Bacterial Transformation and Plasmid Purification PDF - Biotechnology Lab Course

Here is the transcription of the document and conversion to a structured markdown format. # Bacterial Transformation and Plasmid Purification ## Summary Discovered by Joshua Lederberg in the 1950s, the small extragenomic circular loops of DNA called plasmids have revolutionized the world of biote...

Here is the transcription of the document and conversion to a structured markdown format. # Bacterial Transformation and Plasmid Purification ## Summary Discovered by Joshua Lederberg in the 1950s, the small extragenomic circular loops of DNA called plasmids have revolutionized the world of biotechnology and become a fundamental tool in genetic engineering Plasmids allow a gene to be moved from one organism and expressed in another. Plasmids occur naturally, but are engineered by scientists to serve many research and medical goals. They can be used with bacterial, plant, and even mammalian cells. For example, plasmids are the starting point for recombinant protein production in drug development. In fact, the gene that codes for human insulin was initially genetically engineered into a plasmid and expressed in bacteria in 1978 by scientists at Genentech. This was the first-ever recombinant protein developed for a pharmaceutical application. Genetic engineering requires that scientists introduce new DNA into cells. Plasmids and viruses are used to introduce DNA using various techniques, such as transformation, transfection, and infection. Scientists amplify (propagate) plasmids by growing them in E. coli, purify them using various purification techniques, and then assess the quantity and quality of the plasmids produced. Production of plasmids on a small scale is commonly referred to as a miniprep. Activities in this chapter include genetically engineering bacteria to express a jellyfish gene, performing minipreps to purify plasmid DNA, and quantitating plasmid DNA. **Chapter 5: Overview** * 5.1 History of Bacterial Transformation and Plasmids * 5.2 Transforming Cells * 5.3 Plasmid Purification and Quantitation **Chapter 5: Laboratory Activities** * 5.A Bacterial Transformation with S3 Plasmid * 5.B Bacterial Transformation with pGLO™ Plasmid * 5.C Purification of S3 and pGLO Plasmids * 5.D DNA Quantitation ## 5.1 History of Bacterial Transformation and Plasmids Bacteria naturally transfer DNA using a variety of mechanisms, including transformation, conjugation, and transduction. These mechanisms were discovered in the last century, starting with transformation. In the 1920s, pneumonia was frequently fatal and many scientists were studying Streptococcus pneumoniae (pneumococci) to understand more about these deadly bacteria. A British medical officer named Frederick Griffith found that there were two forms of pneumococci bacteria. One form of the bacteria was encapsulated in a polysaccharide coat and was referred to as smooth, or S, strain. The second form of the bacteria was not encapsulated with a coat and was referred to as rough, or R, strain. The R strain was not lethal and did not cause the disease when injected into mice. In contrast, when the S strain was injected into mice, the mice died. When the S strain was killed by boiling, it no longer killed the mice. However, when the dead S strain bacteria were mixed with live R strain bacteria and then injected into mice, they died and their blood contained live encapsulated S strains. Griffith referred to the new encapsulated bacteria as being "transformed." However, the mechanism by which the bacteria were transformed was not determined until the 1940s when Oswald Avery, Colin MacLeod, and Maclyn McCarty discovered that the "transforming principle" responsible for the phenomenon observed by Griffin was DNA from the dead S strain bacteria being transferred into the live R strain and expressed by the R strain to form a capsule. Bacterial conjugation (see Figure 5.1) was discovered in 1946 by Joshua Lederberg and Edward Tatum. In bacterial conjugation, E. coli shuttle DNA across a bridge that forms between the cells. This is dependent on a fertility, or F, factor, and only F+ bacteria can transfer DNA in this manner. Transduction was discovered in 1951 by Lederberg and Norton Zinder. Lederberg and Zinder were testing whether salmonella bacteria could conjugate like E. coli. They discovered that the bacteria did not need to physically contact each other to transfer genetic information, indicating that bacteria do not build a bridge as they do as in conjugation. After further investigation, these researchers discovered that DNA was transferred by viruses called bacteriophages that infect bacteria. Plasmids were discovered by scientists investigating the process of bacterial conjugation. In conjugation, it was discovered that it is not chromosomal DNA that is transferred from