Transformer protocol Student’s guide THE
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THE Transformer protocol Student’s guide INTRODUCTION Bacteria everywhere Bacteria are the commonest living things on Earth. Despite the fact that they are microscopic, together they weigh more than all the planet’s plants and animals combined. Because some bacteria can cause disease they tend to receive a bad press. However, most bacteria are harmless and they perform a vital role by helping to recycle elements, breaking down dead organisms to simpler materials so that they can be used again. They also make nutrients available to other living things by, for example, extracting nitrogen from the air for protein production by plants. Even some of the bacteria that inhabit our intestines are useful — for example, Escherichia coli (E. coli for short) provides us with essential vitamins. Inside a living cell, plasmids can be copied, but they rely upon the molecular machinery of the cell carrying them to do this job. So, in the right conditions, a test tube of bacteria will grow and multiply, but a test tube of plasmids will just sit there. Neither plasmids nor the DNA from which they are made are alive. Plasmids are duplicated separately from the bacterial chromosome, and not just when the cell divides. Consequently one bacterial cell may hold many identical copies of plasmids. old strand new strand Cells express themselves Bacterial cells are enclosed by cell membranes containing the cytoplasm. Most bacteria also have a cell wall surrounding the membranes. Some have one or more thread-like hairs (flagella) to propel themselves along. Inside the cell there’s just one chromosome, in the form of a ring. The chromosome is made of DNA, which carries the instructions needed for making proteins. A set of instructions for making all or part of a protein is called a gene. The genes in bacteria are arranged one after the other, like beads on a string, along the chromosome. The genetic ‘recipe’ of E. coli is 4.6 million base pairs long — that’s enough information for making about 3 000 proteins. Fortunately, every E. coli has about 15 000 proteinmaking ‘factories’ (ribosomes) where the genetic instructions can be translated and the genes ‘expressed’. Just before bacteria divide to form two new cells (which, in favourable conditions, can happen every 30 minutes or so), the whole chromosome is copied so that each new cell ends up with a full set of instructions. Chromosome Plasmids cytoplasm Ribosomes sites of protein synthesis Cell wall Not to scale Cell membranes Some of the main components of a bacterial cell. 2 old strand new strand Nucleotide 5' 3' Phosphate Deoxyribose sugar 3' 5' Complementary base pairs DNA structure and function. The structure of DNA can be likened to that of a twisted rope ladder. At the sides are chains of sugar and phosphate molecules. In the centre are the bases: adenine (A), guanine (G), cytosine (C) and thymine (T). Hydrogen bonds between the bases hold the two helices together—A always pairing withT, and C always pairing with G.Through the double helix, genetic information is copied from one cell or generation to the next.When DNA is duplicated, the two complementary strands are untwisted, and nucleotides are brought into place alongside each of the old strands to form new ones.The new copies are checked to ensure accurate copying. Several different enzymes perform these tasks. The order of bases in the DNA specifies the sequence of the amino acids in proteins. Three bases in a row (a triplet) specify each amino acid. A particular gene — a length of DNA — determines the structure of all or part of a specific protein. A little extra DNA helps Bacterial ‘sex’ Sometimes other smaller rings of DNA, called plasmids, are also found in bacteria. Plasmids carry just a few genes. They are not essential for the bacteria, but they may help them to survive in some environments. For instance, some plasmids help the bacteria that carry them to resist the toxic effects of heavy metals, or to live on particular nutrients. Plasmids are sometimes transferred between bacterial cells in a natural ‘mating’ process called conjugation. This is as close as bacteria get to sex (bacteria don’t actually reproduce by sexual means). Some bacteria can also pick up extra DNA from their surroundings. This is not as easy as it sounds, as DNA molecules can be very large and they have to cross the bacterial cell membrane somehow. Special proteins are needed to help ferry the DNA across and since only a few species have