Genetic Engineering Notes PDF

University of KwaZulu-Natal Department of Biochemistry RDNA202 - Molecular DNA Technology Genetic Engineering 1. INTRODUCTION Genetic engineering is a broad scientific term that refers to artificial manipulation (by removal, addition or modification) of an organisms’ nucle...

University of KwaZulu-Natal Department of Biochemistry RDNA202 - Molecular DNA Technology Genetic Engineering 1. INTRODUCTION Genetic engineering is a broad scientific term that refers to artificial manipulation (by removal, addition or modification) of an organisms’ nucleic acid content. It differs from genetic recombination in that it does not occur through natural processes within the cell, but is human-made. Organisms whose genes have been artificially altered for a desired purpose (either making new metabolites or enhancing function) is often called genetically modified organism (GMO). Recombinant DNA (rDNA) is a form of artificial DNA that is created by combining two or more sequences that would not normally occur together (from different or heterologous sources). In certain instances, it has provided the means to produce large amounts of highly purified normal and mutant proteins for detailed analysis of their function in the organism. rDNA is also referred to as chimeric DNA. The latter term is derived from chimera, a monster in Greek mythology that consisted of a lion’s head, goat’s body and a serpent’s tail. A recombinant protein is a protein that is derived from recombinant DNA. In biology, a clone is a group of genes, cells, or organisms derived from a single common ancestor. The terms DNA cloning, molecular cloning and gene cloning all refer to the same process: the transfer of a DNA fragment (insert or foreign DNA) of interest from one organism to an autonomous- or self-replicating genetic element (vector) such as a bacterial plasmid. The DNA of interest can then be propagated in both high quantities in a foreign host cell and isolated in pure form so as to facilitate the study of genes. This technology has been around since the 1970s, and it has become a common practice in molecular biology laboratories today. The basic principles of recombinant DNA, like the structure of DNA itself, are surprisingly simple. 2 Figure 1: By fragmenting DNA of any origin (human, animal, or plant) and inserting it in the DNA of rapidly reproducing foreign cells, billions of copies of a single gene or DNA segment can be produced in a very short time. The DNA fragment of interest from an organism such as a human is incorporated into the 'plasmid DNA' of a bacterial cell. A plasmid is a circular self-replicating DNA molecule that is separate from the bacterial DNA. When the recombinant plasmid is introduced into bacteria, the newly inserted segment will be replicated along with the rest of the plasmid. 3  Recombinant DNA technology is used to achieve the following: i. Study the arrangement, expression and regulation of genes ii. Modification of gene expression either to enhance or suppress a particular product iii. Artificially produce multiple copies of a nucleic acid segment iv. Introduction of genes from one organism to another, thus creating a transgenic organism with desirable or altered characteristics. v. Creation of transgenic organism so that they can be commercially employed as cell factories. 2) ENZYMES USED IN CLONING 2.1) Restriction endonucleases (RE) Restriction enzymes (RE) are endonucleases that recognize a specific, rather short, nucleotide sequence on a double-stranded DNA molecule, called a restriction site, and cleave the DNA at this recognition site or elsewhere. They are of bacterial origin These enzymes were so named because of their ability to ‘restrict’ growth of bacteriophages in certain bacterial strains. There are three major classes of restriction endonucleases. Their grouping is based on the types of sequences recognized, the nature of the cut made in the DNA, and the enzyme structure. Type I and III restriction endonucleases are not useful for gene cloning because they cleave DNA at sites other than the recognition sites and thus cause random cleavage patterns. In contrast, type II endonucleases are widely used for mapping and reconstructing DNA in vitro because they recognize specific sites and cleave just at these sites (Table 1). 