would no longer be useful so the human protein is far superior to the previous available therapeutic protein and so if we go to the final slide here microorganisms like bacteria are really very useful to clone DNA and to express foreign proteins I've used bacteria as an example but since the days when E. coli was the pioneering organism and still the most popular organism any number of microorganisms other bacteria yeast and fungi as well as multicellular organisms have been engineered indeed you probably hear a lot of discussion about cloning of humans and that's a big debate at this point in society although cloning of sheep was done first and cloning of mice and other mammals certainly has been established as in principle can be done the bacterial plasmids are very highly adaptable so bacterial plasmids and small circular molecules of DNA from other organisms that can be manipulated are the most useful ways to move DNA around from one organism to another so gene cloning has enabled scientists to go in and take DNA and basically design it as they see appropriate whether it's to express a gene to introduce new features or to make deletions or remove genes bacteria like the E. coli we've used are the most popular but it's important to keep in mind that many other microorganisms and actually many higher multicellular organisms have also been used to engineer with gene with gene therapy genetic engineering recombinant DNA methods as we've used the example are really very effective ways at allowing a variety of human proteins to be made in a form where they're abundant to produce bacterial control control signals so this is an area that I think is important to understand as so many different types of genes are expressed in E. coli as so many different forms of this method are applied in the laboratory and these have been coming I think in increasing frequency into the courtroom as questions that are debated as to which approach was the similar or different from another approach what I've hoped to accomplish in this lecture is some basic information some concepts on how genetic engineering, recombinant DNA gene therapy are used in their core approaches. Thank you very much for your attention.