Gene Cloning and Genetic Engineering: Concepts and Techniques

1. Unit I Lecture 2 B. Tech. (Biotechnology) III Year V th Semester EBT-501, Genetic Engineering

2. EBT 501, Genetic Engineering Unit I Gene cloning -concept and basic steps; application of bacteria and viruses in genetic engineering; Molecular biology of E. coli and bacteriophages in the context of their use in genetic engineering, Cloning vectors: Plasmid cloning vector PBR322, Vectors for cloning large piece of DNA; –Bacteriophage-l and other phage vectors; Cosmids, Phagemids; YAC and BAC vectors, Model vectors for eukaryotes – Viruses, Unit II Restriction modification, enzymes used in recombinant DNA technology endonucleases, ligases and other enzymes useful in gene cloning, PCR technology for gene/DNA detection, cDNA, Use of Agrobacterium for genetic engineering in plants; Gene libraries; Use of marker genes. Cloning of foreign genes: DNA delivery methods -physical methods and biological methods, Genetic transformation of prokaryotes: Transferring DNA into E. coli –Chemical induction and Electroporation, Unit III Gene library: Construction cDNA library and genomic library, Screening of gene libraries – screening by DNA hybridization, immunological assay and protein activity, Marker genes: Selectable markers and Screenable markers, nonantibiotic markers, Gene expression in prokaryotes: Tissue specific promoter, wound inducible promoters, Strong and regulatable promoters; increasing protein production; Fusion proteins; Translation expression vectors; DNA integration into bacterial genome; Increasing secretions; Metabolic load, Recombinant protein production in yeast: Saccharomycescerevisiae expression systems; Mammalian cell expression vectors: Selectable markers; Unit IV Origins of organismal cloning in developmental biology research on frogs; nuclear transfer procedures and the cloning of sheep (Dolly) & other mammals; applications in conservation; therapeutic vs. reproductive cloning; ethical issues and the prospects for human cloning; Two-vector expression system; two-gene expression vector, Directed mutagenesis; transposon mutagenesis, Gene targeting, Site specific recombination Unit V General principles of cell signaling, Extracellular signal molecule and their receptors, Operation of signaling molecules over various distances, Sharing of signal information, Cellular response to specific combinations of extracellular signal molecules; Different response by different cells to same extracellular signal molecule, NO signaling by binding to an enzyme inside target cell, Nuclear receptor; Ion channel linked, G-protein- linked and enzyme-linked receptors, Relay of signal by activated cell surface receptors via intracellular signaling proteins, Intracellular signaling proteins as molecular switches, Interaction between modular binding domain and signaling proteins, Remembering the effect of some signal by cells.

3. Unit I • Gene cloning -concept and basic steps • Application of bacteria and viruses in genetic engineering • Molecular biology of E. coli and bacteriophages in the context of their use in genetic engineering • Cloning vectors: Plasmid cloning vector pBR322, • Vectors for cloning large piece of DNA • Bacteriophage-l and other phage vectors • Cosmids • phasmids • Phagemids • BAC vectors • PAC vectors • YAC vectors • Model vectors for eukaryotes - Viruses,

4. Bacteriophage Vectors- phage  • Most bacteriophage cloning vectors have been constructed from the phage chromosome. • phage  is extensively studied virus of E. coli • The DNA of phage λ, in the form in which it is isolated from the phage particle, is a linear duplex molecule of about 48.5 kbp. • The entire DNA sequence has been determined • At each end are short single-stranded 5′ projections of 12 nucleotides, which are complementary in sequence and by which the DNA adopts a circular structure when it is injected into its host cell, i.e. λ DNA naturally has cohesive termini, which associate to form the cossite.

5. phage  • It is possible to insert foreign DNA into the chromosome of phage-λ derivative and, in some cases, foreign genes can be expressed efficiently via λ promoters. • The central one-third (about 15 kb) of the  chromosome contains genes required for lysogeny but not for lytic growth. • This portion of the chromosome can be excised and replaced with foreign DNA. • The foreign DNA inserted must be 10-15 kb.

6. It is possible to insert foreign DNA into the chromosome of phage-λ derivative and, in some cases, foreign genes can be expressed efficiently via λ promoters. • Functionally related genes of phage λ are clustered together on the map, except for the two positive regulatory genes N and Q. • Genes on the left of the conventional linear map (Fig. 4.10) code for head and tail proteins of the phage particle. • Genes of the central region are concerned with recombination (e.g. red) and the process of lysogenization, in which the circularized chromosome is inserted into its host chromosome and stably replicated along with it as a prophage. • Much of this central region, including these genes, is not essential for phage growth and can be deleted or replaced without seriously impairing the infectious growth cycle.

7. phage  • To the right of the central region are genes concerned with regulation and prophage immunity to superinfection (N, cro, cI), • followed by DNA synthesis (O, P), • late function regulation (Q), and • host cell lysis (S, R). • Next Figure illustrates the Map of the λ chromosome, showing the physical position of some genes on the full-length DNA of wild-type bacteriophage λ. • Clusters of functionally related genes are indicated.

8. The central one-third (about 15 kb) of the  chromosome contains genes required for lysogeny but not for lytic growth. • This portion of the chromosome can be excised and replaced with foreign DNA.

10. phage  • The lytic cycle, λ transcription occurs in three temporal stages: early, middle, and late. • Basically, early gene transcription establishes the lytic cycle (in competition with lysogeny), middle gene products replicate and recombine the DNA, and late gene products package this DNA into mature phage particles.

