1. What is Genetic Engineering?
Genetic engineering typically involves inserting a new gene into an organism. The gene may be newly synthesized or transferred for another organism. In the case of bacteria, genetic engineering turns the bacteria into “living factories” for the production of whatever protein the gene codes for.
Various other terms have been used to describe such activities. These include “genetic manipulation”, “genetic modification”, “recombinant DNA technology” or “modern biotechnology”
The term recombinant DNA refers to a new combination of DNA molecules that are not found naturally and often from 2 different sources. Recombinant DNA technology has at its core the process of gene cloning.
2. Gene Cloning
Enzymes caller restriction endonucleases or restriction enzymes generate specific DNA fragments by recognizing and cutting DNA molecules at specific nucleotide sequences.
A fragment of DNA containing the gene to be cloned, produced from restriction endonuclease treatment, is inserted into a circular DNA molecule called a vector, also digested with the same restriction endonuclease to linearise it. This results in a chimera or recombinant DNA molecule.
The vector acts as a vehicle that transports the gene into the host cell, which is usually a bacterium, although some eukaryotic cells can also be used.
Within the host cell, the vector multiplies, producing numerous identical copies of itself and the gene it carries.
When the host cell divides, copies of recombinant DNA molecules are passed to the progeny and further vector replication occurs.
After a large number of cell divisions, a colony or clone of identical hot cells is produced. Each cell in the clone contains one or more copies of the recombinant DNA molecule; the gene carried by the recombinant molecule is said to be cloned.
3. Elements involved in Recombinant DNA Technology
3.1 Restriction Enzymes and DNA Ligase
The cornerstone of recombinant DNA technology is a class of enzymes called restriction endonucleases. These enzymes, isolated from bacteria, are so named because they protect the bacteria against intruding DNA from viruses or other bacterial cells. They work by cutting up the foreign DNA in a process called restriction.
The bacterial cell protects its own DNA from restriction by adding methyl groups to adenines or cytosines within the sequences recognized by the restriction enzyme.
Most restriction enzymes are very specific, recognizing short nucleotide sequences in double stranded DNA molecules and cutting at specific points within these sequences. Many restriction enzymes recognize hexanucleotide target sites, but others cut at 4 or even 8 nucleotide sequences.
Almost all restriction sequences are palindromes; that is, when both strands are considered, they read the same in each direction.
Many restriction enzymes make a double stranded cut in the middle of the recognition sequence, resulting in a blunt end or flush end. However, quite a number of restriction enzymes cut DNA in a staggered manner, so that the resulting DNA fragments have short single-stranded nucleotide overhangs at each end. These are called sticky end or cohesive ends.
These short extensions will form hydrogen bonded base pairs with complementary single stranded stretches on other DNA molecules cut with the same enzyme. The unions formed this way are only temporary because only a few hydrogen bonds hold the fragments together.
The DNA fusions are made permanent by enzyme DNA ligase, which seals the sugar phosphate backbone by catalyzing the formation of phosphoester bond between adjacent nucleotides. This process is called DNA ligation.
3.2 Vehicles for Gene Cloning; Plasmids and Bacteriophages
Vectors are carrier DNA molecules. To serve as a vector, a DNA molecule must have the following properties.
It must be able to independently replicate itself and the DNA segment it carries within the host cell, so that numerous copies of the recombinant DNA molecules can be produced and passed to the daughter cells.
It must be relatively small, relatively less than 10kb in size as large molecules are more difficult to manipulate.
It should contain a number of restriction enzyme cleavage sites that are present only once in he vector. One site is cleaved with a restriction enzyme and used to insert a DNA segment cut with the same enzyme.
It should be easy to recover from the host cell.
3.2.1 Basic Features of Plasmids
Plasmids are circular extra-chromosomal molecules of double stranded DNA that lead an independent existence in the bacterial cell. All plasmids possess at least one DNA sequence that can act as an origin of replication, so that they are able to multiply autonomously within the cell, independent of the main bacterial chromosome.