4  Table 1: Major classes of restriction endonucleases. Use in Class Abundance Recognition site Composition recombinant DNA research Type I Less common than Cut both strands at a Three-subunit complex: Not useful type II nonspecific location > individual recognition, 1000 bp away from endonuclease, and recognition site methylase activities Type II Most common, Cut both strands at a Endonuclease and Very useful approximately 240 specific, usually methylase are separate, enzymes commercially palindromic, recognition single-subunit enzymes available site (4-8 bp) Type III Rare Cleavage of one strand Endonuclease and Not useful only, 24-26 bp methylase are separate downstream of the two-subunit complexes 3′recognition site with one subunit in common 2.1.1) Restriction endonuclease nomenclature Restriction endonucleases are named for the organism in which they were discovered, using a system of letters and numbers. For example, HindIII (pronounced “hindee-three”) was discovered in Haemophilus influenza (strain d). The Hin comes from the first letter of the genus name and the first two letters of the species name; d is for the strain type; and III is for the third enzyme of that type. SmaI is from Serratia marcescens and is pronounced “smah-one”. EcoRI (pronounced “echo-r-one”) was discovered in Escherichia coli (strain R). BamHI is from Bacillus amyloliquefaciens (strain H). Over 3000 type II restriction endonucleases have been isolated and characterized to date. Approximately 240 are available commercially for use by molecular biologists. 5 2.1.2) Recognition sequences for type II restriction endonucleases In the presence of the essential cofactor Mg2+, the enzyme cleaves the DNA on both strands at the same time within or in close proximity to the recognition sequence (restriction site). Each orthodox type II restriction endonuclease is composed of two identical polypeptide subunits that join together to form a homodimer. These homodimers recognize short symmetric DNA sequences of 4–8 base pairs. Six base pair cutters are the most commonly used in molecular biology research. Usually, the sequence read in the 5′ → 3′ direction on one strand is the same as the sequence read in the 5′ → 3′ direction on the complementary strand (palindrome). The enzyme cuts the DNA duplex by breaking the covalent, phosphodiester bond between the phosphate of one nucleotide and the sugar of an adjacent nucleotide, to give free 5′-phosphate and 3′-OH ends (Figure 2). The exact mechanism by which restriction endonucleases achieve DNA cleavage has not yet been proven experimentally for any type II restriction endonuclease. Some enzymes, such as EcoR1, generate a staggered cut, in which the single- stranded complementary tails are called “sticky” or cohesive ends because they can hydrogen bond to the single-stranded, complementary tails of other DNA fragments. Other type II enzymes, such as SmaI, cut both strands of the DNA at the same position and generate blunt ends with no unpaired nucleotides when they cleave the DNA. PO4− PO4− 5’ sticky or cohesive ends Figure 2: Cleavage patterns of some common restriction endonucleases. 6  PO4− PO4− Blunt ends PO4− PO4− 5’ sticky or cohesive ends PO4− PO4− 5’ sticky or cohesive ends 5’--G-A-G-C-T-C--3’ SacI 5’--G-A-G-C-T-OH PO4−-C--3’ 3’--C-T-C-G-A-G--5’ 3’--C-PO4− OH-T-C-G-A-G--5’ 3’ sticky or cohesive ends Figure 2 continued: Cleavage patterns of some common restriction endonucleases. 2.2) Ligases DNA ligases catalyze formation of a phosphodiester bond between the 5′- phosphate of a nucleotide on one fragment of DNA and the 3′-hydroxyl of another (Figure 3). This joining of linear DNA fragments together with covalent bonds is called ligation. Unlike the type II restriction endonucleases, DNA ligase requires ATP as a cofactor. If restriction-digested fragments of DNA are placed together under appropriate conditions, the DNA fragments from two sources can anneal to form recombinant molecules by hydrogen bonding between the complementary base pairs of the sticky ends. However, the two strands are not covalently bonded by phosphodiester bonds. 7  DNA ligase is required to seal the gaps, covalently bonding the two strands and regenerating a circular molecule. The DNA ligase most widely used in the lab is derived from the bacteriophage T4. T4 DNA ligase will also ligate fragments with blunt ends, but the reaction is less efficient and higher concentrations of the enzyme are usually required in vitro. Source one DNA Source two DNA 5’--G-G-A-T-C-C--3’ 5’--G-G-A-T-C-C--3’ 3’--C-C-T-A-G-G--5’ 3’--C-C-T-A-G-G--5’ Cleavage with BamHI to generate fragments with sticky ends 5’--G G-A-T-C-C--3’ 3’--C-C-T-A-G G--5’ Base pairing of complimentary tail regions Gap 5’--G G-A-T-C-C--3’ 3’--C-C-T-A-G G--5’ Gap T4 DNA Ligase 5’--G-G-A-T-C-C--3’ 3’--C-C-T-A-G-G--5’ Ligated source one-two DNA Figure 3: Recombinant DNA molecules can be formed from DNA cut with restriction endonucleases that sticky or cohesive ends, such as BamHI. The sticky tails allow DNA fragments from two different sources to anneal. “Source 1” DNA and “source 2” DNA are then covalently linked by treatment with DNA ligase to create a recombinant DNA molecule. Note that the BamHI site is regenerated in the process. 