these proteins, this sort of DNA transfer (called ‘transformation’) is thought to be fairly rare in nature. Entry restrictions Even if DNA (a plasmid or smaller fragments) makes it across the cell membrane, many bacteria can destroy the incoming genetic message. This is particularly important if the DNA is from a bacteriophage — a type of virus that preys upon bacteria. The bacterial defence mechanism consists of enzymes — restriction enzymes — whose job is to ‘restrict’ the invasion of viruses. There are many hundreds of different restriction enzymes that ‘recognise’ particular sequences of DNA and cut it up, like very precise molecular scissors. The bacteria’s own DNA escapes damage by ‘disguising’ the sites where the restriction enzymes cut — by adding methyl (–CH3) groups to the DNA bases. A computer model of restriction enzyme EcoRI. This enzyme is obtained from Escherichia coli, strain RI. The DNA is shown at the top of the picture, as a ball-and-stick model, looking down the axis of the double helix. The enzyme wraps around the DNA and moves along it,‘searching’ for a site where the enzyme can cut. EcoRI cuts at G AATTC. ➔ Bacteria perform useful tasks It’s not only bacteria that benefit from restriction enzymes. They’re now an important tool used by medical researchers and biologists of all kinds. Parts of the enzyme’s structure are shown here: flat sheets (betapleated sheets) in yellow; coils (alpha helices) in pink and turns in blue and white. With restriction enzymes almost any section of DNA, and consequently any single gene, may be cut out at will. The end of one DNA molecule can be joined to another that’s been cut with the same enzyme. The combined DNA can then be put into a cell in which it may be expressed and duplicated so that it passes from one cell division to the next. For microorganisms, one of the most successful methods for transferring genes is to ‘paste’ them into plasmids. The result is a ring of ‘recombinant’ DNA that can be put into a bacterium. Specialised plasmids can be used to ferry genes from bacteria into yeast cells or even plants. The data for this image was obtained from the Nucleic Acid Database (http://ndbserver.rutgers.edu/NDB/ndb.html) where you can view the computer model in three dimensions. (Search for the protein with the ID code PD0055.) A cause for concern? Genetic modification of this type has already given us new and improved medicines and vaccines, and altered crop plants with new characteristics. As a research tool, genetic modification has helped us to study our own genes and those of other species, and this work will have major consequences for human health. In this way, microbes can be ‘re-programmed’ to make a wide variety of useful substances including vaccines, insulin and other hormones and materials such as plastics, helping to reduce the consumption of valuable oil reserves. Microbes can be altered to clean up toxic wastes, protect us from food poisoning and help in biological research. However, many people are concerned about the potential dangers of ‘meddling with genes’ and doubts have been raised about the wider economic, social and environmental impact that such work may have. How bacteria are genetically modified. First, the gene of interest is cut out, using carefully selected restriction enzymes. Next, some plasmid DNA is cut with the same enzymes. The two fragments are joined using DNA ligase to form a recombinant plasmid. The plasmid DNA is put into a suitable host strain of bacteria.The transformed (genetically-modified) cells are selected. The plasmid DNA is copied within the bacteria, and the proteins it encodes are produced. DNA Restriction enzyme cuts DNA at specific sites Gene isolated So that you can better understand and assess this technology, this kit provides a practical activity which will allow you to investigate some of the key techniques, coupled with a discussion task so that you can begin to think about and critically assess some of the wider implications of genetic modification. DNA ligase bonds DNA fragments New protein made by bacteria Plasmid put into bacterium Plasmid copied Gene spliced into plasmid Cell divides Transformed cell Bacterial chromosome 3 ­ ­ α ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ β ADVANCED INFORMATION Transformation Transformation is the uptake and expression of DNA by cells. Although some bacterial species can take up DNA from their environment naturally, most cannot. Cells that can take up DNA in this way are termed ‘competent’. However, natural transformation is a