Genetic Engineering Notes PDF

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 ha...

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