one cell to another, but a type of extrachromosomal DNA. This discovery was made independently by William Hayes and Lederberg, and, in 1952, Lederberg proposed the name "plasmids" for these extrachromosomal pieces of DNA. In 1961, Tsutomu Watanabe and Toshio Fukasawa were investigating Shigella, a bacterium that was causing dysentery in Japanese hospitals. They found that some Shigella strains carried plasmids that conferred resistance to antibiotics and that these plasmids could be transferred to non-drug-resistant strains. During this early research, plasmid DNA was assumed to be linear; however, in 1962, Allan Campbell correctly proposed that plasmid DNA was circular. After the discovery of restriction enzymes in the late 1960s, the concept of using restriction enzymes and plasmids to recombine DNA was proposed by Peter Lobban, a graduate student at Stanford University, Palo Alto, CA. Prior to this proposal, research into plasmids had been curiosity-driven and focused on the interesting natural phenomena. In 1973, Stanley Cohen, Annie Chang, Herbert Boyer, and Robert Helling published a paper describing how to artificially construct a biologically functional plasmid and began the age of recombinant DNA technology. Herb Boyer and venture capitalist Robert Swanson founded Genentech in 1976 and used recombinant plasmids to produce insulin. Roche purchased Genentech in 2009 for $46 billion. Bacterial transformation and plasmid technology have been used to clone countless genes for the production of thousands of proteins, which have affected all aspects of life, from laundry detergents to dairy farms to breast cancer treatments. These technologies have also been used to sequence thousands of whole genomes, from archaea in the deepest parts of the ocean to pufferfish and people. ### Plasmid Structure Most plasmids are extrachromosomal circles of DNA that can be replicated in the cytosol of bacteria. Plasmids are represented using plasmid maps, and the name of each plasmid usually starts with a lower case "p," which stands for plasmid. Plasmid maps can be simple or complex and show different levels of detail (see Figure 5.2 for a simple plasmid map). ## Bioethics: A World without Antibiotics? Antibiotics have revolutionized medical practice. The first major use of commercially manufactured antibiotics was to treat injured soldiers in World War II, a little more than a decade after Alexander Fleming noticed that Penicillium mold inhibited bacterial growth and named the first antibiotic penicillin. Prior to the discovery of antibiotics, infections were frequently fatal, and surgeries carried huge risks of infection and subsequent complications. Postoperative infections became rare with the use of antibiotics. Antibiotic-resistance genes, or R-factors, are often carried on plasmids and evolve in bacteria under selective pressure when antibiotics are used. These R-factors are naturally passed between bacteria, generating bacterial strains that are resistant to antibiotics. The increase in antibiotic-resistant pathogenic bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), is raising the question of how long we have until pathogens evolve to become resistant to all known antibiotics. We have come one step closer to this frightening future with the emergence of gram-negative enterobacteriaceae (gut bacteria), such as pathogenic forms of E. coli that are resistant to almost all of the last line of powerful antibiotics. Most worrying is that the resistance gene is carried on a natural plasmid that can be easily transferred between enterobacteriaceae. This gene is called New Delhi metallo-ẞ-lactamase (NDM-1) and is named after the Indian city where it was first isolated. A recently identified contributor to antibiotic resistance is the use of antibiotics intended for humans to treat and prevent diseases among chickens, cattle, pigs, and other livestock to treat and prevent diseases in the animals, and to help them gain weight. The FDA has issued guidelines in recent years to limit the use of antibiotics in farm animals. What should the medical community do with the small number of antibiotics that remain effective against enterobacteriaceae that carry NDM-1? Should the use of these antibiotics be restricted in an attempt to slow down the evolution of pathogenic bacteria that will eventually become resistant to even the most effective antibiotic treatments? Who would be eligible for treatment in such a case and how would such eligibility be regulated in the global community? Pharmaceutical companies will likely continue the race to produce new antibiotics as bacteria evolve resistance, but there is not much profit to be made investing billions of dollars to develop new antibiotics. People use them only for short-term treatments, and once bacteria evolve to be drug resistant, there is no longer a market