11. Cosmid Vectors • Hybrids between plasmids and the phage  chromosome. • Replicate autonomously in E. coli. • Can be packaged in vitro into phage  heads. • Accept inserts of 35-45 kb.

13. There are two basic types of phage lamda vectors:insertional vectors and replacementvectors • Wild-type λ DNA contains several target sites for most of the commonly used restriction endonucleases and so is not itself suitable as a vector. • Derivatives of the wild-type phage have therefore been produced that either have a single target site at which foreign DNA can be inserted (insertional vectors) or have a pair of sites defining a fragment that can be removed (stuffer) and replaced by foreign DNA (replacement vectors). • Since phage λ can accommodate only about 5% more than its normal complement of DNA, vector derivatives are constructed with deletions to increase the space within the genome. The shortest λ DNA molecules that produce plaques of nearly normal size are 25% deleted

15. Cosmids are plasmids that can be packaged into bacteriophage lamda particles • Concatemers of unit-length λ DNA molecules can be efficiently packaged if the cos sites, substrates for the packaging-dependent cleavage, are 37–52 kb apart (75–105% the size of λ+ DNA). • Only a small region in the proximity of the cos site is required for recognition by the packaging system • Plasmids have been constructed which contain a fragment of λ DNA including the cos site. • These plasmids have been termed cosmids and can be used as gene-cloning vectors in conjunction with the in vitro packaging system.

16. With a cosmid vector of 5 kb, we demand the insertion of 32–47 kb of foreign DNA – much more than a phage-λ vector can accommodate. • Note that, after packaging in vitro, the particle is used to infect a suitable host. The recombinant cosmid DNA is injected and circularizes like phage DNA but replicates as a normal plasmid without the expression of any phage functions. • Transformed cells are selected on the basis of a vector drug resistance marker. • Cosmids provide an efficient means of cloning large pieces of foreign DNA (50kb). Because of their capacity for large fragments of DNA, cosmids are particularly attractive vectors for constructing libraries of eukaryotic genome fragments. • Partial digestion with a restriction endonuclease provides suitably large fragments

20. Aims for using Phage vectors • rapid and efficient genomic or complementary DNA (cDNA) library construction • increase the capacity for foreign DNA fragments, preferably for fragments generated by any one of several restriction enzymes to devise methods for positively selecting recombinant formation. • • To allow RNA probes to be conveniently prepared by transcription of the foreign DNA insert; this facilitates the screening of libraries in chromosome walking procedures. An example of a vector with this property is λZAP

21. To develop vectors for the insertion of eukaryotic cDNA such that expression of the cDNA, in the form of a fusion polypeptide with β- galactosidase, is driven in E. coli; this form of expression vector is useful in antibody screening. An example of such a vector is λgt11

22. BACs and PACs • Bacterial artificial chromosomes (BACs) have been constructed from bacterial fertility (F) factors. • Bacteriophage P1 artificial chromosomes (PACs) have been constructed from bacteriophage P1 chromosomes. • BACs and PACs accept 150-300 kb inserts and are less complex than YACs.

23. Phagemid Vectors • Contain components from phage chromosomes and plasmids. • Replicate in E. coli as double-stranded plasmids. • Addition of a helper phage causes the phagemid to switch to the phage mode of replication, resulting in the packaging of single-stranded DNA into phage heads.

24. The Life Cycle of Bacteriophage M13

26. Phagemids pUC118 and pUC119

27. Replication as Double-Stranded Plasmids

28. Replication as Single-Stranded Phage DNA

29. BACs and PACs are vectors that can carrymuch larger fragments of DNA than cosmidsbecause they do not have packagingconstraints • Phage P1 is a temperate bacteriophage which has been extensively used for genetic analysis of Escherichia coli because it can mediate generalized transduction. • Sternberg and co-workers have developed a P1 vector system which has a capacity for DNA fragments as large as 100 kb. • Thus the capacity is about twice that of cosmid clones but less than that of yeast artificial chromosome (YAC) clones.

30. P1 vector contains a packaging site (pac) which is necessary for in vitro packaging of recombinant molecules into phage particles. • The vectors contain two loxP sites. These are the sites recognized by the phage recombinase, the product of the phage cre gene, and which lead to circularization of the packaged DNA after it has been injected into an E. coli host expressing the recombinase (Fig. 5.4). • Clones are maintained in E. coli as low-copy-number plasmids by selection for a vector kanamycin-resistance marker. • A high copy number can be induced by exploitation of the P1 lytic replicon (Sternberg 1990). • This P1 system has been used to construct genomic libraries of mouse, human, fission yeast, and Drosophila

32. Eukaryotic and Shuttle Vectors • Because different organisms use different origins of replication and regulatory signals, different cloning vectors must be used in different species. • Special cloning vectors can replicate in other prokaryotes and in eukaryotes. • Shuttle vectors can replicate in E. coli and in another species.

33. An E. coli-Yeast Shuttle Vector

34. Yeast Artificial Chromosomes (YACs) • Genetically engineered yeast minichromosomes. • Accept foreign DNA inserts of 200-500 kb. • Contain a yeast origin of replication, yeast centromere, two yeast telomeres, a selectable marker, and a polycloning site.

35. The PAC Mammalian Shuttle Vector pJCPAC-Mam1

36. Episomes • An episome is a genetic element that is not essential to the host and that can either replicate autonomously or be integrated into the bacterial chromosome. • Integration depends on the presence of IS elements.