relatively rare event. Most bacteria have not evolved the membrane proteins that allow foreign DNA to be ‘recognised’ and absorbed. Without these special mechanisms, gene-sized DNA molecules are too large to diffuse or be transported through the cell membranes. Chemical transformation Escherichia coli (the common gut bacterium) is the best-understood and most studied organism on Earth. It is not naturally competent. However, in 1970 a method was developed to artificially transform this and other bacteria. Today, such transformation is a key process used in the genetic modification of microbes. The original method involved suspending rapidly-dividing cells in a ‘transformation buffer’ of cold calcium chloride solution then subjecting them to a brief heat shock in the presence of the DNA to be taken up. Later it was found that other divalent cations (such as magnesium, Mg2+, manganese Mn2+, and barium Ba2+) all had a similar effect to that of calcium ions. The exact mechanism of DNA uptake is poorly understood. One hypothesis is as follows: ● ● ● ● Cooling the cells to 0 °C stabilizes the normally fluid bacterial cell membranes. Positively-charged ions in the transformation buffer (e.g., Ca2+) are then able to bind to the negatively-charged phosphate groups in DNA and the phospholipids of the cell membranes, shielding their negative charges. This allows the plasmids to approach the membrane and the channels through it that are formed where the outer and inner cell membranes meet. The heat shock helps to force the plasmids through the channels by creating a thermal imbalance on either side of the cell membranes. Other transformation methods In the last decade, other methods of transforming bacteria have been introduced. These include, for example, subjecting the bacteria to brief pulses of very high voltages. This technique — called electroporation — punches holes in the bacteria through which DNA can enter. This is the most efficient method devised so far. A procedure called particle bombardment or ballistic impregnation is also sometimes used to introduce DNA into cells. With this method, the DNA is first stuck onto minute tungsten or gold beads. Using a ‘gene gun’, the DNAcoated particles are fired into the cells. This technique is often used for transforming plant cells. ‘Chemical transformation’ remains popular however, because it is simple, inexpensive and does not require specialist equipment or materials. 6 Plasmid DNA Plasmid DNA entering a bacterial cell. Both DNA and the bacterial cell membrane normally have a negative charge, so they repel one another. However, solutions of positive ions can be used to neutralise both the DNA and the cell surface, allowing the plasmid DNA to enter cells through pores in the bacterial membranes.This is a schematic diagram, showing the general principle involved. In reality, the relative sizes of components and the distribution of charges will differ from that shown here. Selectable marker genes The transformation process is very inefficient and only a small proportion of the cells treated will take up plasmid DNA. Therefore a means of selecting those cells that have been transformed is needed. Antibiotic resistance markers are often used for this purpose. The p2k plasmid includes a kanamycin resistance gene (called APH(3')-I) put there specifically to act as a genetic marker. The strain of Escherichia coli used here is incapable of hydrolysing the sugar lactose, because it lacks the gene for the enzyme lactase (β-galactosidase). However, the p2k plasmid carries this gene, and if a bacterium takes up the plasmid it gains the ability to hydrolyse lactose. The colourless compound X-Gal is hydrolysed by lactase, yielding galactose and an insoluble indigo dye. The dye is precipitated within the bacteria, enabling X-Gal to be used as an indicator of lactase activity (transformed cells are blue). Why can’t lactase action and this blue colour alone therefore be used to select transformed cells? Cells with resistance plasmids are normally disadvantaged compared to their neighbours without them. In the presence of appropriate antibiotics however, such plasmid-bearing cells thrive while their less well-endowed neighbours perish. In this way, selection pressure is applied to maintain the plasmid in the population of cells. Without that pressure, the few transformed cells would be swamped by their untransformed neighbours, and the plates would be covered by a uniform ‘lawn’ of ordinary bacterial cells rather than individual blue colonies. How kanamycin acts on bacteria