for these drugs. What should be the government's role? Should we invest in research into nonantibiotic-dependent antimicrobial therapies such as phage therapy? Consider this the next time you have a minor infection – in a world without antibiotics, it could be fatal! ### Features of Plasmids To copy themselves in the cytosol, plasmids must have a starting point for replication, which is called the origin of replication, or "ori" (see Figure 5.2). The ori has recognition sites for DNA polymerases that replicate DNA and enable the plasmid to be cloned (copied) as the bacterium divides. When bacteria divide during fission, each new bacterium gets a share of the plasmids. The number of plasmids per bacterial cell is referred to as the copy number and can vary from 5 to more than 1,000. The ori determines the copy number of plasmids. Plasmids that are present in relatively low numbers in the cells are referred to as low copy number plasmids, while plasmids that are present in much higher numbers are referred to as high copy number plasmids. Plasmids also contain genes. Many natural plasmids have genes that code for enzymes that confer resistance to antibiotics. For example, ẞ-lactamases are a group of enzymes that have been found to break down antibiotics that have a ẞ-lactam ring as part of their molecular structure, including penicillin and ampicillin. The B-lactamase gene is abbreviated as bla or amp (for ampicillin resistant) (see Figure 5.2). If bacteria contain a plasmid with an antibiotic-resistance gene, they will grow in the presence of that antibiotic, while bacteria without the plasmid will not grow. Using an antibiotic to allow only bacteria that contain a plasmid to grow is called selection. Early genetic engineers used the genes and regulatory sequences of natural plasmids as the basis for recombinant plasmids to produce recombinant proteins, including insulin. Antibiotic- resistance genes were vital to these recombinant plasmids because they provide a mechanism for scientists to separate bacteria containing recombinant plasmids from those that do not. *Figure 5.2. PGLO plasmid map. The plasmid map shows the location of the origin of replication (ori) and the green fluorescent protein (GFP), B-lactamase (bla), and araC genes.* Genes are found on either strand of the plasmid DNA double helix. RNA polymerases read in opposite directions for genes on opposite strands. To indicate the strand the genes are on, a plasmid map often has arrows. If the arrows point in opposite directions, the genes are on opposite strands. If the arrows point in the same direction, the genes are on the same strand of the double helix. To express a gene, a plasmid must have a promoter DNA sequence before the coding region of the gene. A promoter is a sequence of DNA that is located before the protein-coding if a gene is to be expressed from a plasmid (that is, a protein is to be made from the gene), the DNA sequence that encodes the protein needs to be cloned downstream of a promoter and upstream of a terminator. The protein-coding sequence also needs to be cloned in frame, meaning that the first codon of the mRNA is positioned to be read correctly by the ribosome and is not shifted by one or two bases, which would result in a different amino acid sequence. Transcription and translation are discussed further in Chapter 7. These details are not important if the gene is only being housed in a cloning plasmid and is not to be expressed. ### Transcriptional Regulation of Plasmids Transcribing and translating genes takes up energy; therefore, only necessary proteins are expressed by cells at any one time. This means that cells must regulate when and to what level they transcribe their genes. Some genes need to be expressed all the time, while others need to be expressed only at certain times or in certain environments. Genes that are always expressed are referred to as constitutive genes, while genes that are transcribed only when needed are referred to as facultative genes. Bacteria regulate expression of some of their facultative genes using operons. Operons are naturally occurring control units in bacterial chromosomal DNA that consist of one promoter, multiple genes, and a single terminator. One mRNA molecule that encodes multiple proteins is produced from an operon. This means that all the proteins encoded by the operon are made in exactly the same proportions and are all made at the same time. This is beneficial for bacteria that can instantly produce multiple proteins with a single switch for fast use of a new food source. Only prokaryotes have operons. Eukaryotes have more complex transcriptional regulation in which each gene is thought to be regulated individually. The mode of action of the lac operon was discovered by François Jacob and Jacques Monod in 1961. This discovery was important because it was the first time that scientists found out how genes are regulated. The lac operon controls the production of three enzymes *Figure 5.4. lac and araBAD operons. Each operon has three genes regulated by a single promoter. In the absence of the sugar inducer( either arabinose or lactose), the repressor protein binds near the promoter and blocks the RNA polymerase from binding to the promoter. In the presence of the sugar, the repressor protein either moves or changes shape and allows the RNA polymerase to transcribe the genes.