Unit I Lecture 3

B. Tech. (Biotechnology) III Year V th Semester

EBT-501, Genetic Engineering

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: Saccharomyces cerevisiae 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.

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,

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.

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.

The Life Cycle of Bacteriophage M13

DNA cloning with single-strandedDNA vectors

• M13, f1, and fd are filamentous coliphages containing a circular single-stranded DNA molecule.

• These coliphages have been developed as cloning vectors, for they have a number of advantages over other vectors, including the other two classes of vector for E. coli: plasmids and phage λ

Filamentous bacteriophages have a number

of unique properties that make them suitable

as vectors

Unique properties of M13, f1, fd• Contain a single-stranded circular DNA molecule, which

is 6407 (M13) or 6408 (fd) nucleotides long.

• The complete nucleotide sequences of fd and M13 are available and they are 97% identical

• The filamentous phages only infect strains of enteric bacteria harboring F pili. The adsorption site appears to be the end of the F pilus

• Replication of phage DNA does not result in host-cell lysis. Rather, infected cells continue to grow and divide, albeit at a slower rate than uninfected cells, and extrude virus particles.

• Up to 1000 phage particles may be released into the medium per cell per generation

Vectors with single-stranded DNA genomes have specialised uses

• Sequencing by the original dideoxy method required single-stranded DNA

• oligonucleotide-directed mutagenesis

• probe preparation.

• can be purified and manipulated in vitro just like a plasmid.

• the size of the phage particle is governed by the size of the viral DNA and therefore there are no packaging constraints

Phagemids pUC118 and pUC119

Replication as Double-Stranded Plasmids

Replication as Single-Stranded Phage DNA

BACs and PACs are vectors that can carrymuch larger fragments of DNA than cosmids

because 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.

• 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

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.

An E. coli-Yeast Shuttle Vector

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.

The PAC Mammalian Shuttle Vector pJCPAC-Mam1

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.