amino acid Kanamycin kills bacteria by stopping protein synthesis at the ribosomes. Unlike several other antibiotics (e.g., penicillin or ampicillin) kanamycin kills all cells, rather than just those that are actively growing. Consequently, bacteria transformed with p2k require a short ‘recovery period’ before they are transferred onto kanamycin-containing plates. This allows the resistance marker gene to be expressed, and ensures that the transformed cells are not killed on contact with kanamycin-containing growth medium. Transfer RNA small (30S) subunit AUG UAC large (50S) subunit While it would be more practical to use a marker (like ampicillin resistance) that did not need a recovery period, there are several compelling reasons for using kanamycin and kanamycin resistance (see below). How the resistance mechanism works There are very many different mechanisms that confer resistance to the effects of kanamycin and related antibiotics. At least seven of these mechanisms work by transferring a phosphate group onto the kanamycin, altering its structure. The APH(3')-I gene encodes an enzyme that catalyses the transfer of a phosphate group from ATP onto a hydroxyl (–OH) group of kanamycin. The modified antibiotic which results is unable to bind to the bacterial ribosome, so the antibiotic is inactivated. This particular resistance mechanism has been found to occur in about 50% of gram-negative bacteria. The enzyme which confers the resistance is relatively unstable, and it is inactivated by increased temperatures or pH changes. Its requirement for ATP means that this enzyme can only function in environments where that compound is abundant (e.g., inside cells). Use of kanamycin and the resistance gene Many transformation experiments use plasmids that confer resistance to the antibiotic ampicillin. However, in the construction of p2k we have chosen to incorporate a kanamycin resistance marker for several important reasons: ● ● ● ● ● unlike ampicillin, kanamycin is very seldom used to treat human disease, having been superseded by other drugs; it is needed in small amounts in culture plates (a tenth of the concentration normally used for ampicillin); unlike ampicillin, kanamycin is not absorbed by the gut (in clinical use, it has to be injected). Therefore the safety hazard posed by accidental ingestion is reduced; ampicillin resistance enzymes (β-lactamases) often provide resistance to many other similar antibiotics whereas this particular kanamycin resistance gene affects a lesser range of antibiotics of limited use. APH(3')-I confers resistance mainly to kanamycin and neomycin; for several other technical reasons, the use of kanamycin resistance markers is now widely accepted as safe, even in food. Scientists disagree, however, about the wisdom of using ampicillin resistance markers in such products. This is mainly because of the slight risk that the marker gene will pass into other organisms, giving them the ability to withstand not only ampicillin, but other antibiotics too. anticodon CCA 5' GGC AUGCCGGGUUACUUA AUGCCGGGUUACU AUGCCGGGUUAC 3' Messengerr RNA NA codon Bacterial ribosome Protein synthesis at a bacterial ribosome. Successive transfer RNA (tRNA) molecules, each carrying an amino acid, are brought to the ribosome according to the genetic code of the messenger RNA (mRNA). The amino acid residues are strung together to make a protein. Kanamycin interferes with this process by binding irreversibly to the 30S sub-unit of the bacterial ribosomes. The kanamycin/ribosome complex is able to start protein synthesis by binding to mRNA and the first tRNA. However, the second tRNA cannot bind, and the mRNA/ribosome complex dissociates. Further investigations There are numerous variations on the technique described here which can be attempted to try to improve the efficiency of transformation. You could investigate the effect of changing: • • • • the age of the host cells used; the amount of plasmid DNA used; the duration of the heat shock; the intensity of the heat shock (i.e., its temperature); • the duration and/or temperature of the recovery period. To determine the effect of altering these factors, it is useful to calculate the transformation efficiency. This is expressed as the number of transformed colonies produced per µg of plasmid DNA. The transformation efficiency can be calculated as follows: 1. Calculate the mass, in µg, of plasmid DNA used in Step 4. Concentration of the plasmid DNA x Volume of plasmid DNA solution used = Mass of plasmid. 