Plasmids carry 1 or more selectable markers (genes) e.g. antibiotic resistance genes. The ability of a bacterium to survive in normally toxic concentrations of antibiotics such as chloramphenicol or ampicillin is often due to the presence of a plasmid carrying antibiotic resistance genes in the bacterium.
3.2.2 Useful Properties of 2 Plasmid Containing Vectors
3.2.2.1 Plasmid pBR322
It only consists of 4363 base pairs of DNA, thus able to accommodate DNA inserts. It carries 2 sets of antibiotic resistance genes. Either ampicillin or tetracycline can be used as the selectable marker.
It has unique restriction sites that fall within the antibiotic resistance genes. Insertion of a foreign DNA into pBR322 restricted with Pst1 inactivates the ampicillin resistance gene and insertion using BamH1 inactivates the tetracycline resistance gene (insertional inactivation). It has a reasonably high copy number, about 15 molecules present in a host cell.
3.2.2.2 Plasmid pUC18
It is small (2686 bp) which allows it to carry relatively large inserts of DNA. It contains the ampicillin resistance gene. In a host cell, it replicates 500 copies per cell, producing many copies of the inserted DNA. A large number of restriction enzyme sites have been engineered into pUC18, and these are conveniently clustered on one region called the polylinker site.
3.3 Host Cells
Recombinant DNA molecules are replicated to produce many copies. This is accomplished by transferring recombinant DNA molecules into host cells where replication occurs. One of the most commonly used prokaryotic hosts is a laboratory strain of the bacterium E.coli. The yeast Saccharomyces cerevisiae is extensively used as a host cell for the expression of eukaryotic genes.
4. How to obtain a Clone of a Specific Gene
The problem faced by a molecular biologist wishing to obtain a clone of a single specified gene is analogous to looking for a needle in a haystack. Even the simplest organisms such as E.coli contain several thousands of genes and a restriction digest of total chromosomal DNA produces not only the fragment carrying the desired gene, but also many other fragments carrying all other genes. There are 3 methods to obtain a copy of the gene required.
4.1 Chemical synthesis of a gene
The base sequence of a gene can be found directly, or a suitable base sequence can be worked out from the amino acid sequence of the protein it makes. A gene can then be constructed using nucleotides and joining them in the right order. This is only possible for short genes. It has been used for the synthesis of the proinsulin and somatostatin genes.
4.2 Isolation of total mRNA and conversion to cDNA, followed by screening for the desired gene
A characteristic of most multi-cellular organisms is specialization of individual cells. Each cell contains the same complement of genes, but in different cell types, different sets of genes are switched on while others are silent.
A cloning method that uses mRNA as the starting material would be particularly useful of the desired gene is expressed at a high rate in an individual cell type. Being single-stranded, mRNA cannot be ligated into a cloning vector. However, mRNA can be converted to DNA. Complementary DNA (cDNA) synthesis using the enzyme reverse transcriptase, degradation of mRNA using ribonucleases and lastly, synthesis of the 2nd strand of DNA using DNA polymerase.
4.3 Shotgun approach which involves construction of a genomic library and screening it for the required gene
A genomic library is a collection of clones sufficient in number to be likely to contain all the DNA sequences in an individual’s genome. A genomic library is prepared by purifying total DNA from cells and then making a partial restriction digest with a particular restriction enzyme resulting in DNA fragments of varying lengths to be inserted into a suitable vector which is digested with the same restriction enzyme.
Methods 4.1 and 4.2 have an advantage over the shotgun approach in that the gene that is made is not a split gene. Split genes contain one or more sections of DNA called introns which are not part of the code for the final protein. If a eukaryotic gene is placed into a bacterium, the bacterium does not have the necessary enzymes to remove the introns from the mRNA and a non-functional protein will be made.
4.3.1 Isolation and Digestion of Total Genomic DNA
Total genomic DNA is purified from cells. DNA is partially digested with a particular restriction enzyme e.g. BanH1, to generate sticky ended fragments with varying lengths.
4.3.2 Ligation of Fragments with a Vector
Sticky ended fragments restricted with BamH1 are mixed with the plasmid vector pBR322, also restricted with BamH1. DNA ligase is then added. 3 different types of products are formed. The foreign DNA fragments which have annealed to themselves, the plasmid reannealed and the recombinant plasmid.