8 2.3) Terminal deoxynucleotidyl transferase (terminal transferase) These enzyme catalyses the addition of deoxynucleotides to the 3′OH ends of DNA molecules. How is it different to the requirements of DNA polymerase III? Source one DNA Source two DNA 5’--C-C-C G-G-G--3’ 3’--G-G-G C-C-C--5’ Add poly (dT) tails Terminal deoxynucleotidyl transferase Add poly (dA) tails 5’--C-C-C-T-T-T G-G-G--3’ 3’--G-G-G A-A-A-C-C-C--5’ Base pairing of complimentary tail regions Gap 5’--G-G-G-T-T-T G-G-G--3’ 3’--C-C-C A-A-A-C-C-C--5’ Gap T4 DNA Ligase 5’--G-G-G-A-A-A-G-G-G--3’ 3’--C-C-C-T-T-T-C-C-C--5’ Ligated source one-two DNA Figure 4: Recombinant DNA molecules can be formed from DNA cut with restriction endonucleases that leave blunt ends, such as SmaI. Without end modification, blunt end ligation is of low efficiency. The efficiency is increased through using the enzyme terminal deoxynucleotidyl transferase to create complementary tails by the addition of poly(dA) and poly(dT) to the cleaved fragments. These tails allow DNA fragments from two different sources to anneal. Source 1 DNA and source 2 DNA are then covalently linked by treatment with DNA ligase to create a recombinant DNA molecule. Note that the SmaI site is destroyed in the process. 9 2.4) Phosphatases In cloning, the ligase reaction is used to covalently join passenger DNA to the vector DNA. However, it must be noted that the vector DNA can also self- anneal and be ligated without any foreign or passenger DNA inserts. This is not desirable. A 10: 1 ratio of passenger DNA to vector DNA to increase the possibility of passenger DNA – vector DNA annealing reactions and subsequently being ligated. Alternatively, calf intestinal phosphatase is employed to remove the 5′ phosphate from linearized vector DNA. Thus vector DNA will not ligate to itself since the 5′ phosphate is required for this reaction. Since the passenger DNA has 5′ phosphate, there will be some ligation due to the two 5′ phosphates provided passenger DNA. However no ligation will take place at the two other ligation points where the 5′ phosphates are provided by the vector DNA. This chimeric DNA with two unsealed gaps are still able to circularize and can therefore be transformed into cloning host where repair enzymes seal the gaps. 2.5) Large fragment of DNA polymerase I (Klenow fragment) This 76 kD enzyme is produced by cleavage of intact DNA polymerase I with subtilisin. This enzyme component has 5′-3′ polymerase activity and 3′-5′ exonuclease activity but lacks 5’-3′’ exonuclease activity of the intact enzyme. The Klenow fragment is employed for a variety of purposes including: - DNA sequencing using the Sanger dideoxy system. - Filling the recessed 3′ termini created by digestion of DNA with restriction enzymes. - Labeling the termini of DNA fragments using [32P] dNTPS in end-filling reactions. - Second strand synthesis in cDNA cloning. 10 2.7) RNA dependant-DNA polymerase (reverse transcriptase) The reverse transcriptase (RT) catalyzes the synthesis of a single-stranded DNA from the mRNA template. Like a regular DNA polymerase, reverse transcriptase also needs a primer to get started. Further it can have 5′-3′ exoribonuclease activity and 3′-5′ exoribonuclease activity that specifically degrades RNA in DNA-RNA hybrid molecules. This enzyme which is purified from RNA tumour viruses is mainly used to transcribe mRNA into dsDNA which can then be inserted into prokaryotic vectors. First CDNA is synthesized and then the RNA is degraded by alkali or ribonuclease H. Second strand synthesis is then carried out using Klenow fragment of DNA polymerase I or RT itself. In this synthesis cDNA acts as its primer and template through formation of a hairpin. It must be highlighted that RT it has no proofreading ability 11 3) SOURCES OF FOREIGN DNA CLONING a) Chromosomal DNA b) mRNA converted to cDNA c) PCR-amplified DNA 4) CLONING AND EXPRESSION VECTORS 4.1) Plasmid vectors Plasmids are named with a system of uppercase letters and numbers, where the lowercase “p” stands for “plasmid.” In the case of pBR322, the BR identifies the original constructors of the vector (Bolivar and Rodriquez), and 322 is the identification number of the specific plasmid. These early vectors were often of low copy number, meaning that they replicate to yield only one or two copies in each cell. pUC18 and pUC19 are derivatives of pBR322. These are “high copy number” plasmids (> 500 copies per bacterial cell). A high copy number occurs only with those plasmids whose replication in the host is under “relaxed control” as opposed to that under “stringent control”. Stringent control means that plasmid replication is coupled to that of host chromosome so that only one or at most a few copies are present in the bacterium. An ideal bacterial plasmid vector contains the following essential properties: i. An origin of replication (ori), so that they can independently replicate themselves and the foreign DNA segments they contain. ii. Selection marker/s that correspond to gene/s missing in the host cell. These are employed to distinguish clones that bear the recombinant vector from untransformed host cells do not contain a vector. a) Single or twin antibiotic resistance b) Blue-white screening iii. A multiple cloning site (polylinker region) that contains a number of unique restriction endonuclease cleavage sites that occur only once in the plasmid. The restriction sites must not be in regions of the plasmid that are required for replication iv. Efficient and simple extraction from the host cell. 12 Figure 6: pBR322 and pUC18 plasmid vectors. Modern cloning vectors are similar to the pUC19 plasmid, which has a polylinker constructed in between the lac I gene and the lac Z′ gene which is a modified lac Z gene. The polylinker is a multiple- cloning site (MCS). Note that the polylinker is downstream of promoter for the lac Z′ gene but does not affect its transcription. 