* that are involved in the metabolism of lactose in E. coli, while the araBAD operon controls the production of enzymes involved in the metabolism of another sugar, arabinose. Operons are controlled by repressor proteins that are encoded by an additional gene. Repressor proteins bind to the operator region near the operon and block RNA polymerase from transcribing. To turn the operon on, the sugar (lactose or arabinose, depending on the type of operon) binds to the repressor and relieves the block, thereby allowing transcription (see Figure 5.4). The sugar is called an inducer. Inducible operons can turn on genes that are normally off, while repressible operons can turn off genes that are normally on. ### Genetically Engineering the pGLO Plasmid Scientists took inducible operons from bacterial chromosomal DNA and genetically engineered them into plasmids. In the case of the pGLO plasmid, a team of scientists made a plasmid called PBAD18 by cloning the araBAD promoter and the gene for the AraC repressor protein into a parental plasmid containing an ori and the antibiotic-resistant bla gene. Another team cloned the green fluorescent protein (GFP) gene from the jellyfish Aequorea victoria into a different plasmid. A third group of scientists then used restriction enzymes to clone the GFP gene into the pBAD18 plasmid downstream of the araBAD promoter. As a result, the araBAD operon and the AraC protein regulate the expression of GFP. Using this system GFP is expressed only in the presence of arabinose (see Figure 5.5). ### Plasmids for Eukaryotic Expression Bacteria and eukaryotic cells use different types of promoters. Therefore, there are different expression plasmids depending on the plasmid's uses. Plasmids are generally made and amplified (propagated) in bacteria. If the plasmids are destined to be used in eukaryotic cells, they must carry features that allow them to be used in both bacteria and eukaryotes. Plasmids that have both properties are called shuttle plasmids. For example, a yeast shuttle plasmid would have features that allow for replication and selection in both *Figure 5.5. PGLO plasmid regulation. In the original operon, the AraC repressor protein regulates the expression of the araB, araA, and araD genes. The pGLO plasmid contains a modified araBAD operon that expresses GFP instead.* E. coli and yeast cells. For propagation in E. coli, the yeast shuttle plasmid would have an origin of replication and a selectable marker (for example, the bla gene). For expression in yeast, the yeast shuttle plasmid would have the sequences necessary for replication in yeast and a different selectable marker. Since the gene of interest contained in the plasmid is destined to be expressed in yeast, a yeast promoter would be used to drive the gene. Shuttle plasmids also exist for expression in mammalian cells. For propagation in E. coli, the mammalian shuttle plasmid would contain an ori and a bla gene, similar to the yeast shuttle plasmid. For expression in mammalian cells, the main shuttle plasmid would have a selectable marker, such as a neomycin-resistance gene, and a promoter from a mammalian virus to drive gene expression, such as the CMV promoter from the human cytomegalovirus. Viral promoters are ideal for driving expression in many cell types because viruses evolved to use the resources in the cells they infect. Mammalian shuttle plasmids also have a polyadenylation sequence at the end of the gene of interest, so a poly A tail is added to the mRNA when it is transcribed. Polyadenylation is discussed in Chapter 7. Interestingly, mammalian shuttle plasmids do not replicate independently in mammalian cells; they are either expressed transiently or stably integrated into the mammalian genome. A naturally occurring plasmid called Ti (for tumor-inducing) occurs in plants. This plasmid is derived from a pathogenic plant bacterium called Agrobacterium tumefaciens that causes crown gall disease (see Figure 5.6). When a plant is infected with A. tumefaciens, a specific fragment of the plasmid DNA called T-DNA gets integrated into the plant's genomic DNA (gDNA). In the laboratory the Ti plasmid is engineered to remove the tumor-inducing genes and foreign genes are inserted into the T-DNA. The modified plasmid is then propagated in A. tumefaciens using the natural regulatory sequences of the bacterium, and the genes of interest are engineered with plant viral promoters driving their expression. Terminator sequences are also required and are frequently cloned from genes that are found in the natural Ti plasmid. *Figure 5.6. Crown gall disease.