4.3.3 Introduction of Ligation Mixture into Host cells (bacterial transformation)
The ligation mixture is added to E.coli cells that are made competent (ready to take up DNA from the solution) by pre-treating the cells with calcium chloride solution. E.coli cells take up circular plasmids more efficiently than linear ones. Each E.coli cell is likely to take up only one particular DNA molecule.
4.3.4 Identification of E.coli Transformant cells that carry Recombinant Plasmids
Plating onto a selective medium (containing the antibiotic ampicillin) enables transformants to be distinguished from non-transformants. Since E.coli cells are killed by ampicillin, cells that have not taken up the plasmid that confers ampicillin resistance cannot grow on ampicillin-containing nutrient agar i.e. they are sensitive to ampicillin.
Cells that have taken up the reannealed insert DNA fragment do not survive on ampicillin since the vector that carries the ampicillin resistant gene is not present.
Ampicillin-resistant transformants are seen as individual E.coli colonies; each colony arises from one individual E.coli cell that has taken up one circular DNA molecule and is a clone of identical cells.
Ampicillin-resistant transformant colonies comprise cells that contain recombinant DNA molecules and cells that contain self-ligated vector molecules.
To identify recombinants, the colonies on ampicillin (master plate) have to be replica plated onto nutrient agar containing tetracycline. After incubation, some of the original colonies re-grow but others do not. Those that re-grow on tetracycline agar consists of cells that carry the normal relegated pBR322 vector with no insert DNA and thus a functional tetracycline resistance gene. Cells that carry non-recombinant plasmids are resistant to both ampicillin and tetracycline. The colonies that do not grow on tetracycline agar are recombinants, since the foreign DNA inserted into the BamH1 site disrupts the tetracycline resistance gene, and the recombinant plasmid is n longer able to confer tetracycline resistance to the host. Cells that carry recombinant plasmids are resistant to ampicillin but susceptible to tetracycline.
This is known as insertional inactivation of an antibiotic resistance gene, the main method of recombinant selection with pBR322.
4.3.5 Identification of E.coli transformant cells that carry the gene of interest by colony hybridization
How do we distinguish the colony containing the gene of interest from those containing the other recombinant plasmids?
All the ampicillin resistant and tetracycline susceptible colonies are collected from the ampicillin master plate, transferred onto nitrocellulose filter and lysed to release the DNA. This treatment also results in the denaturation of DNA.
Single stranded DNA bound tightly to the filter is hybridized with a single-stranded DNA sequence complementary to that of the gene of interest. It tags the correct clone by hydrogen bonding to its single stranded DNA complement on the filter.
The position of the hybridization signal on the autoradiograph will indicate which colony contains the gene of interest (target DNA)
5. Use of Recombinant DNA Technology in Biotechnology: Production of Recombinant Pharmaceuticals
A number of human disorders can be traced to the absence or malfunction of a protein normally synthesized in the body. If the defect can only be corrected by administering the human protein, then obtaining sufficient quantities will be a major problem, unless donated blood can be used as a source.
Gene cloning can be used to obtain large amounts of recombinant human proteins. How are these techniques being applied to the production of proteins for use as pharmaceuticals?
If a foreign (non-bacterial) gene is simply ligated into a standard vector and cloned in E.coli, it is very unlikely that a significant amount of foreign protein will be synthesized. This is because expression is dependent on the gene being surrounded by a collection of signals that can be recognized by the bacterium. These signals, which are short sequences of nucleotides, provide instructions for the transcriptional and translational apparatus of the bacterial cell.
The 3 most important signals for gene expression in E.coli are the promoter sequence, ribosome binding site and the transcriptional terminator sequence.
5.1 Recombinant Insulin
Insulin, synthesized by beta cells of the islets of Langerhans in the pancreas, controls the level of glucose in he blood. Insulin deficiency manifests itself as diabetes mellitus. This can be alleviated by a continuing programme of insulin injections, thus supplementing the limited amount of hormone synthesized by the patient’s pancreas.