13 4.1.1) Insertion of foreign DNA into plasmid vector 4.1.1.1 Single Restriction Transformation of E. coli Clones resistant to ampicillin contain recombinant DNA Figure 7: Insertion of genomic EcoRI restriction fragments into the pUC19 plasmid vector, which contains a MCS (polylinker). This approach is associated with the following disadvantages; i. The restricted plasmid has compatible sticky ends which can ligate. ii. The restricted genomic DNA fragment can be inserted in both orientations. iii. There is a possibility of multiple fragments inserting into the excised plasmid. 14  4.1.1.2 Double Restriction BamHI and EcoRI Ligation colonies colonies Figure 8: Insertion of foreign DNA restriction fragments into the pUC19 plasmid vector, which contains a MCS (polylinker). In this strategy, two restriction enzymes (BamHI and EcoRI) are employed to cleave both plasmid and genomic DNA components. 15  Double-restriction of both the plasmid vector and foreign DNA components as illustrated in Figure 8 eliminates to a large extent the problems that are usually associated with the single-restriction approach. Directional cloning is very important in expression systems where one wants to ensure that the DNA insert is of the correct orientation in respect to the expression vector’s transcriptional and translational control sequences. If the vector and the foreign DNA were cut with one restriction enzyme then approximately 50% of the foreign DNA would be inserted backwards which is highly undesirable. 4.1.2 Bacterial Transformation The recombinant plasmid is then taken into the bacterial cell by transformation. This involves making bacterial membrane transiently porous so that the recombinant plasmid can enter. Two methods are routinely employed for bacterial transformation; a) Transformation effected by treating a mixture of recombinant plasmid and a suspension of bacterial cells with calcium phosphate. The calcium co- precipitates with the DNA as particles which are taken up by the cell. b) Electroporation can also be used for transformation. The mixture or recombinant plasmids and bacteria are subjected to a high voltage discharge of approximately 2000-4000 volts. This creates reparable holes in the cell membrane through which foreign DNA can enter. After transformation there will be three types of bacterial cells: those without plasmids; those with plasmids having no foreign DNA inserts; and those with plasmids having foreign DNA inserts. The recombinant clones are selected on the basis of growth on an antibiotic containing medium or by blue–white screening. The lac Z gene encodes β-galactosidase in E. coli while lac Z′ encodes the α- peptide from the N-terminus of this enzyme. The form of the enzyme encoded by the chromosome of the host, E. coli, is inactive since it lacks the α- peptide. The plasmid and the host genes therefore direct the synthesis of two 16  complementary components of β-galactosidase that result in an active enzyme. Cloning DNA into the polylinker inactivates the lac Z′ gene. The enzyme β- galactosidase is therefore active only in E. coli that have plasmids without any cloned DNA within the polylinker. A functional β-galactosidase is detected by its ability to liberate a blue chromophore from a colorless chromogenic substrate 5-bromo-4-chloro-3- indolyl-β-galactoside (X-gal) which is hydrolyzed to galactose and 5-bromo-4- chloro-3-hydroxyindole which is blue. Isopropyl β-D-1-thiogalactopyranoside (IPTG), which functions as the inducer of the Lac operon, can be used in some strains to enhance the phenotype, although it is with many common laboratory strains unnecessary. This provides an easy to score selectable marker. When colonies grow on agar plates that contain X-gal, those transformants with plasmids having DNA inserts will be white, those having plasmids without DNA inserts will be blue. White colonies indicate insertion of foreign DNA and loss of the cells' ability to hydrolyse the marker. Figure 8: β-galactosidase mediated hydrolysis of X-Gal. 4.2) Bacteriophage vectors Bacteriophages are viruses that infect bacteria. They can be used as cloning vectors because of their greater efficiency in introducing foreign DNA into bacteria when compared to transformation with plasmids. 