* ## 5.2 Transforming Cells To genetically engineer a cell, foreign DNA must be placed into the cell. The cell receiving the foreign DNA is said to be transformed *see Figure 5.7*. There are many ways to introduce foreign DNA into a cell, depending on the cell type and the purpose of the experiment. The most common methods used to transform bacteria are calcium chloride transformation and electroporation. The success of a transformation procedure is measured by determining the transformation efficiency, which is calculated by quantitating the number of bacteria that were successfully transformed per microgram (µg) of DNA. The number of transformed bacteria is determined by counting the number of colonies (CFU) the bacteria form when spread on an agar plate. In calcium chloride transformation, actively dividing bacteria are repeatedly washed in an ice-cold calcium chloride solution. The calcium chloride makes the cells "chemically competent" and more permeable to DNA. Chemically competent cells can be flash frozen and stored at -80°C for several months. Chemically competent bacteria produced in the laboratory have transformation efficiencies ranging from 104-106 CFU/µg of DNA. Alternatively, highly competent cells with efficiencies of 107-109 CFU/µg of DNA are commercially available. To transform chemically competent bacteria, plasmid DNA is added to the cells and the mixture is incubated on ice. The cells are heat shocked in a 42°C water bath for 50 seconds and then immediately placed back on ice. Heat shock is thought to temporarily open up, or fracture, the cell membranes, allowing the plasmid DNA to enter the bacterial cells. Non-selective growth medium is then added to the cells and they are incubated and allowed to recover at 37°C with shaking for 30 minutes to 1 hour. The cells are then plated and grown overnight on a selective medium. A modified version of the calcium chloride transformation method will be used in Activities 5.A and 5.B. Electroporation, another common transformation method, uses electricity to disrupt cell membranes and temporarily increase the permeability of the cells, which allows plasmid DNA to enter the cells. Electroporation of bacteria is described in transform E. coli Using Electroporation. Electroporation can be used to transform both bacteria and eukaryotic cells. Eukaryotic cells can also be transformed using a method called transfection. In chemical-based transfection, plasmid DNA is first sealed in tiny oil bubbles called lipid vesicles and incubated in the culture medium. The vesicles then fuse with the cell membranes and deliver the DNA into the cells. Plants have cell walls that create a barrier to most delivery methods. To penetrate plant cell walls, biolistics is the method of choice. Biolistics uses small gold or tungsten particles coated with DNA that are shot into cells under helium pressure. A handheld device called a Helios gene gun was developed for delivery of DNA into plant cells. A larger system, the PDS-1000/He™ was also developed for transfecting cells, tissues, or organelles *see Figure 5.8*. Biolistics is also used to deliver DNA into other eukaryotic cell types, including animal cells and fungi. ### How To... Transform E. coli Using Electroporation Electroporation uses an electric pulse between two metal plates to disrupt cell membranes, increasing their permeability. Bacteria, yeast, and mammalian cells are commonly transformed by this method. Electroporation of bacteria is much faster and often more efficient than calcium chloride transformation and must be done in an electroporator, such as Bio-Rad's MicroPulser™ electroporator *see Figure 5.9*. ### Plasmid selection process Electroporation requires electrocompetent bacteria. Electrocompetent cells are commercially available from various suppliers. Alternatively, electrocompetent cells can be prepared by washing actively growing E. coli cells multiple times with ice-cold nonionic solutions such as 10% glycerol. (Electroporation requires that all reagents be ice-cold.) Electrocompetent cells can be flash frozen and stored at -80°C. ### Bacterial Transformation To electroporate E. coli, thaw the electrocompetent E. coli cells on ice and pipet them into chilled microcentrifuge tubes on ice. Add salt-free plasmid (1-50 ng/µl) and incubate for 1 minute. It is important to add the smallest volume possible of DNA since buffer from the DNA can affect the resistance of the electroporation solution and reduce transformation efficiency. Transfer the mixture to a chilled electroporation cuvette and keep the cuvette on ice until ready to electroporate.