Traditionally, insulin has been obtained from the pancreas of pigs and cows slaughtered for meat production. Slight differences between animal and human protein may lead to problems such as side effects and allergic responses in humans. Purification procedures are also difficult, and potentially dangerous contaminants cannot always be completely removed.
Insulin displays 2 features that facilitate its production by recombinant DNA techniques.
Human insulin is not modified after translation by addition of sugar molecules; recombinant insulin synthesized by bacteria should therefore be active. Insulin is a relatively small protein, comprising 2 polypeptides, one of 21 amino acids (A chain) and one of 30 amino acids (B chain).
In humans, the chains are synthesized as a precursor called preproinsulin, which contains the A and B segments linked by a third C chain and preceded by a leader sequence. The leader sequence is removed after translation and the C chain excised, leaving the a and B polypeptides linked to each other by 2 disulphide bonds.
5.1.1 Synthesis and Expression of Artificial Insulin Genes
Based on known amino acid sequences, trinucleotides representing all the codons were synthesized and joined together in the order dictated by the amino acid sequences of the A and B chains.
The artificial genes may not have the same nucleotide sequence as the real gene segments coding for the A and B chains, but they would specify the correct polypeptide.
2 artificial genes were constructed, one carrying the artificial gene for A chain and one gene for the B chain.
Each artificial gene was placed under the control of the strong Lac promoter and a part of the beta-galactosidase structural gene. This was done so that the Lac promoter, being a bacterial promoter, could be recognized by bacterial transcription machinery. Transcription from the Lac promoter was switched on when the E.coli cells were grown in the presence of lactose.
Both recombinant plasmids containing the 2 artificial genes were transformed separately into E.coli to produce large amounts of the A and B polypeptides.
The 2 artificial genes were expressed independently as fusion proteins, consisting of the first few amino acids of beta-galactosidase, followed by the A or B polypeptide. The correct signal for initiation of translation in bacteria is provided by the region immediately upstream of the beta-galactosidase coding region, where the binding site is found.
Each gene was designed so that its beta-galactosidase and insulin segments were separated by a methionine residue, so that the insulin polypeptides could be cleaved from the beta-galactosidase segments by treatment with cyanogens bromide.
The purified A and B chains were attached to each other by disulphide bond formation in he test tube to obtain the functional insulin.
5.2 Recombinant Somatostatin (Growth Hormone Inhibiting Hormone)
Somatostatin was the first human protein to be synthesized in E.coli. Being a very short protein, only 14 amino acids in length, it was ideally suited for artificial gene synthesis. The strategy used was the same for recombinant insulin, involving insertion of an artificial gene into a LacZ vector, synthesis of a fusion protein and cleavage with cyanogens bromide.
5.3 Recombinant Somatotrophin (Human Growth Hormone)
The human growth hormone is a small protein molecule produced in the pituitary gland. It affects almost all tissues of the body. Abnormally low levels of growth hormone in childhood result in dwarfism.
Treatment has relied on growth hormone excreted from the pituitary glands of dead humans, and the supply was not large enough to meet the demand. Moreover, extracts from pituitary glands were occasionally contaminated with infectious protein that caused Creutzfeldt-Jakob disease.
5.3.1 Synthesis of Human Growth Hormone in Bacteria
This protein is 191 amino acids long, equivalent to almost 600 bp, a difficult prospect for DNA synthesis capabilities. In fact, a combination of artificial gene synthesis and complementary DNA cloning was used to obtain a somatotrophin-producing E.coli strain.
mRNA was obtained form the pituitary. A cDNA library was produced. The somatotrophin cDNA contained a single site for restriction enzyme HaeIII, which cuts the cDNA into 2 segments. The longer segment consisting of codons 24 to 191, was retained for use in construction of the recombinant plasmid. The smaller segment was replaced by an artificial DNA molecule that reproduced the start of the somatotrophin gene (codons 1 to 23) and provided the correct signals for translation in E.coli. The modified gene was then ligated into an expression vector carrying the Lac promoter. The hormone was secreted from the bacterium into the surrounding medium after its manufacture, which mage purification a lot easier.