17  Bacteriophage vectors can be used to clone DNA fragments of more than 10 kbp. One of the first bacteriophages to be used in cloning was bacteriophage lambda (λ). This bacteriophage infects E. coli. Bacteriophage lambda (λ) can grow lysogenically or lytically. When it grows lysogenically it inserts its DNA into the host chromosome so that it is replicated together with the chromosome. When lambda grows lytically it makes copies of its genome and packages these into phage particles. It then lyses the host cell to release the new phages. The genome of bacteriophage lambda is 48.5 kbp of linear dsDNA. About 15 kbp of DNA in the middle of this genome are not essential for lytic growth as they contain genes for such functions as integration into the host genome and immunity. They can be removed and replaced with cloned DNA. In practice up to 18 kbp can be cloned since up to 52 kbp can be packaged into the head of lambda. On the other hand if the DNA being packaged into the lambda head is less then 38 kbp it will not package. Any cloning procedure involving λ has to take into account these constraints. After cutting out the non essential region in λ using an appropriate restriction enzyme, it is separated from the other DNA. A 15 kbp fragment of foreign DNA that has been cut with the same restriction enzyme is mixed with the phage DNA and ligation reaction performed. In vitro, this chimeric DNA is packaged into the phage head which infects the bacterium. A selectable marker is also required. The transformants with lambda containing the insert will form plaques. These are zones of clearing on a “lawn” of bacteria on a plate of agar. Another selection method involves the insertion of the lac Z′ gene into the non essential region. Since this is removed by the restriction enzyme when this region is excised, colonies of bacteria that have taken up lambda with a DNA insert will be white. Those that still have a non essential region that has not been replaced by the insert will have blue colonies. 4.3) Cosmids At each end, the linear λ DNA has 12 bp single-stranded 5′overhang or sticky end (Fig 8). These overhangs are actually cohesive due to complementary base-pairing. When the λ genome enters E. coli the cohesive ends anneal and 18  are ligated. The sealed cohesive ends give rise to what is called a cos site. The circular form of λ is essential for replication. Cosmids are plasmids which have a cos site inserted into them. As long as the distance between the cos ends is at least 38 kbp these cosmids will be packaged into phage heads as if they were λ DNA. Since the cosmid vector is about 5 kbp inserts of 33-47 kbp can be made. The cosmids therefore combine the advantages of cloning in a plasmid which include ease of cloning and propagation with those of cloning in a phage vector which include efficiency of transformation and capacity of the phage vector. 4.4) Bacterial Artificial Chromosomes (BACs) These vectors are based on the fertility plasmid (F plasmid). The F plasmid contains partition genes that allow for even distribution of plasmids between two daughter cells after bacterial cell division. F1 helps one bacterium to give its genes to another hence it can hold large DNA pieces from another bacterium. pBACs have an Ori site, a lac Z′ gene insert for selection and a cloning site for the gene of interest. They are designed for cloning large (>50 kbp) DNA sequences. Inserts of up to 300 kbp can be accommodated. pBACs are accommodated only as single copies in each bacterium and have been very useful in mapping of chromosomes. 5) EXPRESSION VECTORS Expression vectors are designed for expression of a target gene with the result that the relevant protein is produced in high quantities that can be up to 40% of the total cellular protein. They are mostly plasmid based and use the highly efficient and tightly controlled phage T7 RNA polymerase gene expression system. 19  A cloned structural gene is inserted into an expression vector which is a plasmid that contains properly positioned transcriptional and translational control sequences for the protein’s expression. The recombinant vector is transformed into an E. coli host that has a T7 RNA polymerase gene within its chromosome. Switching on the T7 RNA polymerase gene leads to high level synthesis of the target structural gene. This in turn results in high levels of synthesis of the protein encoded by this gene. Table 2: Features and applications of different vector cloning systems. Vector Basis Size limits of insert Composition Plasmid Naturally occuring ≤ 10 kb Subcloning and downstream multicopy plasmids manipulation, cDNA cloning and expression assays Phage Bacteriophage λ 5-20 kb Genomic DNA cloning, cDNA cloning, and expression libraries Cosmid Plasmid containing a 35-45 kb Genomic library bacteriophage λ cos construction site BAC Escherichia coli F 75-300 kb Analysis of large genomes (bacterial factor plasmid artificial chromosome) YAC (yeast Saccharomyces 100-1000 kb (1 Mb) Analysis of large genomes, artificial cerevisiae YAC transgenic mice chromosome) centromere, telomere, and autonomously replicating sequence 20