2. Determine how much of the cell suspension you spread, in µL, onto the LB/antibiotic/X-Gal plate. Volume of suspension spread / Total volume of suspension = Fraction of cell suspension spread. 3. Calculate the mass of plasmid contained in the cell suspension spread onto the LB/antibiotic/X-Gal plate. Mass of plasmid x Fraction of cell suspension spread = Mass of plasmid spread. 4. Determine the number of colonies per µg of plasmid DNA. Colonies counted / Mass of plasmid spread (µg) = Transformation efficiency. 7 SAFETY PRECAUTIONS Resistance to the effects of some important antibiotics is now widespread in several species of disease-causing microbes. Special steps have been taken to ensure that the procedures followed in this practical exercise do not contribute to this problem. Antibiotic resistance has evolved to give bacteria the ability to thrive in environments containing antibiotics secreted by other microorganisms. Resistant bacteria often produce proteins that inactivate specific antibiotics or stop them from working in some way (e.g., by preventing their transport into bacterial cells). Resistance genes are often carried on plasmids, which can pass from one bacterial cell to another of the same or a related species by a natural ‘mating’ process called conjugation. During conjugation, a tube or pilus is formed between adjacent cells, through which the plasmid passes. The genes required for the formation of the pilus are also carried on a plasmid (an F or fertility plasmid). The bacterial strain provided in this kit does not carry an F plasmid. Nor does it carry any bacteriophages which may pick up DNA and transfer it to other cells. Deleted genes For a plasmid to travel through a pilus, two additional requirements must be met. The plasmid must possess a gene encoding a mobility protein (mob) and have a nic site. The mobility protein nicks the plasmid at the nic site, attaches to it there and conducts the plasmid through the pilus. p2k has neither a nic site nor the mob gene. This means that once it has been introduced into a bacterial cell by artificial means (transformation) the plasmid cannot naturally transfer (by conjugation) into other cells that do not possess it. 1 F plasmid unwound and replicated The F plasmid has about 30 genes. Bacterial chromosome Sex pilus These include genes for making the pilus and for a mobility (mob) protein. The mob protein conducts the plasmid through the pilus. F plasmid 2 Single-stranded DNA passes into recipient cell through pilus Recipient F- strain 3 Complementary DNA synthesised, forming new F plasmid Conjugation between bacteria Conjugation involves one-way transfer of DNA from a donor (‘male’) to a recipient (‘female’) strain, through a tube called a sex pilus.The pilus is made by the donor cell using genes encoded by a specialised F plasmid. The F plasmid can also temporarily become part of the bacterial chromosome.There it can pick up extra genes that are carried with it when it later passes into another cell by conjugation. F plasmids with these extra genes are called F' plasmids. Biological containment The bacterial strain used in this procedure is non-pathogenic. It has been selected for its suitability for work of this type and its inability to survive outside the laboratory. Over many years of laboratory use and millions of generations, changes have also occurred in the lipopolysaccharides on the outer membrane of this bacterial strain. These changes mean that it is not possible for this strain of E. coli to colonise the mammalian gut. Physical containment In addition to the biological containment measures described above, the practical procedure requires that good microbiological practice is followed to ensure that the microorganisms are physically contained during the investigation and destroyed afterwards. Mass Volume 1 gram (g) = 1 000 milligrams (mg) 1 milligram (mg) = 1 000 micrograms (µg) 1 microgram (µg) = 1 000 nanograms (ng) 1 litre (L) = 1 000 millilitres (mL)* 1 millilitre (mL) = 1 000 microlitres (µL) Nucleic acid 1 kilobase (kb) = 1 000 bases 1 megabase (Mb) = 1 000 kilobases (kb) 8 Donor F+ strain Transfer of antibiotic resistance NOTE Some people prefer to use the cubic decimetre (dm3) and cubic centimetre (cm3) in preference to the litre and millilitre, as S.I. units for volume are derived from those for length. National Centre for Biotechnology Education,The University of Reading, PO Box 228,Whiteknights, Reading, RG6 6AJ. Telephone: 0118 987 3743 Fax: 0118 975 0140 eMail: NCBE@reading.ac.uk Web: www.ncbe.reading.ac.uk Copyright © Dean Madden, 2000 ISBN: 0 7049 1372 0
